Partnership between the GFFCC and WHO at the start of the1st Palliative Training Course in the Gulf Region

Honorary editor

Abdul Rahman Al-Awadi

editor – in – CHief

Khaled Al-Saleh

Senior editorS

Abdullah Behbehani

Farid Saleh

Ibrahim Al-Sheneber

Muhyi Al-Sarraf

regional editorS

The Gulf Journal of Oncology

Abdel Salam Othman ---------------------KuwaitAbdulwahab Andijani --------------------Saudi ArabiaAbraham Varughese -----------------------KuwaitAdel Henayan ------------------------------KuwaitAdel Khedar -------------------------------KuwaitAdnan Azzat -------------------------------Saudi ArabiaAhmed Al-Sharhan ------------------------KuwaitAhmed Ragheb -----------------------------KuwaitAmany El-Basmy -------------------------KuwaitAmany Abdo Bouathy --------------------YemenAmy C. Hessel -----------------------------USAAshok R. Shaba ----------------------------USABadawi Hathout ----------------------------KuwaitBeth S. Edeiken ----------------------------USADahish Ajaram -----------------------------Saudi ArabiaEduardo M. Diaz --------------------------USAEyad Al-Saleh ------------------------------KuwaitFahed El-Enezi ----------------------------KuwaitFalah Al-Khateeb --------------------------OmanH.S.Hooda ----------------------------------KuwaitHamdi Jad ----------------------------------KuwaitHassan Y.M. Al-Idrissi -------------------Saudi ArabiaIman Al-Shammeri ------------------------KuwaitIsmail Helmy -------------------------------QatarIssam M. Francis --------------------------KuwaitJanet Wilson -------------------------------UKJaroslav Nemec ----------------------------KuwaitJatin Shah ----------------------------------USAJean Yves Bobin ---------------------------FranceKamal Al-Ghamrawy ---------------------OmanKhalda Bouarki ----------------------------KuwaitLamk M. Al-Lamki -----------------------OmanLouis. A. Gaboury -------------------------Canada

M. Salah Fayaz -----------------------------KuwaitMagdy El-Shahawi ------------------------EgyptMedhat Faris --------------------------------Oman Michelle D.Williams ----------------------USAMohamed A. El-Massry ------------------KuwaitMohamed Al-Jarallah ---------------------KuwaitMohamed Al-Shahri -----------------------Saudi ArabiaMohamed Amen Al-Awadi ---------------Bahrain Mohammed Sherif ------------------------KuwaitMonir Abol – Ela --------------------------EgyptMostafa M. Elserafi -----------------------EgyptMoustafa A. Manieh -----------------------EgyptNajib Haboubi ------------------------------U.KNasser Behbehani -------------------------KuwaitPawel Kukawski ---------------------------PolandRadfana A. Al-Rayashi --------------------YemenRakesh Mittal -------------------------------KuwaitRamesh Pandita ----------------------------KuwaitRanda Hamada -----------------------------BahrainReham Safwat ------------------------------KuwaitRoman Skoracki ---------------------------USASalem Al-Shemmari ----------------------KuwaitSalha Boujassoum -------------------------QatarSadeq Abuzallouf --------------------------KuwaitSami Al-Badawy --------------------------EgyptShafeka Al-Awadi --------------------------KuwaitSuad Al-Bahar ------------------------------KuwaitTim Whelan --------------------------------CanadaYasser Bahader -----------------------------Saudi ArabiaYousri Gouda -------------------------------EgyptZaidan Al Mazidi --------------------------Kuwait

Abdul Azim Hussein ............... QatarAbdelhamid El-Jazzar............. Kuwait Abdelrahman Al- Jassmi ......... UAE Abdel Rahman Fakhro ............ BahrainAbdullah Al-Amro .................. Saudi Arabia

Bassim Al-Bahrani ...........................OmanFahd Al-Mulla ..................................KuwaitJamal Al- Sayyad .............................BahrainMahmoud Shaheen Al-Ahwal ..........Saudi ArabiaNadim Mohammed Sayed ................Yemen

editorial adviSory Board

The Gulf Journal of

OncologyISSUE 13 JANUARY 2013

TABlE oF CoNTENTS

original articles / Studies

Dosimetric consideration of transient volume enlargement induced by edema in prostate brachytherapy seed implants ...... 06I. Ali, O. Algan, S. Thompson, P. Sindhwani, S. Ahmad

Assessment of an existing and modified model for predicting non sentinel lymph node metastasis in breast cancer patients with positive sentinel node biopsy ....................................................................................................................................... 15M. Al-Masri, G. Darwazeh, M. El-Ghanem, B. Hamdan, M. Sughayer

Docetaxel in advanced or metastatic endometrial cancer: Clinical outcome ............................................................................... 23R.H. Hamed, S.A.Elkhalk, S. Roshdy

Dosimetric comparison between bone marrow sparing intensity-modulated radiation therapy and conventional techniques in the treatment of cervical cancer: a retrospective study ........................................................................................... 30T. Sundaram, V. Nagarajan, M. Nagarajan, S. Jayakumar, K.N. Govindarajan, SS Supe, M. Balasubramaniam, P. Joshi, T.P. Chellapandian

Trends in oesophagus and Stomach cancer incidence in Bangalore, India ................................................................................... 42BR Gopala Krishnappa, CR Vijay, C Ramesh, PP Bapsy, MU Kumar, M Vijayakumar, SS Supe

Clinical significance of telomerase genes (hTERC and hTERT) amplification in patients with acute myeloid leukemia ........ 51M.M. Eid, N.A. Helmy, I.M. Omar, A.A. Mohamed, D. El Sewefy, I.M. Fadel, R.A. Helal

review articles

Management of metastatic breast cancer (MBC) ............................................................................................................................ 61A. AL-Amri

Extensive review in the diagnosis of the malignant transformation of pleomorphic adenoma ................................................... 67Tarakji, K. Baroudi, S. Hanouneh, M.Y. Kharma. M.Z. Nassani

Case reports

Primary adenoid cystic carcinoma of the breast: Case report and review of the literature ........................................................ 83M.A. Naseer, S.S. Mohammed, R. Alyusuf, R. Al Marzooq, S.K. Das Majumdar, A. Al Hammadi

Approaches to management of Adenocarcinoma following Colocystoplasty ................................................................................ 87R. Ramamurthy, S. Susikar

Primary Non-Hodgkin Lymphoma of Frontal Sinus diagnosed by Fine needle aspiration cytology ......................................... 92J.K.S.S. Philip, A. Al- Jassar, I.S. Naquib, S. Usmani, M. El- Kabani, S.M. Refaat

Conference Highlights /Scientific Contribution

• Conference Highlights – The Regional Training of the Trainers Palliative Care Workshop .................................................96• News Notes ....................................................................................................................................................................................101• Advertisements .............................................................................................................................................................................103• Scientific events in the GCC and the Arab World for the 1st Semester of 2013 ...................................................................104

Trends In Oesophagus And Stomach Cancer, BR Gopala Krishnappa, et. al.

5151

Clinical Significance Of Telomerase Genes (hTERC And hTERT) Amplification In Patients With

Acute Myeloid LeukemiaM.M. Eid1, N.A. Helmy1, I.M. omar2, A.A. Mohamed2, D. El Sewefy2, I.M. Fadel1, R.A. Helal1

1Human Cytogenetics, National Research Center, Cairo, Egypt. 2Faculty of Medicine, Ain Shams University, Clinical Pathology Department, Egypt

Corresponding author: Maha Mohamed Eid, Human Cytogenetics, National Research Center, Cairo, Egypt. Email: mahaeid67@gmail.com

Abstract:Acute myeloid leukemia (AML) describes

a heterogenous group of hematological disorders. Cytogenetic and molecular assays have allowed patients’ follow up aiming for detection of minimal residual disease, prediction of patients’ outcome, in addition to providing the rationale for designing novel molecular-targeted therapeutic strategies. Human telomerase reverse transcriptase (hTERT), encoded by the hTERT gene and the telomerase RNA component (hTERC) genes are frequently amplified in human tumors, which may indicate that the hTERT and the hTERC genes may be target for amplification during the transformation of human malignancies including hematological malignancies. This genetic event has implications in diagnosis, prognosis and therapeutics of cancer.

To evaluate the hTERC and hTERT genes as a prognostic marker in patients with AML, hTERC and hTERT gene amplification was studied in 20 adult AML patients using a commercial FISH probes (Kreatech) designated to detect the copy numbers of the genes. They were 12 males and 8 females. Their ages ranged from 16 to 67 years. The patients were further divided into two groups; group I (12 patients) includes newly diagnosed AML patients and

IntroductionAcute myeloid leukemia is a malignant,

genetically heterogeneous disorder. Different prognostic factors are currently available to predict the treatment outcomes in defined subgroups of patients. Cytogenetic aberrations

group II (8 patients) includes patients taken at 28th day of chemotherapy. The hTERC amplification was detected in 19/21 cases (90.5%). The copy number of the gene ranged from 2-5 copies per interphase cell. For the hTERT gene, the amplification was found in the same percent of the patients. The copy number of the gene ranged from 2-9 copies per interphase cell. On comparing the group I with group II there was a highly statistical significant difference regarding the percent of amplification of both genes. The percent of amplification of hTERT gene was found to be higher among patients with poor outcome of the disease than in patients with good outcome. On the contrary the hTERC gene amplification did not exhibit such a correlation. In conclusion, hTERT and hTERC genes amplification were detected in patients with AML; therefore telomerase can be a good cancer marker which may be involved in carcinogenesis of leukemia. Higher amplification was found in de novo cases than cases in remission which emphasize its role in clinical analysis, disease monitoring and detection of minimal residual disease

KeywordsAcute Myeloid leukemia, telomerase

amplification, hTERC gene, hTERT gene

are among the most important independent prognostic markers. Cytogenetics analysis in AML represents an important tool for both diagnosis and prognosis of AML. Hence, the new World Health Organization (WHO) classification of haematological malignancies based on specific chromosome aberrations and their molecular counterparts together with morphology, immunophenotype and clinical

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Clinical Significance of Telomerase Genes Amplification, M.M. Eid, et. al.

features (1,2).

Among these cytogenetics data, the prognostic value of telomere shortening and telomerase activity has been suggested in various human hematopoietic malignancies(3). Telomerase is a ribonucleoprotein reverse transcriptase composed of two subunits: human telomerase reverse transcriptase (hTERT) and human telomerase RNA (hTERC). These two subunits are encoded by two different genes located on 5p15 and 3q26 respectively, both are essential for the function of the enzyme (4,5).

Expression of significant levels of telomerase can dramatically increase proliferative life span and promote cellular immortality, thereby contributing to the malignant phenotype. It is therefore important that its contribution to the burden of human cancer to be understood (6). Despite the questionable role of telomerase reactivation in cell immortalization and carcinogenesis, telomerase itself may serve as diagnostic marker for tumor development. Several studies have demonstrated that the presence of increased telomerase activity can be used to distinguish malignant tissue from normal one of various organs (7).

To highlight the role of hTERC and hTERT genes amplification in AML, the aim of this study was the detection of hTERC and hTERT genes amplification in adult AML patients using FISH technique and to correlate this amplification to the standard prognostic factors in a trial to deduce their clinical significance.

Patients and methods

Patients The present study was carried out on 21 AML

patients. Their ages ranged from 16 to 67 years. There were 13 males and 8 females with a male to female ratio of 3:2. Patients were classified into two groups as following: Group I: Included 12 newly diagnosed AML cases and 1relapsed case (case 1-13). Group II: Included 8 cases at 28th day of chemotherapy (case 14-21).

Methods All patients were subjected to the following:

1) Clinical Investigations: Thorough clinical examination, complete blood picture, bone marrow examination and immunophenotyping of BM or PB samples by flow cytometry (FCM). 2) Cytogenetics study: a) Conventional cytogenetics study using G-Banding technique according to Verma and Babu 1995 (8). b) Fluorescence in situ hybridization analysis (FISH).

Bone marrow or peripheral blood was cultured using complete culture media without phyohemagglutinin added to it. The FISH technique was applied to the interphase nuclei. The fluorescence in situ hybridization (FISH) technique was carried out according to the manufacturer’s instructions of Poseidon Kreatech Repeat free probe which designed to detect the amplification (copy number 3 or more are considered amplified) at 3q26 (hTERC) and 8q24 (cMyc), and the hTERT (5p15) and EGR1 (5q31) control locus. Each probe was added to separate slide.

Slides were examined using an applied imaging system, i.e. an Olympus BX51 microscope (Olympus, Tokyo, Japan) with a fluorescent attachment and equipped with filter sets. For each case, 100 interphase nuclei were studied. Positive amplification is considered if 3 or more signals were detected. Copy numbers was counted for each case. And percent of cells with amplification were calculated.

Results

i. demographic features of the studied groups: Group I: included 13 AML patients (12 newly

diagnosed and one relapsed case; case No. 1 to case No. 13), the age ranged between 16 and 62 years with a median of 33 years. They were 7 males (53.85%) and 6 females (46.15%), with a male to female ratio of 1.2:1.

Group II: included 8 AML patients at 28th day of chemotherapy (case No. 14 to case No. 21), the age ranged between 16 and 67 years with a median of 44.50 years. They were 6 males (75.0%) and 2 females (25.0%), with a male to female ratio of 3:1.

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ii. Clinical and laboratory features of the Studied groups:The clinical and the laboratory findings of the patients are shown in Tables 1-5.

Table 1: Clinical Data of Studied AMl Patients

M: Male F: Female HM: Hepatomegaly DNV: de novo lN: lymphadenopathy SM: Splenomegaly CM: 28th day post chemotherapy Poor outcome: Died or relapsed cases.

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Table 2: Descriptive statistics of karyotyping, prognosis, FISH analysis and outcome for all studied patients

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Table 3: Different patterns of karyotyping detected among studied AML patients

Table 4: Comparison of clinical data between Group I and Group II NS: Non-significant

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Table 5: Comparison of laboratory data between Group I and Group II patientsS: significant. HS: highly significant. NS: Non significant

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iii. Cytogenetics results of the studied group:

a. Conventional Cytogenetic Analysis (Karyotyping): Conventional cytogenetic analysis of BM or PB samples using G-banding technique was performed for all patients. The cultures were successful in 16/21 (76.2%) cases. The cytogenetics results were shown in (Tables 2, 3) (Fig.1).

b. Fluorescent In Situ Hybridization (FISH): Interphase FISH analysis was successfully performed on the 21 BM /or PB samples. It revealed positive hTERC gene amplification in 19/21 cases (90.5%). The percent of cells with amplification ranged from 2% to 69%. The No. of copies

ranged from 2-5 copies (Fig. 2, 3). On the other hand, hTERT gene amplification was detected in 19/21 cases (90.5%). The percent of cells with amplification ranged from 3% to 76%. The No. of copies ranged from 2-9 copies (Fig. 4).

Figure 2: Interphase FISH analysis showing amplification of hTERC (3-4 red signals) / 2 green signals of cMYC

Figure 5: Interphase FISH analysis showing amplification of hTERT (3-4red signals) / 2 green signals

Figure 4: Interphase FISH analysis showing amplification of hTERT (3-4red signals) / 2 green signals

Figure 3: Interphase FISH analysis showing amplification of hTERC (3 red signals) / 2 green signals of cMYC

Figure 1: Conventional cytogenetic analysis by G-banding (case No. 10) showing complex karyotype in the form of t(9;22)(q34;q11) rearrangement and trisomy 4, 15, 17, 19, 21 case No.10.

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iv. Clinical outcome:According to outcome, cases were classified

into good outcome and poor outcome (death or relapse) as follows:

Group I: Out of the 13 cases; there were 5 cases (38.46%) with good outcome and 8 cases (61.54%) with poor outcome.

Group II: Out of the 8 cases; there were 5 cases (62.5.0%) with good outcome and 3 cases (37.5.0%) with poor outcome.

Comparative Studies

Statistical comparison between group i and group ii regarding all studied parameters.

On comparing Group I versus Group II: there was a statistically significant decrease in the TLC (p=0.02), and a highly statistically significant decrease in the PB and BM blast percent among group II (p=0.001, p=0.004 respectively). In addition, statistically significant increase in the percent of amplification of hTERC and hTERT genes was obtained among Group I patients than Group II with p value (p=0.010 and 0.018 ) respectively.

Using Pearson’s correlation test to compare between the percent of amplification and other prognostic criteria in AML in both de novo and after 28 days chemotherapy revealed; none significant correlation with the total percent of amplification except the peripheral blast number which shows a positive correlation with the total percent of the amplification. Also, none of the other studied parameters showed any significant difference between the two groups (p>0.05).

Comparison between good outcome and poor outcome patients as regard the percent of amplification of hTERC gene revealed statistically non significant difference between both groups however, both cases with no amplification of hTERC gene were of good outcome. On the other hand there was a highly statistically significant increase of hTERT gene amplification among poor outcome patients than good outcome group.

DiscussionAcute myeloid leukemia is a malignant,

genetically heterogeneous disorder. Prognostic factors are needed in order to be able to better predict treatment outcomes in defined subgroups of patients. Cytogenetic aberrations are among the most important independent prognostic factors. A prognostic value of telomere shortening and telomerase activity has been suggested in various human hematopoietic malignancies (3).

In the present study FISH analysis revealed positive hTERC and hTERT genes amplification in 90.4% of AML patients. However percent of cells showing amplification was significantly lower in patients at 28 days post chemotherapy. These findings can be explained by the decrease in blasts post chemotherapy.

In the study conducted on hematological malignancies by Nowak et al., 2006 (7), they found hTERC and hTERT genes amplification in all samples tested (16 AML cases) and that leukemic cells possess variable number in a range of 2 to 12 copies 2 to 60 respectively. They suggested that the activation of telomerase in leukemic cells is connected with amplification of hTERC and hTERT genes and that the high expression and activity of telomerase found in leukemic cells can be explained by the amplification of hTERC and hTERT genes. They have demonstrated that the presence of telomerase activity can be used to distinguish malignant from normal cells and that telomerase itself may serve as diagnostic marker for tumor development and could be useful in diagnosis of acute myeloid leukemia.

These results were confirmed by Ying et al., 2008 (9), when they reported also that over expression of both hTERT and hTERC and subsequently increased telomerase activity, whereas over expression of either hTERC or hTERT alone induced telomerase activity to a lesser extent. This finding could support our results, where the 4/8 patients of good outcome have shown amplification of either hTERC or hTERT gene, which led to elevation of the telomerase activity to a lesser values than when both genes are amplified.

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However, Serakinci et al., 2002 (10) found no evidence of amplification of telomerase genes (hTERT and hTERC) in AML and suggested that up-regulation of telomerase activity in this type of cancer might be different from up-regulation of telomerase activity in solid tumors concluding that amplification is a property of solid tumors, not of hematological cancers.

A comparison between Group I and group II with regards to % of amplification was done and revealed a statistically significant increase in the percent of amplification of hTERC and hTERT genes among Group I patients than Group II (p=0.010).

In agreement with our results, Wang et al., 2010 (11) in their work on newly diagnosed group, relapsed group and a complete remission group of acute myeloid leukemia, they found that telomerase activity in AML patients was higher than healthy individuals and telomerase activity level was the highest in the relapsed group, followed by the newly diagnosed group, and then the complete remission group. Same result was reported earlier by Verstovsek et al., 2003(12) and Ghaffari et al., 2008 (13) who observed that telomerase activity was significantly higher during relapse than at diagnosis therefore it was suggested that it may be used to estimate the curative effect and further elevation of telomerase activity is linked to disease recurrence.

Regarding the clinical outcome, when we compared between good outcome and poor outcome patients as regarding the percent of amplification of hTERC gene, there was no statistically significant increase among poor outcome patients than good outcome. However the hTERT amplification revealed a high significant difference between good and bad outcome. This finding is in agreement with Asfour et al. 2008 (14), who reported high levels of telomerase activity in AML blasts predict unfavorable outcome, resistance to therapy and short survival. With exception to karyotype, other studied parameters showed no statistical significant difference between poor and good outcome.

Interestingly, it was observed in our study

that some patients with normal karyotype and poor outcome had high % of hTERC and hTERT amplification. Similar results had been reported by Hartmann et al. 2005 (3), who suggested that telomerase activity and hence hTERC and hTERT amplification could be used to follow up minimal residual disease in cytogenetically normal AML patients which allow the good risk patients to be separated from the bad risk ones within this group in order to improve risk-adapted treatment strategies in AML.

This was in accordance with Asfour et al. 2008 (14), who explained that the presence of normal karyotype does not mean that the cytogenetically normal leukemic blasts harbor no acquired genetic alterations and that the use of molecular genetic techniques in such cases with high telomerase activity may reveal an even stronger association with genetic abnormalities.

In our study, we could not establish a relation between amplification and other prognostic criteria except peripheral blood blasts this was concurrent with Fan et al., 2010 (15) in their work on solid tumor where they found the percent and number of copies of hTERC gene is increased with the grade of malignancy.

In the present work, a case (No. 10) of AML on top of CML had a high % of hTERT amplification at 65% and 35% for hTERC gene amplification in addition to a complex karyotype. The outcome of this patient was poor as she died. This case reflects the impact of both genes amplification and complex karyotype on the clinical outcome which was supported by the above mentioned studies.

In conclusion, although we could not find significant relation of hTERC and hTERT with known clinical prognostic factors, hTERT has clinical utilities for following disease progression and may predict early relapse. Also telomerase amplification may be used as one of the markers for detecting MRD and monitoring effectiveness of chemotherapy in AML.

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References

10. Serakinci N, Koch J (2002) Telomerase activity in human leukemic cells with or without monosomy 7 or 7q. BMC Medical Genetics 3(1): 11.

11. Wang Y, Fang M, SUN X, SUN Y (2010) Telomerase activity and telomere length in acute leukemia: correlations with disease progression, subtypes and overall survival. International Journal of Laboratory Hematology 32(2): 230-238.

12. Verstovsek S, Manshouri T, Smith FO, Giles FJ, Cortes J, et al (2003) Telomerase activity is prognostic in pediatric patients with acute myeloid leukemia. Cancer 97(9): 2212-2217.

13. Ghaffari SH, Shayan-Asl N, Jamialahmadi AH, Alimoghaddam K, Ghavamzadeh A (2008) Telomerase activity and telomere length in patients with acute promyelocytic leukemia: indicative of proliferative activity, disease progression, and overall survival. Annals of Oncology 19(11): 1927-1934.

14. Asfour IA, Fayek MH, El-kourashy SA, et al (2008) Correlation of telomerase activity to apoptosis and survival in adult acute lymphoblastic leukemia: an Egyptian single-center study. Ann Hematol 2008; 87: 213-221

15. Fan YB, Wu X, Fu ZM, Wu GP (2010) Amplification of the human telomerase gene in liquid-based preparations is associated with cervical dysplasia and carcinoma. Int J Gynecol Pathol 29(2): 157-164.

1. Vardiman JW, Harris NL, Brunning RD (2002) The World Health Organization (WHO) classification of the myeloid neoplasms. Blood 100: 2292-2302.

2. Ferrara F, Palmieri S , Leoni F (2007) Clinically useful prognostic factors in acute myeloid leukemia. Oncology/Hematology 66: 181–193.

3. Hartmann U, Brümmendorf, T H, Balabanov S, et al (2005) Telomere length and hTERT expression in patients with acute myeloid leukemia correlates with chromosomal abnormalities. Haematologica 90(3): 307-316.

4. Buys CH (2000) Telomeres, Telomerase, and Cancer. N Engl J Med 342(17): 1282-1283.

5. Davison GM (2007) Telomere and telomerase in leukemia and lymphoma. Transfusion and Apheresis Science 37: 43-47.

6. Drummond M W, Balabanov S, Holyoake T L, Tim H, Brummendorf TH (2007) Concise Review: Telomere Biology in Normal and Leukemic Hematopoietic Stem Cells. Stem Cells 25: 1853-1861.

7. Nowak T, Januszkiewicz D, Zawada M, Pernak M, Lewandowski K, Rembowska J, Nowicka K, Mankowski P, Nowak J (2006) Amplification of hTERT and hTERC genes in leukemic cells with high expression and activity of telomerase. Oncology Reports 16(2): 301-305.

8. Verma RS and Babu A (1995) Human Chromosomes. Principles and Techniques, 2nd edn. New York: McGraw-Hill

9. Ying C, Tracy M B , Roger R R (2008) Increased copy number of the TERT and TERC telomerase subunit genes in cancer cells. Cancer Science 99(6): 1092-1099.