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95 The importance of molecular and cytogenetic markers in the prognostic of myelodysplastic syndromes Nicorescu Iuliana-Maria

ORIGINAL PAPERS

99 Cognitive impairment evaluation in children with dystrophinopathiesPop Anita, Cornitescu M., Pleșca Doina Anca

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109 The dual role of activated species of oxygen in angiogenesisAldea C., Aldea Alina Elena, Negoita Valentina, Anghel Rodica, Gruia Maria Iuliana

114 Outcomes in distal hypospadiasBălănescu R.N., Niculescu Laura, Bălănescu Laura, Moga Andreea

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118 Acute ectopic pancreatitis with ileus occurring in gastric ectopic pancreatic tissueTabacelia Daniela, Ilie Mădălina, Ene D., Constantinescu G., Macovei B., Asmarandei R., Stănciulescu Elena- Luminiţa, Diaconu Camelia, Popa B.

121 Neck suppurations in childrenPalade D., Toader Mioriţa, Constantin Anca, Niculescu L., Drăghici M., Vivisenco Iolanda Cristina

124 Liver metastasis with unknown primary originConstantin Alina, Plotogea Oana, Ilie Mădălina, Constantinescu G., Macovei B., Asmarandei R., Stănciulescu Elena-Luminiţa, Diaconu Camelia, Popa B.

Vol. XIX Number III September 2015

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XIX, Vol.19, Number 3/2015 95

Iuliana-Maria Nicorescu40 Ficusului Blvd, 1st District, 013975, Bucharest, Romaniae-mail: [email protected]

Abstract. Myelodysplastic syndromes represent clonal diseases of hematopoietic stem cells that occur predominantly in elderly people, but myelodysplastic syndromes can affect younger patients as well.Myelodysplastic syndromes are characterized by ineffective hematopoiesis, causing different forms of cytopenia in peripheral blood, contrasting normal or hypercellular bone marrow. Although less known, due to its absence from current clinical practice or in the prognostic score, molecular analysis could bring a significant contribution in the early diagnosis of disease. In this respect, the latest studies (2011) show that this type of analysis is of great importance in terms of input added to the pathogenic mechanisms of the Myelodysplastic syndromes subtypes establishment, with high conversion. Furthermore, the analysis serves elect the optimal timing for the initiation of specific therapy.Keywords: myelodysplastic syndromes, acute leukemias, molecular analysis

Nicorescu Iuliana-Maria1

THE IMPORTANCE OF MOLECULAR AND CYTOGENETIC MARKERS IN THE PROGNOSTIC OF MYELODYSPLASTIC SYNDROMES

REVIEWTherapeutics, Pharmacology and Clinical ToxicologyVol XIX, Number 3, September 2015Pages: 95 - 98© Copyright reserved 2015

Introduction

Myelodysplastic syndromes are clinically heterogeneous disorders characterized by

byclonal hematopoiesis, impaired differentiation, peripheral-blood cytopenias, and a risk of progression to acute myeloid leukaemia. Somatic mutations may influence the clinical phenotype but are not included in current prognostic scoring systems[1].

Myelodysplastic syndromes represent a heterogeneous group of hematologic disorders difficult to diagnose, which is more often underdiagnosed or identified in a stage of transformation into acute leukaemia.

Myeloid malignancies are clonal diseases of hematopoietic stem cells or cell progenitors. These diseases also contain chronic disease which includes myelodysplastic syndromes, chronic myeloproliferative diseases, chronic myelomonocytic leukaemia and acute disorders represented by acute leukemia.

Being a heterogeneous disease category, both in terms of clinical, biological, morphological as well as cytogenetic and molecular manifestation, during the recent years research on cytogenetic and molecular-level changes has expanded. The purpose of this research is the establishment of a loyal group identifier score of increased risk of transformation of the myelodysplastic syndromes into acute leukaemia, linking all mentioned prognostic factors.

At this point, molecular analysis in MDS is less known

and has not yet been introduced into prognostic score, but according to the latest studies (since 2011) it has a great importance in terms of obtaining information on pathogenic mechanisms.

Definitions and classificationMDS - represent clonal diseases of hematopoietic

stem cells, which predominantly occur in elderly people, characterized by ineffective hematopoiesis causing different forms of cytopenia in peripheral blood, contrasting normal or hypercellular bone marrow.

Acute leukaemia is a heterogeneous group of malignant proliferation of pluripotent or unipotent stem cells, characterized by clonal expansion of immature cells that lost the ability to differentiate.

MDS classification – WHO classification - 2008 World Health Organization MDS

Classification and Criteria

• Refractory cytopenia with unilineage dysplasia (RCUD);

• Refractory anaemia (RA), refractory neutropenia (RN), refractory thrombocytopenia (RT).Unicytopenia or bicytopenia, No or rare blasts (<1%), Unilineage dysplasia: 10% of the cells in one myeloid lineage;

• Refractory anaemia with ring sideroblasts (RARS): Anaemia, No blasts, 15% of erythroid precursors are ring sideroblasts Erythroid dysplasia only <5% blasts;

• Refractory cytopenia with multilineage dysplasia (RCMD): Cytopenia(s), No or rare blasts (<1%), No Auer rods <1% monocytes Dysplasia in 10% of the cells in two or more myeloid lineages, <5% blasts in marrow No Auer rods 15% ring sideroblasts;

1 Hematology, Neolife Medical Center, Bucharest, Romania

Therapeutics, Pharmacology and Clinical Toxicology96

• Refractory anaemia with excess blasts-1(RAEB-1): Cytopenia(s) <5% blasts No Auer rods <1% monocytes Unilineage or multilineage dysplasia,5%-9% blasts No Auer rods;

• Refractory anaemia with excess blasts-2 (RAEB-2): Cytopenia(s)5%-19% blasts Auer rods,<1 % monocytes Unilineage or multilineage dysplasia 10%-19% blasts Auer rods.

Myelodysplastic syndrome unclassified (MDS-U): Cytopenias 1% blasts Unequivocal dysplasia in <10% of cells in one or more myeloid cell lines when accompanied by a cytogenetic abnormality considered as presumptive evidence for a diagnosis of MDS <5% blasts.

MDS associated with isolated del(5q) Anaemia Usually normal or increased platelet count No or rare blasts (<1%), Normal to increased megakaryocytes with hypolobated nuclei <5% blasts, Isolated del(5q) cytogenetic abnormality No Auer rods.

“The IPSS and the WHO classification system incorporate only the most common chromosomal abnormalities. An international effort is under way to develop a comprehensive cytogenetic scoring system for the myelodysplastic syndromes that incorporates rare cytogenetic subgroups, which will inform an ongoing revision of the IPSS. With the advent of more sensitive techniques already available in the research setting, including next-generation genome and transcriptome sequencing and arrays of single-nucleotide polymorphisms for the detection of copy-number alterations, the rate of discovery will accelerate, and the compendium of genetic alterations in the myelodysplastic syndromes will undoubtedly expand. Detailed analyses of bone marrow samples from a larger number of patients with myelodysplastic syndromes are needed to establish the spectrum and frequency of mutations, the degree of genotypic overlap, and their clinical significance, particularly in predicting outcomes for available therapies”.[2]

At this moment, according to the studies it is estimated that approximately 50% of patients with MDS show caryotype changes (Fig.1.), most common are the partial deletions – del 5q, del 20q; del 7q; del 11q; del 12p; Losses chromosome monosomy 7; y ;17; Extra chromosome – trisomy 8,11,21; translocation;

complex abnormalities.

According to cytogenetic analysis we could obtain:• normal caryotype• balanced chromosomal abnormalities• unbalanced chromosomal abnormalities ➝ ➝ ➝

common by MDS with transformation risk • complex caryotype ➝ ➝ ➝ common by MDS with

transformation risk

5q deletion – the most common chromosomal abnormality identified in half of the isolated cases, often associated with refractory anaemia { AR } in terms of FAB classification, most common in women- in general good prognosis . It is characterized by increased transfusion requirements and associated hemochromatosis. (fig 2)

It can occur in association with other abnormalities, being characterized by recurrent and severe anaemia, accompanied by leucopoenia and thrombocytopenia. In terms of FAB most cases are AREB1 and AREB2 with

increased risk of transformation into acute leukaemia and poor prognosis.[1].

Del 17p: (fig.3.) the rearrangement of chromosome 17 includes del. 17p or translocations t(5;17)(p11;p11), t(7;17)(p11;p11) or i(17p)(q10). In general del17p is associated with other chromosomal mutations. The patient frequently presents TP53 mutation with low or no response to chemotherapy. Primary 17p deletion (de novo) occurs in patients who have undergone chemotherapy or radiotherapy for other neoplasia.

Patients are showing a reserved prognosis and 4 months median survival rate after evolution into acute myeloid leukemia. It was reported in AML and MDS a strong correlation between 17p deletion (a clonal cytogenetic anomaly consisting of a deletion of the short arm of chromosome 17), and a particular form of morphological dysgranulopoiesis, we also found in such cases a strong correlation between 17p deletion and p53 mutation; these correlations suggest that AML and MDS with 17p deletion constitute a mew morphological- cytogenetic- molecular entity, the 17p syndrome.” [5]

MDS and AML de novo or secondary to an exposure to chemical mutagens or to chemotherapy with alkylating agents may probably also be secondary to immunosuppressive therapy for severe aplastic anaemia. “Chromosome 7 anomalies are not rare in acute Fig.1.Distribution of cytogenetic findings in MDS [3]

Fig.2.Deletion 5q [4]


lymphocytic leukemia – ALL. They occur in balanced translocations involving 7p15 or 7q34 in T lineage and 7q22 or 7q32 in B proliferations; monosomy 7 is present in 5 to 6% of ALL, most often as a secondary anomaly of

the t(9,22)”[8].

Deletion 12p (fig. 4): 12 p abnormalities are common in a broad spectrum of haematological malignancies – ALL; AML, MDS, or chronic myeloproliferative syndromes, nonHodgkin’s lymphoma.

5% of AML secondary MDS after prior mutagenic exposure associated with a poor prognosis (karyotypes

mostly complex). Duplication 12 (p1,2p13) described in one MDS case after benzole agent exposure.

Monosomy 7: In adults second, at children first as frequency, is associated with monocytosis, splenomegaly and poor prognosis.

“MDS is rare in childhood and may have a rapidly progressive course with an extremely poor prognosis without hematopoietic stem cell transplantation (HSCT). The disease can arise in a previously healthy child; in this case, it is referred to as de novo or primary MDS. MDS may develop in a child with a known predisposition (egg, previous cytotoxic chemotherapy); this is referred to as secondary MDS (see Etiology). The disease is most common in adults, especially elderly people, and the course varies, ranging from an acute, rapidly fatal illness to a chronic, indolent illness. When a child presents with cytopenias associated with MDS, physicians should administer supportive care until the diagnosis is established. Many patients present with profound cytopenia and a notable risk for infection. Transfusions and broad-spectrum antibiotics may be required to

treat life-threatening anaemia, thrombocytopenia, and infection until definitive therapy can be started. In MDS paediatric patients with refractory cytopenia, hematopoietic stem cell transplantation (HSCT) from a matched related or unrelated donor early in the course of the disease is the treatment of choice.”[6,9,10]

Trisomy 8 is the only amplified chromosomal disorders associated with MDS and other haematological malignancies and solid tumours. Studies that compare normal cells CD34+ with those in MDS associate with trisomy 8, highlight the presence and increasing imbalance between pro and anti-apoptotic genes. Tested in culture the CD34+ cells combined with trisomy 8 proved to be radiation resistant and gained strength, being able to form hematopoietic colonies, despite the apoptotic protein presence, normally associated with rapid aging and cell death. A subset of patients with MDS and trisomy 8 distinguished a clinical response to immunosuppressive therapy. These patients are young, associate refractory anaemia and show HLA -DR15.

Y - : loss of Y chromosome in case of patients with MDS compared to those with normal karyotype, show favourable prognosis according to the IPSS.

X chromosome abnormalities (fig.5) – loss of an X chromosome in female patients with MDS has been with FISS to affect blast cells as well as myeloid elements of the marrow. A typical structural rearrangement has been proposed for some case RARS. Isolated deletion of Xq as

a sole cytogenetic abnormality in MDS is relatively rare

with REAB and unfavourable prognostic.Evolution and prognosisPrognostic factors and framing into acute leukaemia

transformation and / or death risk group represent continuous changing criteria.

MDS were often called "preleukemic states", which highlights increased risk (10=40% depending on the subtype of the WHO classification) of progression to acute leukaemia. Median survival rate varies depending on the subtype: 20-65 months in AR, 21-76 months ARSI t 5-12 months RAEB.

Based on morphological and biological factors different scores were created. The most used prognostic

Fig.3.Deletion 17p [7]

Fig.4.Deletion 12p [7]

Fig.5.X chromosome abnormalities [7]


scoring system is IPSS (table I).xCaryotype: normal - 46XY; god - Y– / 5q– /

20q–isolate; unfavourable - complex Caryotype (>3 abnormality) or abnormalities of chromosome 7; intermediate – all other abnormalities

xxCytopenia:Hb<10 g/dl, PMN <1.500/mmc, Tr<100.000//mmc

By identifying the high-risk genetic mutations and the correlation of subgroups with molecular changes in subgroups with cytogenetic changes, associated to already established markers in the diagnosis and tracking patients with MDS, the assay is attempting to establish a more accurate diagnose and monitoring protocol for these patients.

MDS remains one of the most challenging adult malignancies with varied and complex features at presentation. Immunophenotyping, cytogenetic-molecular studies and, more recently, high-resolution genome-wide screening, characterize MDS as a heterogeneous disease with distinct manifestations and prognostic and therapeutic implications.

ACKNOWLEDGEMENT: This presentation has been elaborated and written by Iuliana-Maria Nicorescu, MD and third year PhD student since 2012 at UMF Carol Davila under coordination of Prof. Dr. Ana-Maria Vlădăreanu, MD, PhD. This paper is supported by European Project code POSDRU/159/1.5/S/141531, entitled “Human Resource Development - PhD students and postdocs - for research excellence in health and biotechnology”.

References1. Rafael Bejar, Benjamin L Ebert: “The genetic basis

of Myelodisplastic syndromes” Elsevier 2010;

2. Rafael Bejar, M.D., Ph.D., Kristen Stevenson, M.S., Omar Abdel-Wahab, M.D et al –„Clinical Effect of Point Mutations in Myelodisplastic Syndromes” TheNew England journal of medicine – jun 2011 - 2946- 2948;

3. Schantz J, Tuchler H, Sole F, Mallo M, Luno E et al „New comprehensive cytogenetic scoring system for primary MDS and oligoblastic acute myeloid leukemia after MDS derived from an international database merge” J ClinOncol 2012, 30: 820-829;

4. Boultwood Blood 20025. Wintrobe’s Clinical Hematology – Twelfth edition

1956-1976.6. A n n e M u r a t i , M a n d y B r e c q u e v i l l e ,

RaynierDevillier, et al: „Myeloid malignancies: mutations, models and management” BioMed Central – 2012;

7. Atlas of genetics and cytogenetics in oncology and Hematology;

8. Grant E, Nybakken and Adam Bagg “The genetic basis and expanding role of molecular analysis in the diagnosis, prognosis and therapeutic design for MDS” The Journal of Molecular Diagnostics – Elsevier –march 2014;

9. Patnaik MM, Hanson CA, Hodnefield JM, et al: „Monosomal karyotype in MDS, with or without monosomy 7 or 5 is prognostically worse than an otherwise complex karyotype” Leukemia 25: 266-270, 2011;

10. Prasad Mathew, MBBS, DCH, FAAP; Chief Editor: Robert J Arceci, MD, PhD „Pediatric Myelodysplastic Syndrome - Monosomy 7” Medscape;

The prognostic value of the risk factors 0 0.5-1.0 1.5-2.0 ≥2.5

Marrow blasts % <5 5-10 11-20 21-30

Caryotype (meaning)x normal good intermediate unfavourable

cytopenia 0 1 2 3

Table I. IPSS prognostic scoring system


Doina Anca Pleșca21 Basarabia Bvd., Bucharest, Romaniae-mail: [email protected]

Abstract. Human dystrophinopathies are X-linked genetic disorders characterized by impairment in the function of dystrophin in a wide range of tissues. The identification of multiple alternative promoters explains the tissue-specific expression of the dystrophin isoforms. Beside the prominent muscular disease, cognitive impairment has been described in dystrophinopathies; however, the genotype – cognitive phenotype relationship is not yet fully clarified. The aim of the present study was to explore the impact of age and genetic lesion on the visuo-motor performance in Romanian pediatric subjects with dystrophinopathies. Duchenne and Becker muscular dystrophies pediatric patients admitted during a 12 months period in “Dr. V. Gomoiu” Children's Hospital, Bucharest were investigated with the use of Raven Progressive Matrices Tests. Most of the studied subjects had exon deletions in the 45-51 range in the dystrophin gene. The obtained results do not support a progressive cognitive impairment in these subjects.

Key words: dystrophinopathies, cognitive function, genetic mutations

Pop Anita1, Cornitescu M1, Pleșca Doina Anca1

COGNITIVE IMPAIRMENT EVALUATION IN CHILDREN WITH DYSTROPHINOPATHIES

ORIGINAL PAPERTherapeutics, Pharmacology and Clinical ToxicologyVol XIX, Number 3, September 2015Pages: 99 - 102© Copyright reserved 2015

Introduction

Human dystrophinopathies are X-linked diseases caused by impairment in the function of the

dystrophin protein. The most evident clinical expression of dystrophin impairment is the progressive muscle disorder, manifested as proximal limb muscle weakness, development of calf pseudohypertrophy, Gowers sign (climbing from the floor only possible by walking up one's own body), muscle contractures, defective body positions [1,2] and respiratory difficulty [3].

In what concerns the muscle function impairment, three dystrophinopathy phenotypes have been described: the Duchenne (DMD) and Becker (BMD) muscular dystrophies and an intermediate phenotype [4]. Of them, DMD is the most frequent, with an incidence of 1 in 3500 liveborn males [5], while BMD has an estimated incidence of around 1 in 18000 liveborn males [6] and the intermediate phenotype is even less frequent.

Cognitive deficits have been frequently observed in subjects with dystrophinopathies. They had already been mentioned in the original description of DMD by Duchenne de Boulogne [7].

Later reports confirmed a decrease of the full scale IQ (FSIQ) in DMD subjects with approximately one standard deviation below population mean [8,9]. The

cognitive impairment seems to be more severe in verbal than performance tests [9,10].

An increased incidence of attention deficit and hyperactivity disorder (ADHD) and of autism spectrum pathology [11,12] were also reported in DMD children. Deficits in working memory and executive function have also been described [1].

The dystrophin gene is located on the short arm of the X chromosome, in the Xp21.2 region, being composed of 79 exons. The existence of multiple alternative promoters explains the differential tissue expression of dystrophin isoforms [13]. Different gene lesions have specific effects on the expressed isoforms and on the induced pathologic phenotypes.

The most frequent lesions of the dystrophin gene are large deletions encompassing exons (approximately 65% of DMD and BMD patients) [13]. The vast majority of large dystrophin gene deletions cluster around two mutation “hotspots”, spanning exons 45-53 (the distal hotspot) and exons 2-20 (the proximal hotspot) [13]. The other types of mutations found in dystrophinopathies are small deletions and point mutations, that generally introduce premature stop codons and are evenly distributed throughout the gene [13].

Besides the 5' promoter that drives the transcription of the largest dystrophin protein isoform Dp427, at least four other internal promoters control the expression of other dystrophin isoforms in the brain (namely Dp260, Dp140 and Dp71) [14-16].

Several reports suggest a role of mutations in Dp140 and Dp71 in the cognitive impairment in subjects with

1 “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania


distrophinopathies [17-20] and a protective role of an intact brain Dp140 isoform against cognitive impairment in dystrophinopathies [19,20].

The differential role of dystrophin isoforms in the brain is further emphasized by the finding that subjects with mutations in the distal portion of the gene seem to have a greater risk for cognitive impairment in comparison with subjects presenting other genetic mutations [11,17,20,21], while subjects with deletion of the full-length dystrophin promoter at the proximal end of the gene have intelligence within the normal range [11,20,21-23].

The transcription of Dp140 is initiated in intron 44 of the dystrophin gene; the transcript has a long 5' untranslated region (5'UTR) of 1.041 Kbp, the transcription continuing with exon 51 of the gene [20]. Dp71 is initiated in intron 62 of the gene [20].

Mutations in the region spanning exons 45-50 and the portion of exon 51 that lies 5' of c.7381 of the dystrophin gene are considered to affect the sequence of Dp427 and Dp260 and to modify the 5'UTR of the Dp140 isoform, but not to affect the expression of Dp71; on the other side, mutations that are located upstream of intron 44 preserve the expression of Dp140, and Dp71, while affecting the expression of Dp427 and Dp260 [20].

Despite the amount of knowledge accumulated regarding the impact of mutations affecting the expression of Dp140 and Dp71 on the cognitive abilities in subjects with dystrophinopathies, a stringent genotype-phenotype correlation has not been established and predictions about the risk of cognitive disabilities based on structural features are not possible.

The aim of the present study was to investigate the influence of the genetic lesion and of age on the cognitive abilities of Romanian Duchenne and Becker muscular dystrophy pediatric subjects.

Materials and method Study groupThe study was performed on Duchenne and Becker

muscular dystrophy pediatric subjects admitted for routine controls at “Dr. V. Gomoiu” Children's Hospital, Bucharest, during a 12 months period (the year 2014).

The diagnosis was established using clinical, biochemical, histological and genetic methods. The genetic lesion was investigated using multiplex ligation-dependent probe amplification (MLPA) and gene sequencing on genomic DNA extracted from peripheral blood lymphocytes.

The study was performed in accordance with the human subjects protection provisions of “Carol Davila” University of Medicine and Pharmacy, Bucharest.

Cognitive function testsThe visuo-motor performances of studied subjects

were studied using the Raven Progressive Matrices Tests [24]. The tests consist of a series of increasing difficulty problems in which the subjects indicate missing elements in graphical matrices based on their inference of rules from already present items. The Raven tests are available in colour and black-and-white versions. The intelligence quotient is calculated based on the

performance of age-matched pediatric populations.Data analysisThe location of mutations in the dystrophin gene

was inspected in order to determine the regions with higher mutation frequency in the study group.

The correlation of IQ and age was investigated using the non-parametric Spearman’s rank correlation test and the parametric Pearson’s product moment correlation test. p values lower than 0.05 were considered significant.

ResultsThe study group included 19 subjects with

dystrophinopathies aged between 4.33 and 16.5 years (mean age 9.01, standard deviation 2.95), Romanian language natives, presenting Duchenne and Becker muscular dystrophy phenotypes. Of the 19 subjects, 17 were male and 2 female; 13 subjects (68% of total) presented Duchenne muscular dystrophy and 6 subjects (32% of total) - Becker muscular dystrophy phenotype (Table I, Figure 1).

The age of subjects in the group with exon deletions in the exon range 45-51 varied between 5.16 and 16.5 years, with a mean age of 10.29, and a standard deviation of 3.24 (Figure 2)

Type of genetic lesions in the study groupOf the studied group, 13 subjects (68%) presented

exon deletions, while the rest presented mutations other than exon deletion, including point mutations.

Exon deletions were identified in 10 (77%) of the Duchenne muscular dystrophy subjects and in 3 (50%) of the Becker muscular dystrophy subjects.

Two regions with increased exon deletion frequency were detected: a proximal region (exons 2-7) and a distal one (exons 44-51) (Figure 1).

The majority of exon deletions subjects presented a DMD phenotype. Surprisingly, a subject with a large exon deletion (exons 2-7) presented a BMD phenotype (Figure 1). Of the 6 subjects with mutations other than exon deletions, 3 presented DMD and 3 presented BMD phenotype.

Relation between the visuo-motor performance and the genetic lesion

The IQ values obtained by applying the Raven Progressive Matrices Tests in the study sample were in the 75 to 120 range, with a mean of 91.21 and a standard deviation of 11.59. For the subjects with exon deletions

Subjects Total Males Females

Sex 19 (100% of total)

17 (89% of total)

2 (11% of total)

DMD phenotype

13 (68% of total)

12 (92% of DMD subjects)

1 (8% of DMD subjects)

BMD phenotype

6 (32% of total)

5 (83% of BMD subjects)

1 (17% of BMD subjects)

Age 4.33-16.5 years 4.33-16.5 years 10.41 years (twins)

Table I. Structure of the study group


in the 45-51 region, the IQ varied between 83 and 95, with a mean IQ value of 89.11 and a SD of 4.67 (Figure 3).

The study of the tendency of linear variation of IQ with age for the group of subjects with exon deletions in the 45-51 range suggested a tendency of minor decrease of IQ with age ([x]=age, [y]=IQ, y=-0,64x+96,22) (Figure 4). However, the coefficient of determination for the linear correlation was weak (R2=0.16) and both the non-parametric and the parametric correlation analyses did not reach significance (Spearman’s rank correlation p=0.15, Pearson’s product moment correlation p=0.34).

Discussion and conclusions The study sample included subjects admitted for

routine controls during a 12 months period in “Dr. V. Gomoiu” Children's Hospital, Bucharest. They were mostly male (89%) as expected from the X-linked character of the pathology. Their age varied between 4.33 and 16.5 years (mean age 9.01, standard deviation 2.95), with a predominance of cases in the 8-11 years old range.

The ratio of DMD to BMD cases in the study sample was 2.16; the difference from the estimated ratio in the general population may be explained by the bias introduced by the different hospitalization necessities of the two phenotypes.

The detected exon deletions in the dystrophin gene were located in two particular regions, namely a proximal region (exons 2 to 7) and a distal region (exons 44 to 51) (Figure 1). The two regions are superposable with the two hotspots already described in the literature [13].

The majority of exon deletions in the 44-51 exon range were accompanied by DMD phenotype (in 9 of 10 subjects). 2 of 3 subjects with exon deletions in the 2-7 exon range presented BMD phenotype (one of them had a longer deletion encompassing the whole 2-7 exon region). Of the group of 6 subjects with mutations other than exon deletions, 3 subjects presented Duchenne and 3 Becker muscular dystrophy phenotype.

The investigation of the visuo-motor performance by Raven Progressive Matrices Tests showed an IQ spanning the interval between 75 to 120 (mean IQ 91.21, SD 11.59) for the entire study sample. The large SD may be accounted for by the varied type of genetic lesions in the study subjects.

For the group of subjects with exon deletions in the exon range 45 to 51 the IQ varied in a narrower range of 83 to 95 (mean IQ 89.11, SD 4.67), suggesting a more uniform group with a stronger genotype-phenotype correlation. The range of IQ values is similar with that reported for subjects with similar mutations in the dystrophin gene located in the region corresponding to the 5'UTR of the Dp140 dystrophin isoform [20].

The slope of the linear model approximating the dependence of the IQ on the age of subjects in the exon 45-51 deletion group was negative (-0.64) suggesting a mild decrease of IQ with age; however, the lack of significance of the results (p higher than 0.05) does not support a correlation of IQ with age. The obtained result is in accordance with published reports suggesting that the cognitive deficits in dystrophinopathies may be

non-progressive, in contrast to the progressive aspect of the muscular degeneration [11].

The data presented in the current work do not support a correlation of cognitive impairment with age in pediatric subjects with dystrophinopathies with deletions in the 45 to 51 exon range.

The location of the genetic defect in exon deletion subjects was pictured. The horizontal axis maps the location of the deleted exons. Darker color and symbol “D” signifies Duchenne muscular dystrophy, while lighter color and symbol “B”- Becker muscular dystrophy. The numbers in parentheses represent numbers of cases with identical genetic defect and phenotype in the studied group.

Number of cases were presented for specific dystrophin gene lesion types: deletions in the exon 44-51 region (D44-51); deletions in the exon 2-7 region (D2-7); other mutations than exon deletions (O).

Number of cases were presented for specific dystrophin gene lesion types: deletions in the exon 44-51 region (D44-51); deletions in the exon 2-7 region (D2-7); other mutations than exon deletions (O).

Fig.1. Repartition of the detected exon deletions in the dystrophin gene

Fig.2. Age distribution of the subjects in the study sample

Fig.3. Intelligence quotients (IQ) distribution of subjects in the study sample


Intelligence quotients (IQ) were measured using Raven tests. Rhombs represent subjects with exon deletions in the region between exons 45 and 51 (D45-51), asterisk- the subject with a deletion of exon 44 (D44), triangles- subjects with exon deletions in the interval between exons 2 and 7 (D2-7), circles- subjects with mutations other than exon deletions (O)

References 1. Darras BT, Miller DT, Urion DK.

Dystrophinopathies. 2000 Sept 5 [Update 2014 Nov 26]. In: Pagon RA, Adam MP, Ardinger HH, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2015. Available from: http://www.ncbi.nlm.nih.gov/books/NBK1119/ (Accessed on February 24, 2015).

2. Kinali M, Main M, Eliahoo J, et al. Predictive factors for the development of scoliosis in Duchenne muscular dystrophy. Eur J Paediatr Neurol. 2007; 11: 160-6.

3. Finder JD, Birnkrant D, Carl J, et al. Respiratory care of the patient with Duchenne muscular dystrophy: ATS consensus statement. Am J Respir Crit Care Med. 2004; 170: 456-65.

4. Darras BT. Clinical features and diagnosis of Duchenne and Becker muscular dystrophy. In: UpToDate, Post TW (Ed), UpToDate, Waltham, MA; www.uptodate.com; 2015 (Accessed on February 24, 2015).

5. Emery AE, Emery ML. The History of a Genetic Disease: Duchenne Muscular Dystrophy or Meryon's Disease 2nd Edition (Oxford Medical Histories). Oxford University Press, 2011.

6. Emery AE. Population frequencies of inherited neuromuscular diseases: a world survey. Neuromuscul Disord. 1991; 1: 19-29.

7. Duchenne (de Boulogne) GB. Recherches sur la paralysie musculaire pseudohypertrophique ou paralysie myo-sclérosique. Archives générales de médecine, 1868; 11: 179-209.

8. Emery A, Muntoni F. Duchenne muscular dystrophy. 3rd ed. Oxford: OxfordUniversity Press; 2003.

9. Cotton S, Voudouris NJ, Greenwood KM. Intelligence and Duchenne muscular dystrophy: Full-scale, verbal, and performance intelligence quotients. Dev Med Child Neurol 2001; 43: 497-501.

10. Hinton VJ, De Vivo DC, Nereo NE, et al. Selective deficits in verbal working memory associated with a known genetic etiology: the neuropsychological profile of Duchenne muscular dystrophy. J Int Neuropsychol Soc 2001; 7: 45–54.

11. Anderson JL, Head SI, Rae C, Morley JW. Brain function in Duchenne muscular dystrophy. Brain. 2002; 125: 4-13.

12. Hendriksen JG, Vles JS. Neuropsychiatric disorders in males with duchenne muscular dystrophy: frequency rate of attention-deficit hyperactivity disorder (ADHD), autism spectrum disorder, and obsessive--compulsive disorder. J Child Neurol. 2008; 23: 477-81.

13. Blake DJ, Weir A, Newey SE, Davies KE. Function and genetics of dystrophin and dystrophin-related proteins in muscle. Physiol Rev. 2002; 82: 291-329

14. Blake DJ, Kroger S. The neurobiology of Duchenne muscular dystrophy: learning lessons from muscle? Trends Neurosci 2000; 23: 92–9.

15. Mehler MF. Brain dystrophin, neurogenetics and mental retardation. Brain ResBrain Res Rev 2000; 32: 277–307.

16. Muntoni F, Torelli S, Ferlini A. Dystrophin and mutations: one gene, several proteins, mutiple phenotypes. Lancet Neurol 2003; 2: 731–40.

17. Moizard MP, Billard C, Toutain A, et al. Are Dp71 and Dp140 brain dystrophin isoforms related to cognitive impairment in Duchenne muscular dystrophy? Am J Med Genet 1998; 80: 32–41.

18. Moizard MP, Toutain A, Fournier D, et al. Severe cognitive impairment in DMD: obvious clinical indication for Dp71 isoform point mutation screening. Eur J Hum Genet 2000; 8: 552–6.

19. Bardoni A, Felisari D, Sironi M, et al. Loss of Dp140 regulatory sequences is associated with cognitive impairment in dystrophinopathies. Neuromuscul Disord 2000; 10: 194–9.

20. Taylor PJ, Betts GA, Maroulis S, et al. Dystrophin gene mutation location and the risk of cognitive impairment in Duchenne muscular dystrophy. PLoS ONE 2010; 5: 1–9.

21. Giliberto F, Ferreiro V, Dalamon V, et al. Dystrophin deletions and cognitive impairment in Duchenne/Becker muscular dystrophy. Neurol Res 2004; 26: 83–7.

22. Desguerre I, Christov C, Mayer M, et al. Clinical heterogeneity of Duchenne muscular dystrophy (DMD): definition of sub-phenotypes and predictive criteria by long-term follow-up. PLoS ONE 2009; 4: 1–10.

23. Rapaport D, Passos-Bueno MR, Takata RI, et al. A deletion including the brain promoter of the Duchenne muscular dystrophy gene is not associated with mental retardation. Neuromuscul Disord 1992; 2: 117–20.

24. Raven J., Raven J.C., Court J.H. Manual Raven, adapt. In lb. Romana: Anca Dobrean. Ed a 2-a. Cluj Napoca: Editura RTS, 2010. ISBN 978-973-1816-37-1.

Fig.4. Relation between the visuo-motor performance and the age of subjects


Beatrice Lucache6 Strap. Silvestru, CL21/16, Iasi 700006, Romania e-mail: [email protected]

Abstract. Chronic low back pain (CLBP) can be treated by quite a large number of treatment meth-ods, each method with its own advantages and drawbacks and comparable from the standpoint of ef-ficiency, side effects, costs, necessary time, easiness, convenience etc. The aim of this paper is to assess and compare the effectiveness and side effects of three of the most common used therapies in CLBP. A prospective case-control study was conducted over o period of six months. A lot of 127 patients divided in three groups, have each followed one of the three mentioned treatments. The effectiveness was assessed through the pain intensity and pressure pain threshold (PPT), while the side effects were evaluated through clinical approach. The results were statistically processed and analyzed, the significa-tion threshold being p=0.05. All the three applied treatments led to an improvement and the differences between the results were not significant. Acupuncture led to better results, but the differences became statistically significant slowly, in the last days of treatment. In what concerns the side effects, these were mostly minor and recorded especially for diclofenac. One major event wsa recorded in the case of bee venom therapy, namely anaphylactic shock was developed by one patient in the sixth treatment session. The results obtained by acupuncture or by bee venom therapy are not significantly , but due to the fact that they less invasive than diclofenac, we conclude that these complementary therapies have to be maintained as cost-efficient alternative procedures in CLBP treatment.Key words: cronic low back pain, diclofenac, acupuncture, bee venom

1. Centre for the Study and Therapy of Pain, Faculty of Medicine, Univeristy of Medicine and Pharmacy "Grigore T. Popa" Iasi, Romania

Beatrice Lucache1, Irina Jaba1, Elena Albu1, Mungiu C.O.1

ACUPUNCTURE AND BEE VENOM THERAPY VS. DICLOFENAC IN THE CHRONIC LOW BACK PAIN TREATMENT


Introduction

Rheumatic diseases express the largest palette of pain, both under pain physiopathologic

mechanisms and the pain intensity level. Among these, the chronic low back pain (CLBP) represents a major health problem having high social impact, characterized by important costs and economics losses, but also an important suffering source at the individual level[1]. In the industrialized countries, back pain affects between 15% and 30% of the population and generates high costs due to the ambulatory care visits[2]. Complementary and alternative medicine (CAM) is commonly used to treat back pain and, among these, acupuncture and bee venom therapy (BVT) are considered one of the most efficient. But, treatment with medicines remains important, mostly because it takes less time and is cheaper for patients.

Despite the multitude of studies and practical evidence, the effectiveness of these CAM therapies in

the treatment of back pain often generates disputes and polemics. However, the health care policy makers from many countries have included acupuncture in the recommended procedures for the incipient chronic rheumatic diseases[3].

The aim of this paper is to assess the relation between effectiveness and side effects for a few common used treatments in CLBP. The prospective case-control study included two complementary therapies (acupuncture and bee venom therapy) versus one pharmaceutical treatment (diclofenac).

Material and methodA prospective case-control study was conducted

to investigate and compare the improvements and side effects obtained from the three proposed therapies in the treatment of CLBP. The study protocol included the patients’ selection, getting the informed consent (approved by the Ethics Commission of the University of Medicine and Pharmacy “Gr.T.Popa” of Iasi, Romania), the patients’ assessment and pain questionnaires.

Three subjects groups were formed by Caucasian adult population, having the age between 20 and 70


years, the initial total number of subjects being 127. They were selected from the totality of the patients that accessed in the time interval October 2011 -March 2012 the services of two medical centres due the lumbar pains. The selected lot for the comparative study consisted from persosns with no psychical affections, unemployed and non-pregnant women. The three subject groups were sequentially completed, as long as patients came and addressed for lumbar pain treatment. Each patient followed a 12 days treatment and was assessed regarding both the pain intensity and sensitivity, before and every three days during the treatment.

The pain intensity was assessed by means of the visual analogical scale (VAS) that ranges from 0 (no pain) to 10 (worst possible pain). The pain sensitivity was measured with a digital algometer (FPIX25 - Wagner, USA), which detects the pressure pain threshold (PPT).

The evaluation of the side effects of the treatment was done through clinical observations every three days during treatments. The clinical evaluation of the patients has been composed by the blood pressure measurement and patient interview.

One group of 52 subjects received the NSAID treatment. The product diclofenac was selected, being a common drug, usually accepted and tolerated by the patients[4]. The treatment consisted in daily oral administration of 100 mg Diclofenac, for a 12 days’ cure.

The second group of 49 subjects received 30 minutes daily session of acupuncture treatment during 12 days. Based on a literature review on acupuncture for low back pain, only standard widely accepted and used acupoints were selected[5,6]. The used standard points were:• in the lumbar region (local points) UB25 and DU3, • in the lower extremity (distal points) were St44 • in the upper extremity (distal points) were LI4 and TF5.

Also other points were used: ST36 (analgesic point), UB17, UB40, UB62, DU20 and auricular points: Shenmen and Lombar spine. In the standard acupuncture disposable stainless steel needles (0,2mm x 40mm, Seirin Co.Ltd) were inserted (to a depth of 20mm) into the muscle, and the “sparrow pecking“ technique (alternate pushing and pulling of the needle) was applied. When the patient felt dull pain or the acupuncture sensation (de qi), the manipulation was stopped, and the needle was set in the site for 20-30 minutes. In the case of auricular acupuncture auricular needles (0,2mm x10mm) were used.

The third group composed initially by 26 subjects received 12 days of bee venom therapy. A solution that contains pure whole dried bee venom was injected intradermally to imitate the bee sting. The first step was to test the patient for any allergic reaction by injecting 0,1 ml of solution. Then, doses of bee venom were injected daily in trigger points and few acupuncture points. The injected doses and the application points were gradually increased, up to 0,2 ml in one specific spot and up to maximum 2 ml in total, identically for all the subjects.

The compositions of the three groups are quite similar regarding age and gender. As expected, the patients suffering of the lumbar diseases are mostly over 40 years old (47% of our subject between 40 and 59, and 35% over 60). Regarding the gender, women are preponderant, their number being approximately two times higher than the number of men. In the 52 subjects

under treatment with diclofenac 14 men (27%) and 38 females (73%) were registered. In the 49 subjects from the acupuncture group we recorded 19 men (39%) and 30 females (61%) and in the 26 subjects of BVT group we recorded 12 men (48%) and 14 females (52%).

ResultsAs already mentioned, the subjects have been assessed

before treatment and every three days = pain intensity, pain sensitivity, blood pressure, visual aspect and verbal complains were measured

The results in the effectiveness assessment show a similar evolution for the two parameters – pain intensity and PPT - for the all compared procedures. The evolutions are represented in Figure 1 for the pain intensity and in Figure 2 for the PPT. It is very important for the study conclusions to notice that, at baseline, the three groups were characterized by almost similar mean values of the parameters and practically it can be considered that they had the same baseline. This similitude is remarkable mostly because the subjects’ collection was a sequential process, which was carried out during a quite long period (18 months).

The experimental results have been also statistically processed and the significance threshold (p<0.05) has been tested. Both Anova and the post-hoc test Bonferoni (multiple comparisons) have been used. The results obtained through the last method are presented in Table I for the pain intensity estimation (VAS) and in Table II for the PPT measurement (pressure-type digital algometer).

The results in the assessment of the side effects are moderate and related to therapy. The most important complains were recorded during treatment with diclofenac. A total number of 13 of 52 patients from this group of subjects, suffered from minor side effects, as

Figure 1. Evolution of the pain intensity

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

DAY 1 DAY 3 DAY 6 DAY 9 DAY 12

Diclofenac

Acupuncture

Venom

Figure 2. Evolution of the pain sensitivity (pressure pain threshold)

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00


Diclofenac

Acupuncture

Venom

Fig.1. Evolution of the pain intensity

Figure 1. Evolution of the pain intensity

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00


Diclofenac

Acupuncture

Venom

Figure 2. Evolution of the pain sensitivity (pressure pain threshold)

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00


Diclofenac

Acupuncture

Venom

Fig.2. Evolution of the pain intensity (pressure pain threshold)


follows: 6 showed higher blood pressure, 6 recorded water retention, 3 complained for abdominal pains, 3 complained for nausea, 2 reported vomiting sensation.

The acupuncture therapy was accompanied by some minor secondary reactions. From the 49 subjects of the group, few recorded local bleeding (4 subjects), minor hematoma (1 subjects), sweating (2 subjects) and weakness (3 subjects).

The BVT was accompanied by local blushes and erythema, 15 from the 25 subjects recorded this normal reaction. One patient recorded palpitations and two other recorded intense sweating. But surprisingly, major reaction occured: one patient (female, 36 years old) developed an anaphylactic shock in the sixth treatment session. In consequence, the subject was forced to stop the treatment (consequently was excluded from the BVT study group).

DiscussionThe results for all three applied treatments show an

improvement for both analysed parameters. It is easy to notice how the parameters’ evolution keeps the same trend (monotony), but is not synchronic. Generally, PPT values vary different rhythms than the ones associated to pain intensity, confirming the subjectivity of the patients’ perception. The conscious experience of pain may be modulated by mental, emotional and sensory mechanisms. Giesbrecht and Battié [8] proved that possible sensory threshold fluctuations can be influenced by neurobiological and biopsychosocial factors.

Comparing the three treatments’ effectiveness, one notices moderately better results in the case of alternative treatments (acupuncture and BVT), suggesting that they have stronger effects on pain relief. But the differences are not so spectacular. It is interesting to notice that during the first 6 days for pain intensity and during the first 9 days for PPT, there were nostatistically significant differences between the three groups. Only after these intervals of time, the differences became statistically significant. The findings match the results of other studies. In a review, Chou and Hoffman9 found good evidence that NSAIDs are moderately effective for short term pain relief, while the same authors in another review[3] – that didn’t take into account the BVT - noticed that efficacy does not clearly differ between acupuncture and massage or analgesic medication. Kaptchiuk et al.[10] admitted that the recent scientific evidence for acupuncture and CLBP has not been so spectacular as the acupuncturists often claim, even if it has provided solid evidence that acupuncture significantly helps patients. In studies dedicated to BVT, like the one of Shin et al.[11], the effectiveness of this kind of therapy for CLBP has been proven. One possible explanation on the efficcacy of CAM could be, as Cherkin et al.[12] mention, the longer time spent by a patient (up to one hour) in a relaxed environment, the ongoing attention and the therapeutic touching. Also, acupuncture might enhance mental health independently from its effects on the physical health.

The assessment of the side effects and toxicity of the three studied treatments was performed through clinical observation (blood pressure, visual aspect and

verbal complains). In the case of diclofenac treatment we recorded

mostly minor side effects like water retention, abdominal pains, nausea and vomiting sensation. In the case of 6 patients we recorded higher blood pressure, even though they took specific medication to keep it within normal limits. However, the relatively short period of study didn’t allow us to discover severe side effects or toxicity events like gastric or hepatic damages.

One of the main advantages of acupuncture is usually claimed to be the lack of the adverse effects compared especially to drugs administration. In our study, foccused on acupuncture treatment we recorded, for 5.6% from the subjects only minor secondary reactions like: local bleeding, minor hematoma, sweating and weakness. These findings are similar to Ammendolia et al.[14] who, in a comprehensive study, identified twelve trials that reported complications or side effects related to acupuncture for CLBP. All of them were minor and most involved local bleeding or hematoma, the reported rate being between 6 and 8% of the patients.

In our study, BVT was accompanied by local blushes and erythema for more than half of the subjects, just one patient recording palpitations and two other -intense sweating. These are considered normal reactions and based on the literature, there should be no strong objections to the use of bee venom. However, while a limited number of stings is considered safe for non-alergic persons, the danger increases along with the number of stings. Mass inoculation of bee venom may induce acute renal failure (ARF), adult respiratory distress syndrome, liver injury, cardiac damage, pancreatitis, skin necrosis, shock hypertension, bleeding, thrombocytopenia, hemolysis, and rhabdomyolysis[20].

The toxicity can be immediate or can manifest itself days or even weeks after the exposure[21]. In our study, a major anaphylactic shock was recorded in the sixth treatment session by a patient (female, 36-years-old). The patient was stabilised with anti-shock adrenaline dose and transferred to emergency service for surveillance and rehabilitation. The patient abandoned the treatment, but contacted later, she confirmed a normal health condition and the absence of any other symptom, following the shock. A similar case of late reaction, was been analysed by Alqutub et al.[22] regarding a 35-year-old female, following bee sting therapy for multiple sclerosis that developed a hepatotoxicity.

Our study has a number of limitations. The first to mention is that the selection of patients was not a randomised trial. Our work was materialized in a cohort study wwhere patients’ preferences were taken into account. The patients that addressed to the family medicine clinic selected medication as a preferred treatment option, while patients that came to the complementary-medicine centre were committed to the alternative treatment methods. No patient was redirected to other kind of treatment outside their option. Even though making changes to the care plan is a key characteristic of maximizing clinical outcomes[23], the study established protocol was strictly followed.

The second limitation is the relatively short period


of time allocated to the study, only 12 days. After this time interval, the majority of patients was lost to follow up, having no possibility to interview and measure their parameters some time after the treatment ended, in order to trace their experience post treatment.

Regarding the results and their generality, it is known that the effectiveness of any manipulation therapy (like acupuncture, BVT, massage etc.) is highly dependent on the therapist’s technique and experience. In our study a single therapist was involved hence, the possible variations in treatment were avoided, enhancing the internal validity. On the other hand, the use of a single therapist is considered a threat to external validity, adding a question about the results’ generality.

Despite the limitations we believe that the findings in our study are important. To our knowledge, this comparative analysis for the three therapies was performed for the first time meaning that results may serve as a baseline for future studies or therapy management for patients with lumbar pains.

ConclusionsThe comparative study has focused on three of

the most used therapies for the CLBP: medication (diclofenac), acupuncture and bee venom therapy. All the applied therapies led to pain amelioration and, even if the differences regarding the effectiveness and side effects are not spectacular, acupuncture - followed by the bee venom therapy - recorded better results. On the other hand, diclofenac administration was lower, in terms of performance and recorded the most important side effects. But in the particular case of our study, it was the cheapest and the less time-consumming therapy. The acupuncture or BVT sessions need more daily time as compared with the simple pill administration. Patients that prefer this kind of treatment are either allergic to drug administration or pursue complementary acupuncture positive effects , that usualy manifest as an improvement of the tonicity and an overall wellness sensation.

References

Dependent Variable (I) tratament (J) tratament

Mean Difference

(I-J)Std. Error p (Sig.)

95% Confidence Interval

Lower Bound Upper Bound

VAS _ day 1 diclofenac acupuncture .00750 .18521 1.000 -.5187 .5337

BVT -.02777 .22639 1.000 -.6710 .6155

acupuncture diclofenac -.00750 .18521 1.000 -.5337 .5187

BVT -.03527 .22863 1.000 -.6849 .6143

BVT diclofenac .02777 .22639 1.000 -.6155 .6710

acupuncture .03527 .22863 1.000 -.6143 .6849

VAS _ day 3 diclofenac acupuncture .13293 .17598 1.000 -.3671 .6329

BVT .12885 .21512 1.000 -.4824 .7401

acupuncture diclofenac -.13293 .17598 1.000 -.6329 .3671

BVT -.00408 .21725 1.000 -.6213 .6132

BVT diclofenac -.12885 .21512 1.000 -.7401 .4824

acupuncture .00408 .21725 1.000 -.6132 .6213

VAS _ day 6 diclofenac acupuncture .55911* .16877 .011 .0796 1.0386

BVT .36662 .20631 .772 -.2196 .9528

acupuncture diclofenac -.55911* .16877 .011 -1.0386 -.0796

BVT -.19249 .20835 1.000 -.7845 .3995

BVT diclofenac -.36662 .20631 .772 -.9528 .2196

acupuncture .19249 .20835 1.000 -.3995 .7845


BVT .50454 .20997 .173 -.0920 1.1011


BVT -.34016 .21204 1.000 -.9426 .2623

BVT diclofenac -.50454 .20997 .173 -1.1011 .0920

acupuncture .34016 .21204 1.000 -.2623 .9426


BVT .58746 .21090 .059 -.0118 1.1867


BVT -.34743 .21299 1.000 -.9526 .2577

BVT diclofenac -.58746 .21090 .059 -1.1867 .0118

.34743 .21299 1.000 -.2577 .9526

Table I. Bonferroni analysis of the pain intensity* The mean difference is significant at the 0.05 level.


1. Chenot JF, Becker A, Leonhardt C, Keller S, et al. Use of complementary alternative medicine for low back pain consulting in general practice: a cohort study. BMC Complementary and Alternative Medicine 2007; 7(42).

2. Kanodia A. Perceived Benefit of Complementary and Alternative Medicine (CAM) for Back Pain: A National Survey. J Am Board Fam Med. 2010; 23:354 –362.

3. Chou R, Huffman LH. Nonpharmacologic therapies for acute and chronic low back pain: a review of the evidence for an American Pain Society/American College of Physicians clinical practice guideline. Ann Intern Med. 2007; 147(7):492-504. PMID: 17909210.

4. Wiegand T, Delenick M, Abbasi A. Nonsteroidal Anti-inflammatory Agent Toxicity, Medscape Reference, © 2011 WebMD http://emedicine.medscape.com/article/816117-overview (accesed 2014 Mar 1).

5. Ionescu-Tîrgoviște C. Teoria și practica acupuncturii moderne. București, Ed. Academiei Române, 1993.

6. Jayasuriya A. Clinical Acupuncture (fifteenth edition). Kalubowila, Sri Lanka, Ed. Medicina Alternativa International, 1994.

7. Giesbrecht J. and Michele Battié, A Comparison of Pressure Pain Detection Thresholds in People With Chronic Low Back Pain and Volunteers Without Pain, Physical Therapy, October 2005, Vol. 85, no. 10:1085-1092. PMID: 16180957.

8. Chou R., Hoffman L.H. , Medications for Acute and Chronic Low Back Pain: A Review of the Evidence for an American Pain Society/American College of Physicians Clinical Practice Guideline, Ann Intern Med. 2007; 147:505-514. PMID: 17909211

9. Kaptchuk TJ, Chen K, Song J. Recent trials of acupuncture in the West: responses from practitioners. Chin J Integr Med. 2010 June, 16(3): 197-203.

10. Shin B.-C. et al. Bee venom acupuncture for chronic low back pain: A randomised, sham-controlled, triple-blind clinical trial, European Journal of Integrative

Dependent Variable (I) tratament (J) tratament

Mean Difference

(I-J)Std. Error p (Sig.)

95% Confidence Interval

Lower Bound Upper Bound

Algometer _ day1

diclofenac acupuncture .16146 2.10211 1.000 -5.8112 6.1341

BVT .92815 2.56961 1.000 -6.3728 8.2291

acupuncture diclofenac -.16146 2.10211 1.000 -6.1341 5.8112

BVT .76669 2.59503 1.000 -6.6065 8.1399

BVT diclofenac -.92815 2.56961 1.000 -8.2291 6.3728

acupuncture -.76669 2.59503 1.000 -8.1399 6.6065

Algometer _ day 3

diclofenac acupuncture .88069 2.03434 1.000 -4.8994 6.6608

BVT 1.11938 2.48678 1.000 -5.9462 8.1850

acupuncture diclofenac -.88069 2.03434 1.000 -6.6608 4.8994

BVT .23869 2.51137 1.000 -6.8968 7.3742

BVT diclofenac -1.11938 2.48678 1.000 -8.1850 5.9462

acupuncture -.23869 2.51137 1.000 -7.3742 6.8968

Algometer _ day 6

diclofenac acupuncture -2.45836 2.03031 1.000 -8.2270 3.3103

BVT .37854 2.48185 1.000 -6.6731 7.4302

acupuncture diclofenac 2.45836 2.03031 1.000 -3.3103 8.2270

BVT 2.83690 2.50639 1.000 -4.2845 9.9583

BVT diclofenac -.37854 2.48185 1.000 -7.4302 6.6731

acupuncture -2.83690 2.50639 1.000 -9.9583 4.2845

Algometer _ day 9

diclofenac acupuncture -5.84714* 2.03928 .046 -11.6413 -.0530

BVT .08454 2.49281 1.000 -6.9982 7.1673

acupuncture diclofenac 5.84714* 2.03928 .046 .0530 11.6413

BVT 5.93167 2.51746 .195 -1.2211 13.0845

BVT diclofenac -.08454 2.49281 1.000 -7.1673 6.9982

acupuncture -5.93167 2.51746 .195 -13.0845 1.2211

Algometer _ day 12

diclofenac acupuncture -6.32072* 2.08635 .028 -12.2486 -.3928

BVT -.22554 2.55035 1.000 -7.4718 7.0207

acupuncture diclofenac 6.32072* 2.08635 .028 .3928 12.2486

BVT 6.09518 2.57557 .190 -1.2227 13.4131

BVT diclofenac .22554 2.55035 1.000 -7.0207 7.4718

-6.09518 2.57557 .190 -13.4131 1.2227

Table II. Bonferroni analysis of the PPT* The mean difference is significant at the 0.05 level


Medicine 4 (2012), Elsevier, pp: e271–e280.11. Cherkin D et al. Randomized trial comparing

traditional Chinese Medical acupuncture, therapeutic massage, and self-care education for chronic low back pain. Arch Intern Med. 2001: 161: 1081-1088. PMID: 11322842

12. Ammendolia C, Andrea D. Furlan, Marta Imamura, Emma Irvin, Maurits van Tulder. Evidence-informed management of chronic low back pain with needle acupuncture. Elsevier. The Spine Journal 8 (2008) 160–172.

13. Kluwer W. Health: Bee Venom - Print Version 2009, http://www.drugs.com/npp/bee-venom.html?printable=1, (Accessed 7/22/2013).

14. Betten DP , Richardson WH , Tong TC , Clark RF. Massive honey bee envenomation-induced rhabdomyolysis in an adolescent. Pediatrics. 2006;117(1):231-235.

15. Alqutub A.N., Masoodi I., et al. Bee sting therapy-induced hepatotoxicity: A case report, World Journal of Hepatology, 2011 October 27; 3(10): 268-270.

16. Maiers MJ, Westrom KK, et al. Integrative care for the management of low back pain: use of a clinical care pathway. BMC Health Service Research 2010. 10:298.


Aldea Ciprian1 I.C.Bratianu Blvd, Bucharest, Romaniae-mail: [email protected]

Abstract. Lately, the expanding accumulation of scientific data has indicated/demonstrated that malignant tumor growth depends on local angiogenesis. This occurs as a result of a process induced by a subgroup of transformed cancer cells which adopt an angiogenic phenotype, resulting in a new local balance of proangiogenic and antiangiogenic factors. The purpose of our work is to explore the hypothesis that acti-vated species of oxygen play an important role in the pathways that stimulate the angiogenesis favouring tumour growth. The study was performed in vivo, on rats with experimental RS-1 tumours treated with bevacizumabum. We made a dynamic analysis of biochemical parameters of oxydative stress (the level of lipid peroxydes, thyolic groups, total antioxydants); apoptosis was measured by flow-cytometry in tumoral and normal hepatic tissues. We registered a decrease in the oxidative status during the antiangiogenic treatment and a significant increase of apoptosis (from 9.12% to 20.14%) in the malignant cells. These data support the hypothesis that activated species of oxygen can initiate the proangiogenic phenotype transformation. In this kind of oxidative circumstances targeted antiangiogenic drugs can reverse the process and induce tumour cells apoptosis, resulting in clinical benefits for cancer patients.Key words: angiogenesis, bevacizumabum, activated oxygen species, experimental tumours, apoptosis

1. Coltea Clinical Hospital Bucharest2. "Prof. Dr. Al. Trestioreanu" Institute of Oncology Bucharest

Aldea C.1, Aldea Alina Elena 1, Negoita Valentina2, Anghel Rodica2

Gruia Maria Iuliana2

THE DUAL ROLE OF ACTIVATED SPECIES OF OXYGEN IN ANGIOGENESIS


Introduction

Angiogenesis is a complex of coordinated cellular and molecular processes having as a result

new vessels formation, departing from the previous vascular net [1]. This is a two step process, with a phase of activation, which results in new vessels formation, followed by a phase of maturation, with vessel stabilization [2].

The activation phase implies basal membrane destruction around the old vessel, followed by migration and proliferation of endothelial cells. A cord of endothelial cells is formed, which becomes tunnelled and connects to another vessel. In these phases the vascular endothelial growth factor (VEGF) and angiopoetin 2 (ANG2) are activated.

The maturation phase of the new vessel makes possible the stabilization by basal membrane reconstruction and perivascular cells (pericytes) recruitment. In this phase several process intervene/act: platelet derived growth factor (PDGF), angiopoetin 1 (ANG1) , transforming growth factor beta (TGFbeta)

and bone morphogenesis protein (BMP) [3].Most of the tests that measure in vitro angiogenesis

investigate only the activation phase (for example protease secretion) offering an incomplete view of the process. Even so these tests led, during the last years, to the discovery of a number of molecules that are essential for the angiogenesis process[4]. The hypoxic signalization pathway which induces, through HIF (hypoxia inducible factor), the expression of proangiogenic factors VEGF and ANG2 is essential for neoangiogenesis[5][6]. Reactive oxygen species (ROS) are known as mediators of angiogenesis and as having a role in metastatic potential of tumour cells[7].

By modulating the activity of different enzymes and critical transcription factors oxidative stress can activate a series of intracellular signalization pathways[8]. In such a scenario, increase transcription factors activated by ROS will translate from cytoplasm to the nucleus binding to the promoter regions of specific genes[9]. As a result, activation of such pathways with consecutive gene expression modulation can heavily determine cells; activities and course of action (cytokine production, proliferation or apoptosis). The balance between ROS production and antioxidant cellular defence, activation of oxidative stress related to signalising pathways with gene transcription products appearance will determine


if a cell or group of cells exposed to ROS increase will survive or die [10].

Bevacizumabum is a relatively new monoclonal antibody, which acts as an anticancer drug by binding to VEGF receptors and blocking them. This prevents the binding of VEGF and blocks the activation phase of angiogenesis. The formation of new vessels is prevented [11]. As a result tumour growth is stopped. Vascular depletion results also in tumour cell death. Normal cells are not or less affected. As a consequence the safety profile is much more favourable than those of the cytotoxic drugs.

Our objective was to identify and characterize the role of ROS in the cascade of angiogenesis signalization with consequent antitumor effects. We developped an experimental in vivo model. Angiogenesis inhibition was obtained using Bevacizumab. The resulting variation of parameters was quantified and followed during the Avastin administration.

Materials and MethodsIn vivo experimental design

Our study was performed in vivo, on animals carrying the experimental RS-1 hepathoma. The used animals (Wistar rats) originated from the Bucharest Institute of Cancer, accredited for biologic material use from 1964 (when it was created).

In conformity with what was stipulated at Bratislava 2002 Conference “Ethics in Scientific Research” and in accordance with the Royal Society for Animal Violence Protection from UK we consider that suffering provoked to the animals during the research, as a consequence of stress reaction can influence the accuracy of scientific data. The principle of pain limiting (to reduce as much as possible the animal suffering provoked by the experiment) and that of number reduction (using the minimal number of animals necessary to attain study objectives) were implemented. Working on small rodents, the experiment design and control methods were conceived having in mind both the importance of the problem and the limits of safety. Evaluation of toxicities was based on behaviour observation/including growing, biochemical tests and also on histological and necropsy reports. Our experimental model respected the relevant legislation (the principles to be followed when working with small animals) and the recommendations of the Ethical Committee of the Institute. The animals were kept in the Institute’s Biobasis, with standard conditions of feeding and hydration. They were separated in two arms, one with no treatment and the second one treated with Avastin (for 3 months, 5 days weekly, 5mg/kg/day, in normal saline solution, 0,2 ml iv). The treatment started 21 days after tumour inoculation.

Tumour implantation The RS-1 hepathoma is a transplantable carcinoma,

chemically induced in rats by 2-acetileaminofluoren. The tumour is maintained to present time by subcutaneous transplantations to Wistar albino rats; it is defined by a latency period of 25-30 days and a rate of successful transplantation of 90%. It does not

metastasise; local invasion can be observed, median survival of porters is 70-80 days. Approximately one million cells/ml were inoculated in the right flank. The tumour can be maintained also by intrahepatic inoculation. Subcutaneous and hepatic greffons are histologically identical (solid carcinoma, with mucinous areas), with a low mitotic activity and a moderate rate of apoptosis and spontaneous necrosis. It is an ovoid, neatly delimited tumour, with a smooth capsula of rose-yellow colour. Necrotic and hemorrhagic areas are alternating in its structure.

Biochemical assays The index of lipid peroxidation was evaluated by

measuring the serum malonaldehyde, the final product of lipid hidroperoxids degradation. The method is based on a new red adduct formation (MDA-TBA2,) with a maximum of absorption at 532 nm, registered on a Analytic Yena Specord 210 spectrophotometer. The solutions are prepared on site using genetic pure water from a Milli-Q device. The substances used (barbituric acid, acethical acid, trichloroacetic acid) are provided by Merck and have the required purity grade[12][13] [14]. Lipid peroxides were measures in tumoral and in normal liver tissue.

Albumin thiols determination Albumin-thiols groups were determined using

the reaction with 5,5`-ditio-bis (2-nitrobenzoic) acid Ellman reactive. Reactives were prepared on site utilizing chemical compounds provided by Merck. They were measured in serum, tumour tissue and normal liver tissue by spectrophotometry.

FRAS (ferric reducing ability of serum)The reaction measured the reduction of Fe3+-

2,4,6-tri(2-piridil)-1,3,5-triazina (TPTZ) to a coloured product. The method which measures the combined antioxidant effect of non-enzyme antioxidants from biologic fluids is used in controlling the index of resistance to oxidative destructions. At low pH, when the above mentioned complex is reduced to Fe2+ by the oxidants from the probe,it results in blue coloration with λmax 593nm. In this method an excess of Fe3+ is used while the limiting factor for the complex (and colour) formation is the antioxidant (the redactors from plasma) [16][17].

Flowcytometry measurements Anexin V a calcium dependent anticoagulant protein

has a big affinity for fosphatidilserine, binding selectively to negatively charged phospholipides. By conjugation to a fluorochrome (for example fluoroscein) it can be used as a marker for apoptosis identification, together with DNA colour reaction with propidium iodure (18).

The detection of hypodiploid DNA by flow cytometry using the quantitative binding reaction of propidium iodure (the most used colorant) to DNA (as a result of cell permeabilisation) is the most common technique for apoptosis determination[19]. Apoptotic cells have a low level of DNA and can be recognised


by their position on DNA content histograms, the low G1 peak, positioned at the left of the peak representing cells from the G1 phase of the cell cycle. The absence of the hypodiploid peak is not always a proof for apoptosis absence. However, the presence of this peak cannot be considered a proof for an apoptotic population in the absence of supplementary proofs [20].

Results

As a reference tissue we chose the normal liver tissue. On measuring one can see a significant increase of lipid peroxidation in the observation arm, a dynamic rise after a decrease to one third of the values registered at five weeks of treatment. The data suggests that the initial inhibition of the oxygen metabolisation is rapid, with subsequent restoring values, even if the treatment with antiangiogenic agent continues. We suppose that this phenomenon is based on low initial concentrations of ROS, gradual elimination of hypoxia and signalization of the events necessary to neovascularization.

In the tumoral tissue one can see the same profile of lipid peroxidation during the antiangiogenic treatment; the rise is more important in the observation (untreated) arm.

SH group during the anti- VEGF treatment in tumoral and liver tissue

Albumin – thiol groups were processed from the same biological materials, tumour tissue versus normal liver tissue, with the same dynamics. Identical final products of oxidative-degradative reaction appear as a result of ROS attack on sul containing proteins. It is a well known fact that tumour tissue can produce an excess of proteins that offer antioxidant protection (metaltioenins, glutation, cistheine,etc). Dynamics rises from the treatment point to the antioxidant defence mechanisms in the tumoral tissue.

11.25

3.57

5.827.17

11.359.88

6.29

9.76

0

2

4

6

8

10

12

3 weeks 5 10 15 weeks

Treated

Control

Fig. 1. Lipid peroxidation reaction in normal liver tissue

In the normal liver tissue the value differs from the tumor tissue; in the treated arm we can see lower registered values for the studied item, comparing to the observational arm.

As a result of ROS, induced signalling endogen antioxidant defence systems were activated; as a result the obtained values were lower in the treatment arm (versus those in the observational arm).

In the normal liver tissue the antioxidant profile is higher. We can interpret this only as a result of the activation of liver metabolism and detoxification after prolonged treatment. These mechanisms involve liver enzymes as those from the cytochrome family, which produce ROS.

The obtained biochemical data suggest that bevacizumabum inhibits the production of ROS the first month of treatment in hypoxic conditions, in a dose-dependent pattern. After that the profile of apoptosis directed oxidative reactions is modified. These results are presented in the following diagrams, constructed on flow cytometry determinations.

10.1713.72

24.31

53.45

10.95

0

10

20

30

40

50

60


Treated

Control

Fig. 2. Lipid peroxidation in tumoral tissue

265 287

782 792

313268

218152

0100200300400500600700800900


Treated

Control

Fig. 3. The value of albuminic thiols in liver tissue

2425

17901594

511

2644

21171829

1509

0

500

1000

1500

2000

2500

3000


Treated

Control

Fig. 4. The value of albuminic thiols in liver tissue

3.136 3.4162.827

1.6672.224

2.983

4.8395.145

0

1

2

3

4

5

6


Treated

Control

Fig. 5. Total antioxidants at the hepatic level

0.7910.709

1.3381.482

0.9340.824 0.775

1.146

00.20.40.60.8

11.21.41.6


Treated

Control

Fig. 6. Total antioxidants in tumour tissue


Conclusions The comparison of data collected through by

measurements on tissue homogenates obtained from animal carriers of experimental RS-1 hepathoma treated with bevacizumabum versus data obtained in the observational, non-treated arm led to the following conclusions:

-lipid peroxidation in the tumour tissue is not significantly changed after antiangiogenic treatment

-one observes a rise of total thiols and of the level of antioxidants suggesting an activation of natural defence systems against ROS, after an oxidative stress

-treatment with the antiVEGF monoclonal antibody bevacizumabum significantly changes the cell phenotype, towards an apoptotic one. After the treatment there is a significant increase of the apoptosis in the tumour tissue

-the data suggest that ROS have a dual role in this process; at lower levels, corresponding to the first exposure to avastin they have a role of signalising hypoxia. This stimulates angiogenic effects. Biochemical mechanism will be redirected to ROS production that be involved in the apoptotic cytotoxicity.

References1. Folkman J., ”Anti-angiogenesis:new concept for

therapy of solid tumors”,Annals of surgery 175:409-416, 1972.

2. Fidler I. J., Kerbel RS, Ellis LM, ”Biology of cancer:angiogenesis “ Cancer:principles and practice of oncology.6-th ed. Philadelphia(PA):Lippincott;p. 137-148, 2001.

3. Plank M.F., Sleeman B.D., Tumour-induced angiogenesis: a review, J. Theoret. Biol., 5, 137-153, 2003

4. Duda D.G., Ancukiewicz M., Jain R.K., "Biomarkers of angiogenic therapy: How do we move from candidate biomarkers to valid biomarkers”, Journal of Clinical Oncology, Volume 28, 2, 183-184, 2010

5. Ihle J.N Signal transducers and activators of transcription, Cell, 84:331-334, 1996

6. Pugh C.W., Ratliffe P.J., Regulation of angiogenesis by hypoxia: role of the HIF system, Nature Medicine, Vol. 9, Number 6, 677-684, 2003

7. Gupta, SC. Patchva D H, Park B, Koh W, and Aggarwal B.B. Upsides and Downsides of Reactive Oxygen Species for Cancer: The Roles of Reactive

Fig. 7. The apotosis diagrams obtained by flow-cytometry in normal liver tissue

Apoptosis determination by flow-cytometry

Fig. 8. The apoptosis diagrams registered during treatment in tumour tissue

In these diagrams one canobserve a rise in apoptosis from 14,3% do 29,6% during the treatment phase. The results suggest an apoptotic treatment- induced mechanism. Data for tumour tissue follow.

They show a higher increase in apoptosis, up to 59,72% thus demonstrating the treatment’s efficacy on the tumor tissue, expressed by apoptosis induction instead of angiogenesis blockage.


Oxygen Species in Tumorigenesis, Prevention, and Therapy Antioxidants & Red. Singn, 16 (11) 2011

8. Kerbel R.S., Tumor angiogenesis, N. Engl.J. Med, 358:2039-2049, 2008.

9. Jouanneau J., Adhesion, motilite et migration cellulaires,Cancerologie fondamentale, Paris, John Libbey Eurotext, p. 53-60, 2005.

10. BissetD., Molecular cancer Biology, Oxford Handbook of Oncology, USA, 2-nd edition, Oxford University press, p. 3-15, 2009.

11. Yang J.C., Haworth L.,Sherry R.M., Hwu P., Schartzzentruber D.J., Topalian S.L., Steinberg S.M.,X.ChenH.,Rosenberg S.A., A Randomized Trial of Bevacizumab, an Anti-Vascular Endothelial Growth Factor Antibody, for metastatic Renal Cancer, The New England Journal of Medicine, Vol. 349, No 5, 427-434, 2003.

12. Halliwell, B , Gutteridge, JMC (1984) Lipid peroxidation, oxygen radicals, cell damage and antioxidants therapy. Lancet. 1, 1396.

13. Carboneau M.A. (1991) Peroxides measurement in blood and plasma. Clinic. Chem. 37, 1422-1428.

14. Janero D.R., Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury Free Rad. Biol. Med. 9(6), 515-540,1990.

15. Romero, Fj et al. The reactivity of thiols and disulfides with different redox states of myoglobin. J. Biol. Chem. 267, 1680. 1992.

16. Cao, G.; Prior, R. L Comparison of different analytical methods for assessing total antioxidant capacity of human serum. Clin. Chem. 1998, 44, 1309–1315.

17. Zwart, L. L.; Meerman, J. H. N.; Commandeur, J. N. M.; Vermeulen, N. P. E. Biomarkers of free radical damage: applications in experimental animals and in humans. Free Rad. Biol. Med. 1999, 26, 202–226.

18. Shapiro H. Practical flow cytometry, 3rd ed. New York: Wiley-Liss, 1995:542.

19. Joensuu H, Klemi PJ. DNA aneuploidy in adenomas of endocrine organs. Am J Pathol 1988;132:145-151.

20. Hedley DW, Shankey TV, Wheeless LL. DNA cytometry consensus conference. Cytometry 1993;14:471.


Laura Niculescu30-32 Iancu de Hunedoara Blvd., sector 1, Buchareste-mail: [email protected]

Abstract. Hypospadias is one of the most common congenital malformations of the external male genitalia, in which the urethra opens ventrally, anywhere from the glans to the perineum, the penis has an incompletely formed prepuce and may exhibit ventral curvature. Many surgical techniques for the reconstruction of this defect have been devised, each one having a different success rate. The aim of our study is to determine the complication rates and predictive factors for Mathieu and tubularized incised plate (TIP) techniques used in the repair of distal hypospadias. We assessed 62 patients diagnosed with distal hypospadias that were subjected to surgical correction by using either Mathieu or TIP urethroplasty during January 2011 and December 2012 in our clinic. The patients were divided into two groups according to the surgical technique which was performed. Urethroplasty complications, defined as fistula, urethral stricture and dehiscence, were statistically analyzed. There were 23 patients operated using the Mathieu technique and 39 patients using TIP repair. Mean age of presentation was 14 months for the Mathieu group and 20 months for the TIP groups. The urethral complication rate was slightly lower for the Mathieu group than in those cases solved by using the TIP technique (17.39% vs. 23.07%). There was just one case who presented with postoperative fistula in a patient belonging to the Mathieu group, while 5 patients operated with the TIP technique presented urethral stricture. No such cases were reported in the Mathieu group. Dehiscence was present in both groups (13.04% in the Mathieu group vs. 10.25% in the TIP group). We conclude that both techniques are acceptable and effective for the treatment of distal hypospadias, with complication rates being similar in the two groups.Keywords: Mathieu, complications, Snodgrass

1. “Grigore Alexandrescu” Clinical Emergency Hospital for Children2. “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania

Bălănescu R.N.1, 2, Niculescu Laura1, Bălănescu Laura1, 2, Moga Andreea1

OUTCOMES IN DISTAL HYPOSPADIAS

Therapeutics, Pharmacology and Clinical ToxicologyVol XIX, Number 3, September 2015Pages: 114 - 117© Copyright reserved 2015

Introduction

Hypospadias is one of the most common congenital malformations of the external

male genitalia, in which the urethra opens ventrally, anywhere from the glans to the perineum, the penis has an incompletely formed prepuce and may exhibit ventral curvature (Figure 1). The overall incidence of hypospadias is between 0.3% to 0.6%, with a higher risk in families where this anomaly has already occurred. Many surgical techniques for the reconstruction of this defect have been devised, each one having a different success rate. The correction of this malformation includes straightening of the penis, urethroplasty, glanduloplasty and ultimately circumcision, with the main goal of surgery being that of obtaining a functionally and cosmetically normal penis. Current literature is dominated by two techniques and attempts to improve them:the tubularized incised plate (TIP) andthe modified Mathieu technique.

The TIP technique, which was popularized by Snodgrass since 1994, consists of a longitudinal incision of the urethral plate and further tubularizing the incised urethral plate [1]. The most common complications of this technique are urethral fistulas, stenosis and wound dehiscence [2]. (Figure 2)

The principle of the Mathieu technique involves the construction of the urethra from a proximal penile

ORIGINAL PAPER

Fig. 1. Hypospadias classification


flap sutured over the urethral plate. The primary complications of this method are also urethral fistula and stenosis [3] as well as a round and flattened

urethral meatus [4].These two techniques have been often compared [3, 5, 6, 7] in order to establish the superiority or the specific indication of one of them, the results so far having been ambiguous. (Figure 3)

This study proposes a comparison of the two techniques by assessing the patients with distal hypospadias operated between January 2011 and December 2012 in the Pediatric Surgical Department of the "Grigore Alexandrescu" Emergency Children’s Hospital in Bucharest, Romania.

Materials and MethodsA number of 62 patients diagnosed with distal

hypospadias (coronal, subcoronal or distal penile) admitted to the Pediatric Surgical Department of the “Grigore Alexandrescu” Emergency Clinical Hospital for Children over a one year period were enrolled in our study, with data being collected from the clinical and operative records.

The admissions that did not end with surgery during hospitalization or surgical correction other than those two studied methods (TIP- Snodgrass and Mathieu) and proximal forms hypospadias were excluded from the study.

In the statistical analysis we included only the patients without history of prior surgery for hypospadias at the time of entry in the study. For the TIP - Snodgrass technique group we selected only the patients operated on from January to December 2011 in order to obtain two comparable groups. Our study’s limitation was the fact that the operations were not performed by the same surgeon.

According to the operative records, the TIP – Snodgrass urethroplasty was performed in one layer with separate or continuous suture on a probe that was maintained postoperatively for an average duration of 8 days. In 50% of cases the neourethra was covered with dartos. The modified Mathieu urethroplasty

was done on a urinary catheter that was maintained postoperatively only for a two days average period. The covering of the neourethra with dartos was used in 75% of cases.

Urethral complications of the two surgical methods were defined as the need for surgical repair of urethral fistula, urethral stenosis or partial/total dehiscence of the urethra. Patient follow-up was for a minimum period of six months after the last intervention; in this respect we included in the statistical analysis the redos from January to July 2013 for the patients enrolled in the study.

Statistical analysis was performed using MedCalc software (version 8.0.0.1, MedCalc Software, Ostend, Belgium). The data collection followed the national legislation concerning the processing of personal data (Law 677/2001 updated in 2013).

ResultsThe study group included 62 patients of whom 23

patients were operated by using the modified Mathieu technique (Group 1) and 39 were treated using TIP - Snodgrass technique (Group 2). The age and the distribution of the clinical forms are shown in Table I.

Age ranged from 6 to 43 months in Group 1 and from 13 to 38 months in Group 2. The two groups showed no statistically significant differences in terms of age at the time of the intervention (p = 0.7). Subcoronal hypospadias prevailed in the group of patients operated by using the modified Mathieu technique, while in the TIP Group there was an equal number of cases with subcoronal and penile hypospadias (46.15%). 8 patients (34.78 %) in the Mathieu group and only 1 patient in group Snodgrass were not stented in the postoperative period (p<0.001).

There were 4 patients (17.39 %) in the Mathieu group with postoperative complications that required surgical reintervention, including one patient with urethral fistula (4.34 %). In the Snodgrass group postoperative complications were found in 9 patients (23.07 %) with

Fig. 2. TIP repair technique

Fig. 3. Mathieu Reconstruction Mathieu TIP - Snodgrass p

No. of patients 23 39Age at the operation

14 (6, 43)

20 (13, 38) 0.7

Hypospadias type

Subcoronal 0 3 (7.69%) 0.45

Coronal 15 (65.21%)

18 (46.15%) 0.23

Penile 8 (34.78%)

18 (46.15%) 0.53

Stenting time 1 (1,2) 8 (7, 9.75) <0.001Age is presented in month as median (percentile 25, 75).Stenting time is presented in days as median (percentile 25, 75).

Table I. Characteristics of patients


a number of 5 cases (12.82 %) with urethral stenosis that required dilatations. The other postoperative complications recorded was total or partial dehiscence that required redo urethroplasty in 3 patients(13.04 %) in the Mathieu group and 4 patients (10.25 %) in the Snodgrass group (Figure 4).

In order to determine the prognostic factors for complications requiring reoperation we performed a logistic regression on the two groups of patients in which the dependent variable was the presence/absence of redo and independent variables (predictors) were age at the time of the operation, surgical technique (modified Mathieu and Snodgrass) and hypospadias type. None of the above-mentioned parameters did significantly correlate with the prognosis (Table II).

Discussions

The aim of this study is to evaluate the results obtained by using the modified Mathieu and the TIP-Snodgrass techniques in the repair of distal hypospadias of patients admitted to the Pediatric Surgical Department of the "Grigore Alexandrescu" Emergency Hospital for Children, Bucharest, Romania. We enrolled 39 patients in the TIP-Snodgrass group and 23 patients in the Mathieu group, both groups being comparable to the data published in literature [3, 4, 5, 7].

Patients included in the study were operated on by several pediatric surgeons, this representing a limit of our study due to the lack of uniformity of technique practiced but at the same time an advantage since it provides an overview of the results.

Optimal age for correction of this malformation is still a controversial topic, with the European Society of Pediatric Urology recommending the correction of hypospadias at 6-18 (24) months [10]. According to the American Association of Pediatrics that age is

optimal for surgery from the point of view of emotional development, separation of parent - child is minimal and shortens the period in which the child "bears" the malformation, thus less affecting his self-perception. The postoperative behavioral problems such as aggressive or regressive attitude, night terror or anxiety appear particularly at the age of 1-3 years [9]. Studies on the influence of age on the operative success exhibit different results, with works arguing that postoperative results are better in children aged 1 than in 5 year olds [11] or in young children compared to adolescents [12], while other studies show that age does not influence prognosis[13]. Analyzing age of patients included in our study, this was not statistically significantly when associated with prognosis (p =0.35).

Looking for the success rate of hypospadias repair in our study, by assessing the need for redo because of urethral complications, we recorded results comparable to those published in literature. In the Mathieu group overall complication rate was 17.39 %, while in the TIP - Snodgrass group the complication rate was 23.07%. A meta-analysis published in 2013 that included 12 studies similar to ours on 690 patients showed an overall urethral complication of 16-17 % for Mathieu technique and 20-21% for TIP - Snodgrass technique [3].Urethral fistula was low for both techniques, 4.34% for Mathieu group and zero for the Snodgrass group, when compared to other studies that recorded similar complications in 10% of patients operated with the modified Mathieu technique [7] and 11.5 % [3] of the patients operated by using the TIP procedure. Postoperative urethral stenosis was recorded only in the TIP - Snodgrass group with a frequency of 12.82 %, a number which was higher than those published in other series[8]. The rate of partial or complete urethral dehiscence that ended with redo urethroplasty was high in both groups, this aspect requiring to be deepened to discover and correct the factors which led to these results.

We performed a logistic regression in order to identify potential predictor factors for the occurrence of complications. The chosen independent parameters (location of the meatus, surgical technique Mathieu versus TIP) did not correlate significantly with the outcome, an aspect which was confirmed by a meta-analysis results [3, 6] but are still disputed in recent individual studies[8].

Conclusions

Success rate of hypospadias repair in the Pediatric Surgical Department of the "Grigore Alexandrescu" Emergency Hospital for Children is comparable to those published in the international literature, with the particularity of a low rate of urethral fistulas but a higher percentage of postoperative dehiscence. Age and hypospadias type (coronal, subcoronal or penile) do not significantly influence the postoperative outcome. Both surgical techniques (modified Mathieu and TIP - Snodgrass) are effective and have similar rates of postoperative complications.

ACKNOWLEDGEMENT: This paper is supported by the Sectoral Operational Programme Human Resources Development (SOP HRD) 2007-2013, financed from the European Social Fund and by the Romanian Government under the contract number POSDRU/107/1.5/S/82839".

Fig. 4. Postoperative complications

coefficient OR CI OR P

Age 0.007 1.007 0.99-1.02 0.35Hypospadias type 0.68 1.97 0.63-6.1 0.24Surgical technique -0.39 0.67 0.17-2.62 0.56

Dependent variable presence/absence of urethral complications (0=without redo; 1=redo)Age – continuous variableHypospadias type: coronal=0, subcoronal=1, penile=2Surgical technique: TIP-Snodgrass=1, Mathieu=2

Tabel II. The influence of age, hypospadias type and technique on prognosis


References1. Snodgrass WT. Snodgrass technique for

hypospadias repair. BJU Int. 2005 Mar;95(4):683-93.2. Warren T. Snodgrass. Tubularized incised plate

hypospadias repair for distal hypospadias. Journal of Pediatric Urology (2010) 6, 408-413.

3. Furan Wang. Systematic review and meta-analysis of studies comparing the perimeatal-based flap and tubularized incised-plate techniques for primary hypospadias repair. PediatrSurgInt (2013) 29:811–821.

4. Ehab O ElGanainy. Combined Mathieu and Snodgrass urethroplasty for hypospadias repair: A prospective randomized study. International Journal of Urology (2010) 17, 661–665.

5. AyhanKarabulut. Retrospective analysis of the results obtained by using Mathieu and TIP urethroplasty techniques in recurrent hypospadias repairs. Journal of Pediatric Urology (2008) 4, 359-363.

6. David J. Wilkinson. Outcomes in distal hypospadias: A systematic review of the Mathieu and tubularized incised plate repairs. J Pediatr Urol. 2012 Jun ;8(3):307-12.

7. Mazin Adday Hasoon. Comparative Study in Anterior Distal Hypospadias Reconstruction Utilizing Different Techniques (Mathieu and Snodgrass): Outcome, Complications and Failure Rate. IOSR

Journal of Pharmacy 2013 May 3(4):53-59.8. Muhammad ShahzadSaleem et al. Comparative

Study between Tubularized Incised Plate (Snodgrass) Urethroplasty and Reverse Flap (Mathieu’s) Repair in Distal Hypospadias. Ann. Pak. Inst. Med. Sci. 2012; 8(2): 96-100.

9. American Academy of Pediatrics. Timing of Elective Surgery on the Genitalia of Male Children With Particular Reference to the Risks, Benefits, and Psychological Effects of Surgery and Anesthesia. PEDIATRICS Vol. 97 No. 4 April 1, 1996, pp. 590 -594.

10. European Society for Paediatric Urology, European Association of Urology 2013. Guidelines on Pediatric Urology. www. Uroweb.org/guidelines.

11. Korvald C, Stubberud K. High odds for freedom from early complications after tubularized incised-plate urethroplasty in 1-year-old versus 5-year-old boys. J PediatrUrol 2008 Dec;4(6):452-6.

12. Dodson JL et al. Outcomes of delayed hypospadias repair: implications for decision making. J Urol 2007 Jul;178(1):278-81.

13. Bush NC et al. Age does not impact risk for urethroplasty complications after tubularized incised plate repair of hypospadias in prepubertal boys. J Pediatr Urol 2013 Jun;9(3):252-6.


Ilie Mădălina8 Calea Floreasca St., Bucharest, Romaniae-mail: [email protected]

Abstract. Ectopic pancreatic tissue is most often an incidental finding of imaging, surgery, or autopsy. Image-guided diagnosis is difficult and definitive diagnosis usually relies on histological analysis. A case of ectopic pancreatic tissue located near the lesser curvature of the stomach complicated with acute ectopic pancreatitis is presented followed by a brief discussion of the clinical management of heterotopic pancreas.Keywords: pancreatitis, heterotopic pancreas, ileus

1. Clinical Emergency Hospital Bucharest, Romania* all authors equally contributed to this work

Tabacelia Daniela1*, Ilie Mădălina1*, Ene D.1*, Constantinescu G.1*, Macovei R.1*, Asmarandei R.1*, Stănciulescu Elena- Luminiţa1*, Diaconu Camelia1*, Popa B.1*

ACUTE ECTOPIC PANCREATITIS WITH ILEUS OCCURRING IN GASTRIC ECTOPIC PANCREATIC TISSUE


Introduction

Ectopic (heterotopic) pancreatic tissue, often referred to as a pancreatic rest is the presence

of pancreatic tissue found outside the normal anatomic position and lacking vascular connection with the body of the pancreas[1] occuring in 0.55% to 13.7% of the population[2]. In most cases, the tissue is functional and is commonly found along the greater curvature of the stomach, duodenum, proximal jejunum and ileum [3.4.5]. Less frequently heterotopic pancreatic tissue can be found in the umbilicus, common bile duct, gallbladder, and Meckel’s diverticulum. Despite its rare clinically significance ectopic pancreatic tissue has been associated with ulceration and bleeding, intussusceptions, and even pancreatitis and adenocarcinoma. When the tissue is symptomatic, treatment consists in surgical resection, but the removal of ectopic pancreatic tissue that is found incidentally remains a controversial matter.

Pathophysiology The pathophysiology of heterotopic pancreas is

not fully understood and multiple theories exist. One explanation proposes that during embryogenesis, pancreatic metaplasia of the endodermal tissues localized in the gastric mucosa occurs [5,6]. Another theory suggests that heterotopic pancreas could be caused by the inhibition of normal cellular signaling during development; inhibiting hedgehog signaling in chick embryos leads to ectopic budding of pancreatic structures in the stomach and duodenum [7].

Presentation Most patients are asymptomatic, but symptoms can

include nausea, vomiting, epigastric pain, dyspepsia, abdominal fullness, and melena [1,3]. The most common symptom is epigastric pain[7]. About a third of symptomatic patients report clinical symptoms that mimic disease related to the organ in which the tissue resides[5]. More serious complications can ensue such as massive gastrointestinal bleeding, gastric outlet obstruction, gastric or duodenal ulceration, pancreatitis, and malignant degeneration[3,8].

DiagnosisDiagnosis of heterotopic pancreas can be very

difficult and is dependent on presentation (aici nu inteleg sensul- depinde de prezentare?). The physical examination rarely provides clues to the diagnosis of pancreatic rests as they are rarely, if ever, large enough to be detected by palpation[2]. CT can sometimes be helpful; the findings usually depict a small, round or oval sharply marginated broad-based mass[3]. Occasionally, it may appear as a mass with an irregular surface resembling an adenomatous polyp or polypoid carcinoma[3]. Five CT criteria have been used with good sensitivity and specificity to help differentiate between ectopic pancreas and gastrointestinal stromal tumor (GIST)[9]. These criteria are as follows: pre-pyloric antrum or duodenum in location, an ill-defined border, an endoluminal growth pattern, a long diameter/short diameter ratio larger than 1.4, and prominent mucosal enhancement. When two or more criteria are met, the sensitivity and specificity for diagnosing ectopic pancreas approaches 100% and 82.5%, respectively[9]. EGD can be useful and findings can be described as a small (around 2 cm) nodular, submucosal mass

CASE REPORT


covered by normal mucosa with or without central umbilication[4,1]. Endoscopic ultrasonography can be helpful for determining the nature of the mass. Findings of indistinct borders, anechoic duct-like structures, mural growth pattern, presence in more than one layer, and indistinct borders suggest ectopic pancreatic tissue (and less likely other tumors) in the stomach[10]. Barium swallow studies reveal a similar appearance. Biopsies done during EGD are frequently non-diagnosted due to the superficial sample; they are usually reported as normal gastric mucosa. Definitive diagnosis is always made histopathologically[3,1]. Specimens can be sent for frozen section[1].

Treatment Treatment of heterotopic pancreas is specific

for the patient with obvious symptomatology. In the asymptomatic patient, maintaining medical supervision with periodic reviews is recommended[5]. The symptomatic patient usually experiences relief when the lesion is removed [1,3,4]. When encountered incidentally during surgery, lesions can be excised to prevent further operations or complications.

PrognosisThe prognosis in patients with surgically treated

pancreatic rests is usually excellent[3,1]. Although most of these lesions remain asymptomatic, it is important to remember that they are still susceptible to the same pathologic conditions as normal pancreatic tissue such as pancreatitis and malignant change[3,1].

Case reportA 38-year-old woman presented to the emergency

room (ER) with moderate, colicky abdominal epigastric pain, nausea and vomiting, absence of stool and gas passage. The symptoms started three days prior to presentation and evolved progressively. The patient had a surgical history for a right oophorectomy due to an extrauterine pregnancy. On presentation the patient denied fever, diarrhea, constipation, black stools, hematochezia, hematemesis, dysuria, or abnormal vaginal bleeding. On physical examination, vital signs were within normal limits. The abdomen was soft, non-distended, but mildly tender in the epigastric quadrant. No rigidity, rebound, or guarding was

present. Her white blood cell count increased to 11.300/ml with a left shift. Comprehensive metabolic profile showed hypercholesterolemia and hypertriglyceridemia, amylase and lipase level 10 xUNL; her urinalysis was within normal limits. Her pregnancy test was negative. Conservative management was implemented for a presumptive diagnosis of pancreatitis. The abdominal ultrasound revealed slow bowel movements and free liquid in the pelvic floor that measures 9mm. The plain radiography described air-fliud levels in the mesogastric quadrant, without free gas in the abdominal cavity (figure 1).

Esophagogastroduodenoscopy (EGD) was advised to confirm there were no changes on the wall of the stomach. The EGD showed an extrinsic/submucosal mass with antral localization, with central depression, measuring 15/5mm suggestive of ectopic pancreas. The overlying gastric mucosa was normal in appearance (figure 2 and 3).

The follow-up CT found the mass to be essentially unchanged. On the external face of the head of the pancreas, in contact with the structure of the pancreas, we describe a round structure with the highest diameter 23/16mm, with the same density with the pancreas, structure that impress the duodenum between the second and the third portion with irregular contour and heterogeneous enhancement(figure 4).

Free fluid in the Douglas pouch measuring 19mm. The diagnosis of acute pancreatits on ectopic pancreas and functional ileus was established.

The patient improved with pain management and intravenous fluids; she was then discharged home without pain; the patient was counseled regarding Fig. 1.

Fig. 2 and 3.

Fig. 4.


treatment; given the risk of the recurrence and the rare risk for malignant transformation and the fact that she elected conservative management and observation.

DiscussionsHeterotypic pancreas is an infrequent diagnosis and

rarely makes its way into differential diagnoses. Symptoms can be variable and the incidence is low enough so that many physicians have never seen a case. In addition, it can manifest clinically in some rare diseases of the pancreas including pancreatitis, islet cell tumor, pancreatic carcinoma, and pancreatic cyst. To date, there have been a few reports describing pancreatitis due to heterotopic pancreas. Therefore, the present case was considered to be a very rare case of this disorder; the incidence is reported to be 0.4%.

Although many modalities are available for workup, definitive diagnosis is always histological. Specific CT findings described above can be helpful for differentiating between ectopic pancreas and GIST. However, the tumor met a part of the criteria (pre-pyloric antrum location, an ill-defined border, an endoluminal growth pattern and prominent mucosal enhancement ), and therefore the diagnosis could be made with CT alone. Frozen section can allow for immediate local excision without further operations but in our case it was recommended that the patient should not undergo invasive procedures because of the risk of pancreatitis recurence. However, for our patient, it was felt the risk of complication with excision outweighed the benefit and it was also the patient’s desire for conservative management. In this circumstance, we felt that monitoring was a more appropriate option.

ConclusionsThe importance of this case is emphasized by the low

frequency of acute pancreatitis on ectopic pancreatic tissue which in our opinion should be actively assessed and treated appropriately; also, it is important for all the specialists involved in the medical management of these patients to distinguish their high potential for relapsing symptoms and very rare life threatening

complications like gastric perforation. Ectopic pancreas with its unusual clinical manifestations, locations or complications is of clinical interest. To conclude, heterotopic pancreas and its complications should always be considered in the differential diagnosis of acute abdomen.

References1. Hsia C-Y, Wu C-W, Lui W-Y. Heterotopic pancreas:

a difficult diagnosis. J Clin Gastroenterology 1999; 28: 144–147.

2. Sleisenger and Fordtran’s Gastrointesstinal and Liver Diseases: Pathophysiology/ Diagnosis/Management Philadelphia, PA 19103-2899, p. 896, 2327.

3. Cho J, Shin K, Kwon S, Kim J, et al. Heterotopic pancreas in the stomach: CT findings. Radiology 2000; 217: 139–144. PMid:11012436.

4. Schwartz S, Shires G, Spencer F. Principles of surgery. 6th ed. New York: McGraw-Hill; 1994, p.1144–1145, 1406.

5. Christodoulidis G, Zacharoulis D, Barbanis S, Katsogridakis E, Hatzitheofilou K. Heterotopic pancreas in the stomach: a case report and literature review. World J Gastroenterol 2007; 13:6098–6100.

6. Chandan VS, Wang W. Pancreatic heterotopia in the gastric antrum. Arch Pathol Lab Med 2004; 128: 111–112. PMid:14692822.

7. Feldman M, Friedman L, Brandt L, et al. Gastrointestinal and liver disease. 8th ed. Philadelphia, PA: Saunders Elsevier; 2006, p. 1180–1183.

8. Zak FG. Aberrant pancreatic carcinoma in a jejunal diverticulum. Gastroenterology 1956; 30:529–534.

9. Kim JY, Lee JM, Kim KW, et al. Ectopic pancreas: CT findings with emphasis on differentiation from small gastrointestinal stromal tumor and leiomyoma. Radiology 2009; 252: 92–100.

10. Park SH, Kim GH, Park DY, et al. Endoscopic findings of gastric ectopic pancreas: a single center experience. J Gastroenterol Hepatol 2011; 26: 1441–1446.

11. http://www.ncbi.nlm.nih.gov/pubmed/20977114


Toader Miorița30-32 Iancu de Hunedoara Blvd., Bucharest, Romaniae-mail: [email protected]

Abstract. In the past, neck suppurations were common causes of morbidity and mortality. Nowadays, the progress of antibiotherapy and rapid isolation of pathogenic bacteria with the antibiogram obtained in proper time, along with better living conditions and increased medical education of the population on hygiene conditions led to a significant decrease in the incidence of these diseases and an increase in the therapeutic success rate. Polybacterial etiology remained unchanged, with different pathogen combinations, often multiresistant to antibiotics. Infections can occur both in healthy individuals without significant local or general personal medical history and in people with existing diseases, which have been neglected or unknown. The authors present the diagnostic and therapeutic approach in one case of a child with neck suppuration, in which unusual pathogens for the paediatric ENT pathology were involved.Keywords: neck suppuration, antibiotherapy, drainage, child

1. University of Medicine and Pharmacy “Gr. T. Popa”, Iaşi2. Emergency Hospital for Children ”Grigore Alexandrescu”, Bucharest3. Dentirad Hospital, Ploieşti4. University of Medicine and Pharmacy “Carol Davila”, Bucharest

Palade D.1, Toader Mioriţa2, Constantin Anca2, Niculescu L.2, Drăghici M.3, Vivisenco Iolanda Cristina2,4

NECK SUPPURATIONS IN CHILDREN


Background

Neck suppurations remain a current topic. The progress of antibiotherapy did not significantly affect the rate of complications in cases of acute upper respiratory tract infections, but using combinations of antibiotics did limit the number of deaths or sequelae[1]. Presentation of the patient to the doctor since the onset of the disease and compliance with the prescribed treatment are prerequisites for the favourable outcome of these diseases.

Case reportA 14 year old girl from the rural environment without

significant pathological personal history was admitted in emergency to the ENT Clinic in the Emergency Hospital for Children "Grigore Alexandrescu", Bucharest for a giant laterocervical mass.

From history we have learned that the onset of the current disease was 10 days prior to the admission date with toothache for which the girl’s mother administered of her own volition painkillers and antibiotic treatment - cefuroxime orally in insufficient dose for the child’s age

and body weight (7 mg / kg / day). In evolution, symptoms persisted, therefore the patient was seen by the local ENT specialist, who established the diagnosis of acute pharingitis and recommended laboratory tests that the mother has neglected, and a new antibiotic - amoxicillin + clavulanate (50 mg / kg / day). Because symptoms worsened after 72 hours, the patient was taken by her parents to an emergency room for adults in Bucharest, from which she was sent by ambulance in our clinic.

On admission, the patient was conscious, but with an altered general condition, low grade fever and a giant swelling on the left hemiface, extended in the laterocervical area and left supraclavicular fossa (Figure 1). She had also trismus and cough with frankly purulent sputum.

CASE REPORT

Fig. 1. Laterocervical giant swelling


ENT examination revealed a painful and fluctuant swelling on palpation, extended from the apex of the mastoid to the supraclavicular fossa, anteriorly to the mediocervical line and submental space and posteriorly beyond the edge of the sternocleidomastoid muscle. Neck movements were possible, but difficult and painful.

It was difficult to perform the oropharyngoscopy because the patient had trismus (Figure 2). We noticed saburral tongue, complete and cared dentition, without cavities or dental abscesses, slightly asymmetric oropharyngeal isthmus, slightly swollen left tonsil, normal pharyngeal mucosal color and pus draining from cavum nasi on the posterior wall of the oropharynx. Bilateral nasal passages and ear meatus were free, tympanic membranes were apparently normal, mastoid points were painless, the hearing was normal and cranial nerve examination did not show any pathological findings.

We immediately performed laboratory tests, which showed insignificant leukocytosis (10.35/mm³) with discrete left shift of leukocyte formula, but with important inflammatory syndrome (ESR = 100 mm/h, fibrinogen = 732mg/dl, C-reactive protein = 8.5 mg/dl). A quantitative immunoglobulin determination was performed, which was within normal ranges.

Ultrasound examination of the swollen region revealed a transonic, impure collection with areas of hyperechoic, heterogeneous fluid, with maximum collection point in the left supraclavicular area, lack of vascularisation and left submandibular ganglia with inflammatory feature.

We completed the investigations with chest and sinus X-rays, which were within normal limits. We decided to explore the craniocervical and mediastinal region by computed tomography imaging (Figure 3), which highlighted the following:• On the coronal section we noticed a fluid, hypodense

collection, which does not capture the contrast dye, with the upper limit near the skull base, medial to the left internal carotid artery; the collection extends from the base of the skull until left supraclavicular fossa, anteriorly to the collarbone, pushing the pharyngo-laryngo-tracheal axis to the right, beyond the median line;

• Left palatine tonsil is edematous and increased in volume, but does not suggest a starting point for the collection (fig. 3.8, 3.10);

• There are multiple laterocervical lymphadenopathies

with inflammatory features, without any change of the petromastoid bony structures (fig 3.9);

• On the transverse section we noticed diffusion of the collection in the retromandibular space, fistulization in the endolaryngeal space and presence of gas bubbles inside the supraclavicular collection, suggestive feature of necrosis and infection with anaerobic germs (fig. 3.1-4);

• The collection diffuses anteriorly to the collarbone, while respecting the superior thoracic aperture and mediastinum; it also diffuses in the submandibular and sublingual areas (fig. 3.5-6);

• Left parotid gland is embedded in the collection and has structural changes, being edematous infiltrated (fig. 3.7);

• The collection is also in contact with the pharynx and the larynx, which are pushed to the right of the midline; the collection fills the entire left parapharyngeal lodge, with maximum collection point in the left supraclavicular space;

No endocranial changes.Fig. 2. Oropharingoscopy on admission

Fig.3. Computed tomography


Based on ENT clinical examination and imaging we established the diagnosis: left peritonsillar phlegmon fused in the laterocervical and supraclavicular space. We began intravenous empirical treatment with ceftriaxone, gentamicin and metronidazole, as well as rebalancing electrolyte infusion and analgesics.The next step was to perform bacteriological sampling, required for the etiological diagnosis. We practiced peritonsillar puncture which was negative, but aspirate was sent to lab, in order to be examined microscopically and inoculated on culture media for aerobic and anaerobic germs. For microscopic examination we used various staining, that have highlighted the following:• Giemsa stain - rich cellularity with 100% neutrophils,

absent fusospirillary bacteria,• Gram stain - polymorphic abundant flora with

gram-positive cocci and bacilli, rare gram-negative bacilli, rare fungi, Ziehl-Nielsen stain – absent acid-fast bacteria.48 hours after admission, we re-examined the oral

cavity under general anaesthesia with spontaneous breathing. We practiced thick needle puncture in the upper pole of the left palatine tonsil which was negative. Subsequently, thick needle puncture was performed at the supraclavicular fossa, which was positive, followed by incision at this level and discharge of pus under pressure in high volume (400ml), with false membranes and bad smelling. We washed the cavity with povidone-iodine solution and we used a suction drainage with a drainage tube sutured to the skin and connected to a vacuum nylon bag.At 72 hours after microbiological sampling, culture results and antibiogram were available. Aerobic cultures isolated the following germs:• Klebsiella pneumoniae (gram-negative, facultative

anaerobe);• Haemophillus parainfluenzae (gram-negative);• Streptococcus mitis (gram-positive, facultative

anaerobe).Anaerobic culture isolated Prevotella melaninogenica (gram-negative, anaerobe).Based on bacteriological results, we decided to change the antibiotic therapy with the combination imipenem/cilastatin, vancomycin and metronidazole. The patient also received supportive treatment with gamma globulins.Parenteral antibiotic therapy was continued for 10 days with favorable outcome. The patient was discharged from the hospital after this period and oral antibiotic treatment with clindamycin was recommended at home.

DiscussionThis case is peculiar and interesting. Studying literature

data on microbial particularities, we notice that Prevotella melaninogenica is usually causing dentoalveolar infections [2]. However, our patient had impeccable teeth and no alveolar changes visible on CT scan. This bacterium plays a major role in subcutaneous infections produced by complex mixtures of indigenous oral bacteria. Prevotella melaninogenica tends to produce hydrogen sulfide, ammonia and cytotoxic substances, inhibiting at the same

time phagocytosis and the ability of the host to kill other bacteria. In addition, the bacterium stimulates the host's immune system in order to produce substances which can cause tissue necrosis [2,3]. This may be an explanation for the rapid extension of the suppurative process in the presented case.

Haemophillus parainfluenzae is usually a commensal germ in oropharyngeal flora, being mainly isolated in the sputum of patients with chronic obstructive pulmonary disease [4,5]. Our patient had no history of acute or chronic pulmonary disease, chest X-ray and computed tomography confirming this. Also, this germ causes specific changes in the immune response, but our patient immunogram was absolutely normal. Haematological changes were predominantly inflammatory rather than infectious in this case.

Because the giant suppurative process was unilocular, all cervical spaces communicating with each other, the collection was completely drained. This facilitated the action of the antibiotics, which were chosen initially on empirical criteria, based on etiological probability, then according to antibiogram.

The severity of the case was given by the appearance of a large collection in a short time. Thus it was necessary to adopt an emergency approach, choosing an empirical association of antibiotics in order to cover both aerobic, as well as anaerobic germs and practicing incision and drainage of the collection within the first 48 hours, without waiting for the bacteriological results.

ConclusionIn conclusion, therapeutic protocol for neck

suppurations must consider the host immune status, associated diseases, polymicrobial etiology and modern imaging investigations. Solving these cases requires working in a multidisciplinary team consisting of ENT physician, surgeon, pediatrician, laboratory physician, radiologist and, not least anesthesiologist.

References1. Songu M, Demiray U, Adibelli Zh, Adibelli H.

Bilateral deep neck space infection in the paediatric age group: a case report and review of the literature. Acta Otorhinolaryngologica Italica. 2011;31(3):190-193.

2. Yanagisawa M, Kuriyama T, Williams DW. Proteinase Activity of Prevotella Species Associated with Oral Purulent Infection. Current Microbiology. 2006; 52(5): 375–8.

3. Ingham HR, Tharagonnet D, Sisson PR. Inhibition of phagocytosis in vitro by obligate anaerobes. Lancet. 1977; ii: 1252–1254.

4. Mitchell JL, Hill SL. Immune Response to Haemophilus parainfluenzae in Patients with Chronic Obstructive Lung Disease. Clinical and Diagnostic Laboratory Immunology. 2000;7(1):25-30.

5. Hill SL, Pye A, Johnson MM. A role for Haemophilus parainfluenzae in chronic lung disease. Am J Respir Crit Care Med. 1997; 155: A105.


Therapeutics, Pharmacology and Clinical ToxicologyVol XIX, Number 2, June 2015Pages: 124 - 126© Copyright reserved 2015

CASE REPORT

Mădălina Ilie 8 Calea Floreasca, 1st District, Bucharest, Romaniae-mail: [email protected]

Abstract. Pancreatic neuroendocrine tumors (pNETs) are usually slow growing tumors and their malignant potential is often underestimated. Most of them are malignant at diagnosis (50-80%, except for insulinoma) and have an aggressive course with liver metastases as well as other sites. These heterogeneous neoplasms are divided into two groups: functioning tumors, which secrete a variety of peptide hormones and non-functioning tumors (up to 90% of pNETs), which often show metastases at the time of diagnosis. We report a case of 44 years old male, non-smoker, recently diagnosed with type II diabetes mellitus, admitted for fatigue, loss of appetite and severe weight loss. Laboratory tests showed discrete inflammatory syndrome, while chest X-ray was normal. Abdominal ultrasound revealed hyperechoic lesions in both liver lobes. Tumor markers (carcinoembryonic antigen, alpha-fetoprotein and CA19-9) were elevated. The upper endoscopy and colonoscopy described no lesions. Abdominal computed tomography (CT) revealed hepatic metastases with unknown primary site, while contrast-enhanced ultrasound (CEUS) highlighted a hypoechoic hypervascular mass in the pancreatic tail. Chromogranin A, urinary 5-hydroxyindoleacetic acid, serotonin and neuron-specific enolase were normal. Ultrasound-Guided Liver Biopsy from the hepatic metastases was performed in order to establish the diagnosis. The histopathological and immunohistochemical results were decisive. Poorly differentiated pancreas neuroendocrine tumor (G3) was the diagnosis supported by the Ki67 index > 20 %.The patient was referred to medical oncology.Keywords: liver metastasis, pancreatic neuroendocrine tumors, ultrasound-guided liver biopsy

Constantin Alina1*, Plotogea Oana1*, Ilie Mădălina1*, Constantinescu G.1*, Macovei R.1*, Asmarandei R.1*,Stănciulescu Elena-Luminiţa1*, Diaconu Camelia1*, Popa B.1*

LIVER METASTASIS WITH UNKNOWN PRIMARY ORIGIN

Introduction

Pancreatic neuroendocrine tumors (pNETs) are relatively rare representing approximately

2% of all pancreatic neoplasms, having an incidence and prevalence that have increased substantially [1]. Their clinical incidence is reported to be of 1-5 new cases/100,000 population per year with a prevalence of 10/100,000 population[2]. These heterogeneous neoplasms can be classified in those associated with a functional syndrome and those that are not associated with a functional syndrome. Those associated with a functional syndrome secrete a variety of peptide hormones (insulin, gastrin, glucagon, vasoactive intestinal peptide, somatostatin or combination of them). Non-functioning tumors may also secrete a number of other substances, for example: chromogranins, neuron-specific enolase,

subunits of human chorionic gonadotropin, neurotensin, and grehlin, although, by definition, nonfunctioning pancreatic NET do not secrete or do not lead to a clinical syndrome. Non-functioning pancreatic tumors often show metastases at the time of diagnosis, most of them being liver metastases; up to 90% of pNETs are non-functioning [3, 4].

Fig.1. Ultrasound image of liver with multiple hypoechoic masses

1. Clinical Emergency Hospital Bucharest, Romania* all authors equally contributed to this work


Case Report We present the case of a 44 years old male patient,

non-smoker, recently diagnosed with type 2 diabetes mellitus, who was admitted to our hospital for fatigue, appetite loss and severe weight loss. He denied alcohol consumption. The clinical exam did not reveal any abnormalities, except for a low body mass index. Laboratory tests showed only a discrete inflammatory syndrome, while chest X-ray was normal. We performed an abdominal ultrasound which revealed multiple hyperechoic masses in both liver lobes (Figure 1)

Following ultrasound, we decided to test digestive tumor markers - carcinoembryonic antigen, alpha-fetoprotein and CA19-9 which were elevated. Subsequently, we performed an upper endoscopy and colonoscopy, both showing no lesions. Our suspicion of liver metastases was then confirmed by computed tomography (CT) which revealed hepatic metastases (Figure 2), but without identifying the primary site. For a better description of the masses, we decided to repeat the ultrasound using SonoVue – a contrast substance. This time, the contrast enhanced ultrasound (CEUS) not only highlighted the metastases, but more important it revealed a hypoechoic hypervascular mass in the pancreatic tail (Figure 3).

Fig.2. CT image of hepatic metastasesmasses

Fig.3. CEUS image of hypoechoic hypervascular mass in the pancreatic tail

In order to have a precise diagnosis we performed an ultrasound-guided biopsy from the liver masses (Figure 4). The histopathological and immunohistochemical results were decisive and the final diagnosis was poorly differentiated pancreas neuroendocrine tumor (G3), supported by the Ki67 index which was positive in more than 20% of tumoral cells. Biochemical markers showed normal levels of Chromogranin A (CgA), urinary 5-hydroxyindoleacetic acid, serotonin and neuron-specific enolase. The patient was referred to medical oncology.

Discussions As a consequence of the long delay between the onset

of symptoms and the final diagnosis, many patients have advanced disease at the time of diagnosis. 32% up to 73 % of pNETs are metastatic at diagnosis. Nonfunctioning pNETs present with non-specific signs and symptoms like abdominal pain, weight loss, anorexia and nausea [5]. Our patient presented only with fatigue, appetite loss and weight loss, symptoms which might have occur in a variety of catabolic diseases.

Imaging techniques [i.e., ultrasound or contrast-enhanced computed tomography (CT), magnetic resonance (MRI) or contrast-enhanced ultrasound (CEUS)] are necessary to detect both the primary tumor and metastases. CT scan is usually the first imagistic method used for evaluating pNETs, with great accuracy; however, sensitivity is decreased for tumors smaller than 2 cm in diameter [6]. These small tumors can often appear as vascular, hypodense or cystic lesions.

Endoscopic ultrasonography (EUS) is considered highly accurate for detecting primary pancreatic NETs with high sensitivity (82%) and specificity (95%), and it can detect lesions as small as 2 to 3 mm in diameter (6). One report showed that EUS had higher sensitivity than helical, contrast-enhanced CT scan (92% versus 63%) [6], having the possibility to perform fine-needle aspiration (FNA) that provides a non-operative histologic diagnosis of pNETs. Another benefit of EUS is the capability to use elastography, which is a method for real-time evaluation of tissue stiffness. The disadvantages of this method are represented by cost and availability.

In functioning neuroendocrine tumors, somatostatin receptor scintigraphy (SRS/Octreoscan) is considered the gold-standard to detect metastases,

Fig.4. Ultrasound-Guided Liver Biopsy


including extrahepatic ones, although there are studies which show that positron emission tomography (PET) using 68 Ga appears to be more sensitive, particularly in detecting small lesions.

Biochemical markers including CgA, pancreatic polypeptide and specific hormones depending on clinical presentation should be performed in all patients at the diagnosis and during follow-up [7,8].

Prognostic factors include histological grading, tumor differentiation and tumor staging. According to the recent World Health Organization (WHO) 2010 classification, three classes of tumors are identified (G1, G2, and G3): well-differentiated NETs can be classified as G1 tumors, when they express <2 mitoses/10 HPF and ≤2% Ki-67 index; as G2 tumors, when they express 2-20 mitoses/10 HPF and 3-20% Ki-67, whereas neuroendocrine carcinomas (NECs) usually belong to G3 category, with >20 mitoses/10 HPF and >20% Ki-67 index [9]. However, the presence of metastases, mainly liver metastases, represents one of the most important negative prognostic factor. In European and US referral centers, patients with pNETs often present with distant metastases at initial diagnosis (10). The occurrence of liver metastases is related to tumor extent (T-stage), differentiation, and grading (G1-G3); approximately 50% of poorly differentiated neuroendocrine carcinomas (NEC G3) are metastatic at initial diagnosis versus only 21% of well-differentiated and and 30% moderately differentiated neuroendocrine tumors (NETs G1 and G2), respectively[10]. The site of the primary tumor also has prognostic significance, since pNETs are usually characterized by a more severe clinical course when compared with gastrointestinal tumors, with a 5-year survival rate of 30-60% versus a rate of 60-90% for carcinoids [11, 12].

At present, a variety of therapeutic options exist for metastatic pNETs, including surgery, loco-regional therapies, chemotherapy, biotherapy with somatostatin analogues (SSAs) and interferon (IFN) and, more recently, the novel molecular targeted therapies and the systemic peptide receptor radionuclide therapy. Also liver transplantation (OLT) may be evaluated in highly selected patients [12].

According to ENETS Consensus guidelines, systemic chemotherapy using various cytotoxic agents (i.e., streptozotocin, doxorubicin, 5-fluorouracil, cisplatin, etoposide, dacarbazin) is recommended in pNETs, metastatic foregut NET G2, and in NEC G3 of any site [10].

Conclusion The majority of pancreatic neuroendocrine tumors

(PNET) are metastatic at diagnosis. Even if we don’t have a primary site from the first evaluations, repeating ultrasound more accurately and adding contrast enhanced US can establish the diagnosis. Liver biopsy

in our case, ultrasound guided, conferred a good specimen for certifying the pathology diagnosis.

References1. Franko J, Feng W, Yip L, et al. Non-functional

neuroendocrine carcinoma of the pancreas: incidence, tumor biology, and outcomes in 2,158 patients. J Gastrointest Surg 2010;14:541-8.

2. Oberg K. Pancreatic endocrine tumors. Semin Oncol 2010;37:594-618.

3. Cusati D, Zhang L, Harmsen WS, et al. Metastatic nonfunctioning pancreatic neuroendocrine carcinoma to liver: surgical treatment and outcomes. J Am Coll Surg 2012;215:117-24; discussion 124-5.

4. Jensen RT, Cadiot G, Brandi ML, et al. ENETS Consensus Guidelines for the management of patients with digestive neuroendocrine neoplasms: functional pancreatic endocrine tumor syndromes. Neuroendocrinology 2012;95:98-119.

5. Fesinmeyer MD, Austin MA, Li CI, et al. Differences in survival by histologic type of pancreatic cancer. Cancer Epidemiol Biomarkers Prev 2005; 14:1766.

6. Rösch T, Lightdale CJ, Botet JF, et al. Localization of pancreatic endocrine tumors by endoscopic ultrasonography. N Engl J Med 1992; 326:1721.

7. Falconi M, Bartsch DK, Eriksson B, et al. ENETS Consensus Guidelines for the management of patients with digestive neuroendocrine neoplasms of the digestive system: well-differentiated pancreatic non-functioning tumors. Neuroendocrinology 2012;95:120-34.

8. Massironi S, Sciola V, Peracchi M, et al. Neuroendocrine tumors of the gastro-entero-pancreatic system. World J Gastroenterol 2008;14:5377-84.

9. Rindi G, Arnold R, Bosman FT, et al. Nomenclature and classification of neuroendocrine neoplasms of the digestive system. In: Bosman FT, Carneiro F, Hruban RH, et al. eds. WHO classification of tumors of the digestive system. Lyon: International Agency for Research on Cancer (IRAC), 2010:13-145.

10. Pavel M, Baudin E, Couvelard A, et al. ENETS Consensus Guidelines for the management of patients with liver and other distant metastases from neuroendocrine neoplasms of foregut, midgut, hindgut, and unknown primary. Neuroendocrinology 2012;95:157-76.

11. Panzuto F, Nasoni S, Falconi M, et al. Prognostic factors and survival in endocrine tumor patients: comparison between gastrointestinal and pancreatic localization. Endocr Relat Cancer 2005;12:1083-92.

12. Tomassetti P, Campana D, Piscitelli L, et al. Endocrine pancreatic tumors: factors correlated with survival. Ann Oncol 2005;16:1806-10.


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