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Equivocal Cytology in Lung

Cancer DiagnosisImprovement of Diagnostic Accuracy Using Adjuvant Multicolor FISH,DNA-image cytometry, and Quantitative Promoter Hypermethylation Analysis

Martin Schramm, MD1; Christian Wrobel1; Ingmar Born1; Marietta Kazimirek1;

Natalia Pomjanski, MD1; Marina William, MD2; Rainer Kappes, MD3; Claus Dieter Gerharz, MD, PhD4;

Stefan Biesterfeld, MD, PhD1; and Alfred Bocking, MD, PhD1

BACKGROUND: Sometimes, cytological lung cancer diagnosis is challenging because equivocal diagnoses

are common. To enhance diagnostic accuracy, fluorescent in situ hybridization (FISH), DNA-image

cytometry, and quantitative promoter hypermethylation analysis have been proposed as adjuncts.

METHODS: Bronchial washings and/or brushings or transbronchial fine-needle aspiration biopsies were pro-

spectively collected from patients who were clinically suspected of having lung carcinoma. After routine

cytological diagnosis, 70 consecutive specimens, each cytologically diagnosed as negative, equivocal, or

positive for cancer cells, were investigated with adjuvant methods. Suspicious areas on the smears were

restained with the LAVysion multicolor FISH probe set (Abbott Molecular, Des Plaines, Illinois) or according

to the Feulgen Staining Method for DNA-image cytometry analysis. DNA was extracted from residual liquid

material, and frequencies of aberrant methylation of APC, p16INK4A, and RASSF1A gene promoters were

determined with quantitative methylation-specific polymerase chain reaction (QMSP) after bisulfite conver-

sion. Clinical and histological follow-up according to a reference standard, defined in advance, were avail-able for 198 of 210 patients. RESULTS: In the whole cohort, cytology, FISH, DNA-image cytometry, and

QMSP achieved sensitivities of 83.7%, 78%, 79%, and 49.6%, respectively (specificities of 69.8%, 98.2%,

98.2%, and 98.4%, respectively). Subsequent to cytologically equivocal diagnoses, FISH, DNA-image

cytometry, and QMSP definitely identified malignancy in 79%, 83%, and 49%, respectively. With QMSP, 4 of

22 cancer patients with cytologically negative diagnoses were correctly identified. CONCLUSIONS: Thus,

adjuvant FISH or DNA-image cytometry in cytologically equivocal diagnoses improves diagnostic accuracy

at comparable rates. Adjuvant QMSP in cytologically negative cases with persistent suspicion of lung

cancer would enhance sensitivity. Cancer (Cancer Cytopathol) 2011;119:177-92. VC 2011 American Cancer

Society.

KEY WORDS:lung cancer, cytology, FISH, DNA-image cytometry, promoter hypermethylation.

Received:October 7, 2010; Revised: December 31, 2010 and January 24, 2011; Accepted: January 25, 2011

Published online March 16, 2011 in Wiley Online Library (wileyonlinelibrary.com)

DOI: 10.1002/cncy.20142, wileyonlinelibrary.com

Corresponding author: Stefan Biesterfeld, MD, PhD, Institute of Cytopathology, Heinrich Heine University, Moorenstr.5, D-40225 Dusseldorf,

Germany; Fax: (011) 49-211-8118402; [email protected]

1Institute of Cytopathology, Heinrich Heine University, Dusseldorf, Germany; 2Institute of Pathology, Heinrich Heine University, Dusseldorf, Germany;3Department of Pulmonology, Florence Nightingale Hospital, Dusseldorf, Germany; 4Institute of Pathology, Evangelical Bethesda-Johanniter Clinical

Center, Duisburg, Germany

Cancer Cytopathology June 25, 2011 177

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Lung cancer is 1 of the most frequent causes of death

worldwide. It is estimated that in 2008, 161,840 peo-

ple in the United States and 342,000 in Europe died

from lung cancer.1,2 More than 77% of the patients in

the United States are in the late stages of the diseasewith metastases to lymph nodes and distant sites at di-

agnosis, as often early symptoms are missing.3 In recent

years, several attempts have been conducted to improve

an early or accelerated diagnosis because patients in

early lung cancer stages have a better overall prognosis

after therapy.4 Diagnosis with physical examination,

chest x-ray, spiral computed tomography (CT), and

bronchoscopically obtained histological or cytological

specimens represent, in most cases, the first attempt to

confirm suspected lung cancer. Cytological methodsinclude investigation of sputa, bronchial washings,

bronchial brushings, and transbronchial or transtho-

racic fine-needle aspiration biopsies.5 Because lung

cancer is often diagnosed in late stages, operative ther-

apy is not always recommended, and final diagnosis is

not seldom solely based on cytology.6 Sometimes be-

nign and malignant lesions cannot be discriminated by

morphology with certainty, and reactive changes of

bronchial and alveolar epithelium, air-drying artifacts

during smear preparation, and poorly preserved speci-

mens sometimes impede or even render a distinct cyto-

logical diagnosis impossible.7-10 Accordingly, cytology

sometimes leaves an equivocal (2.9% of cases in a single

institution; 8.6% from a single hospital during a 9-

month period in our institution) or inconclusive result

even in the hands of experienced observers.5,11,12 To

prevent repeated diagnostic efforts and potentially

harmful invasive diagnostic procedures, it is essential to

enhance diagnostic accuracy in these cases. The desired

result is a definite positive or negative diagnosis of

malignancy.

Based on the hypothesis that (chromosomal) aneu-

ploidy essentially contributes to tumorigenesis,13-17

numerical chromosomal aberrations can be detected in

cancer cells with fluorescent in situ hybridization (FISH).

The LAVysion (Abbott Molecular, Des Plaines, Illinois)

multicolor FISH probe18 has been previously used for the

early detection of lung cancer on cytological speci-

mens.11,19-26 DNA-image cytometry uses the detection of

DNA aneuploidy via an abnormal cellular DNA content

after stoichiometric staining of DNA according to the

Feulgen staining method.27,28 Diagnostic application in

pulmonary pathology has been reported for identificationof prospective malignant lesions (ie, dysplasia) and predic-

tion of prognosis in manifest cancers.29-35. Both, FISH

and DNA-image cytometry can be performed on the

same specimen subsequent to a cytological diagno-

sis.11,18,21,30,32 There is no need for additional material,

which means no further stress for the patient.

Promoter hypermethylation is a major mechanism

of tumor suppressor gene inactivation in lung cancer and

can be used as a biomarker for early detection.36-39 Panels

of aberrantly methylated gene promoters, investigated

with quantitative methylation-specific real-time polymer-ase chain reaction (QMSP) can be used as biomarkers for

the detection of lung cancer on residual liquid material

from regular diagnostic cytology specimen collection.40

The aim of our prospective cohort study was to

compare the potential benefit for diagnostic accuracy on

pulmonary cytology of LAVysion multicolor FISH,

DNA-image cytometry, and a panel of aberrantly methyl-

ated tumor suppressor genes with QMSP.40 The study

intended to determine a diagnostic algorithm for the

application of these methods, especially in cytologically

equivocal cases. Each method was correlated with a prede-fined reference standard. In a second step, all adjuvant

methods were directly compared on the same specimen

for their diagnostic power.

MATERIALS AND METHODS

Diploidy is defined as a 2-fold chromosomal set. Euploid

polyploidy, in this context, means 2n chromosomal sets

(including tetraploidy). Aneuploidy means a chromo-

somal set =2n, which is because integrated-value multi-

ples of single chromosomal sets, apart from 2n, do not

occur in non-neoplastic tissues.

Patient Selection and Study Design

The study was approved by the local ethics committee.

Bronchoscopically obtained diagnostic material on 843

consecutive patients with suspected lung cancer from the

Florence Nightingale Hospital in Dusseldorf, Germany,

Original Article

178 Cancer Cytopathology June 25, 2011


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was sent to the Institute of Cytopathology during May

2008 and February 2009. These materials included bron-

chial washings, bronchial brushings, and transbronchial

fine-needle aspiration biopsies of peribronchial lesions

and intrapulmonary and mediastinal lymph nodes. A rou-

tine cytological diagnosis was made in accordance with

accepted diagnostic groups (see below). Only the first

cytological specimen of a patient was included in the

study. Patients with manifest lung cancer, whose bron-

chial aspirates were taken for aftercare purposes, were

excluded because this condition is known to bear the risk

of a false-positive diagnosis for methylation analysis.40

After application of these inclusion and exclusion criteria,

3 groups were built consisting of the first 70 patients with

either a cytologically negative, equivocal, or positive diag-

nosis (Fig. 1). FISH, DNA-image cytometry, and QMSP

were applied to each group. The authors had no addi-

tional influence on recruitment of these 210 patients. Six

and 11 months after cytological diagnosis, the follow-up

reference standard was determined by review of patients

charts. The latter interval was chosen to disclose poten-

tially premalignant lesions.32,41,42

Cytological Investigation and Selection of

Smears for Adjuvant Methods

Immediately after bronchoscopy, bronchial washings

were fixed in Saccomanno fixative (50% ethanol, 2%

polyethylene glycol 1.500, and 60 mg/L rifampicin). An

aliquot of each sample was used for preparation of 4 routine

FIGURE 1. Modified STARD diagram illustrates a patients way through the study.

Improving Equivocal Lung Cancer Cytology/Schramm et al



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smears. Residual material was stored at 4C for subsequent

investigation. Bronchial brushings or transbronchial fine-

needle aspiration biopsies were smeared on 2-10 glass

slides and immediately fixed with alcohol-spray (Mercko-

fix; Merck KGaA, Darmstadt, Germany) by the bronchos-

copist. For clinical routine cytology, all specimens were

Papanicolaou stained and interpreted by experienced cyto-

pathologists. All specimens were diagnosed according to

the accepted diagnostic categories as follows: negative

(no tumor cells), doubtful (probability of a malignant

tumor approximately 30%), suspicious (probability of a

malignant tumor approximately 70%), positive (tumor

cells present), or not sufficient (no cells from deeper air-

ways present or severe artifacts). Examples are presented in

Figure 2. Afterward, the smear with the highest amount of

atypical cells was selected for DNA-image cytometry.

Another smear with a lesser amount of atypical cells was

selected for FISH. QMSP was performed on residual, not

smeared, bronchial-washing material.

Follow-Up Reference Standard

The reference standard was obtained by review of patients

charts by reviewers who were blinded to FISH, DNA-

FIGURE 2. (A) These are normal respiratory epithelial cells with minor degenerative changes. (B) Regenerative epithelial cells are

shown with nuclear enlargement, prominent nucleoli, round to oval nuclear outline, and slightly coarse chromatin. (C) Cluster of

poorly differentiated squamous cell carcinoma cells shows irregular nuclear outline, coarse chromatin, and nuclear polymorphism.

(D) Cancer or regeneration? Cytological diagnosis is hampered by air- drying artifacts in this cluster of regenerative epithelial

cells and may lead to an equivocal result. Exclusion of aneuploidy with FISH or DNA-image cytometry helped to resolve this diag-

nostic problem and supported a certain negative diagnosis. Follow-up showed no sign of malignancy in this case. (Papanicolaou

stain; original magnification, 63; oil immersion objective)

Original Article



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image cytometry, or promoter methylation analysis. The

positive reference standard was defined as either diagnosis

of a malignant tumor with histological biopsy and/or

resection specimen from the same pulmonary region and

in chronological context with the bronchoscopicallyobtained material for cytology or cytological diagnosis of

a malignant tumor with a consistent clinical course (ie,

imaging or adequate therapy). The negative reference

standard was defined as a benign histological or cytologi-

cal diagnosis consistent with the overall clinical context or

no proof of a malignant lung tumor within 11 months

after cytological diagnosis. All histological diagnoses were

reviewed by experienced pathologists. In cases of discrep-

ancies in the adjuvant methods and the reference stand-

ard, residual Papanicolaou smears were reviewed without

changing initial cytological diagnosis.

FISH Analysis

For bronchial specimens, the Vysis LAVysion multicolor

FISH probe was used. It consisted of a mixture of 4 directly

labeled DNA FISH probes (chromosomal regions 5p15.2

[green signal], 6p11.1-q11 [blue signal], 7p12 [EGFR, red

signal], and 8q24.12-24.13 [C-MYC, yellow signal]).

FISH analysis was made on 1 of the Papanicolaou-stained

slides previously used for cytological diagnosis after labeling

areas with suspicious cells with a diamond pen on the back-side of a slide and processing as previously described.43

Briefly, the smears were uncovered, rehydrated, and

destained, then digested by using pepsin, washed in phos-

phate-buffered saline, and then fixed in formalin. After

dehydration, denaturation at 73C, hybridization with

the FISH probe mix (7 lL LSI/WCP hybridization

buffer, 2 lL purified water, and 1 lL LAVysion multi-

color probe), and an additional washing step, the smears

were counterstained with 40,6-diamidino-2-phenylindole

dihydrochloride (DAPI) (Vectashield DAPI mounting

medium; Vector, Burlingame, California), cover-slipped,

and sealed with rubber cement.

FISH cases were analyzed by 2 independent observ-

ers, each with knowledge of routine cytology diagnoses but

blinded to DNA-image cytometry and QMSP. In cases of

discrepancy, an additional opinion was obtained from a

third observer, and a decision was made by a majority.

Hybridized areas on the slides were screened for

atypical cells (nuclear enlargement, irregular shape, patchy

DAPI staining) using the DAPI filter. Signals were

recorded from these cells. A cell was defined as chromo-

somally aneuploid with a gain of 2 or more of the 4

probes.18,21 Tetrasomy or even octasomy, defined as the

presence of 4 or 8 signals of 3 or more probes, was notconsidered abnormal (Fig. 3). A specimen was considered

positive for malignancy when 6 or more cells on a slide

exhibited chromosomal aneuploidy.18,21When this num-

ber could not be reached after counting 25 abnormal cells,

the number of cells investigated was extended to 60 in a

first step, or the whole hybridized area on a slide had to be

scanned. In each specimen, normal respiratory epithelial

cells or lymphocytes served as internal controls, and

hybridization efficiency was evaluated in these cells.

DNA-Image Cytometry

DNA-image cytometry was applied to 1 of the smears

used for cytological diagnosis after suspicious cells were

labeled on the coverslip. A photocopy of the slides was

made to preserve labels after uncovering. The slides were

uncovered in xylene and restained according to the

method described by Feulgen.27 Measurements of nuclear

DNA contents were performed as previously described.28

A computer-based image analysis system was used consist-

ing of a Motic BA400 microscope (Motic, Xiamen,

China) with a40 objective, a 12-bit color CCD camerawith a resolution of 13601024 pixels (MoticamPro

285A; Motic, Xiamen, China), and the MotiCyte-DNA-

image cytometry software (Motic, Xiamen, China), which

provides shading and glare correction. The Conformite

Europeene (CE) label as a diagnostic device was available

for the MotiCyte-DNA instrument. In each case, at least

30 normal respiratory epithelial cells, lymphocytes, or

granulocytes were measured as internal reference cells. A

minimum of 70 and optimum of 300 chosen nuclei of in-

terest were measured in the previously labeled areas per

specimen. The relevant parameter for a positive diagnosis

of malignancy was the proof of DNA aneuploidy. Two

algorithms for the identification of DNA aneuploidy were

used: abnormal position of any DNA stemline and/or

occurrence of cells >9 c (c DNA content). DNA stem-

line ploidy was defined as the modal value of a DNA

stemline in c U. DNA stemline aneuploidy was assumed

when the modal value of a stemline was 2.20

c and 4.40 c. Single-cell aneuploidy was




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FIGURE 3. Examples of FISH images with the LAVysion probe set (chromosomal regions 5p15.2 [green signal], 6p11.1-q11 [blue sig-

nal], 7p12 [EGFR, red signal], 8q24.12-24.13 [C-MYC, yellow signal]) demonstrate (A, arrow) disomy, (B) tetrasomy with a 4-4-4-4

pattern, (C) tetrasomy with a 4-4-4-3 pattern, (D) octasomy, and (E) aneusomy. (F) Aneuploid cells are shown with clusters of

epidermal growth factor receptor (EGFR) gene amplification. (Original magnification, 100; planar objective, several planes)

Original Article



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diagnosed when at least 1 cell per slide had a DNA con-

tent >9 c. Rarely, octaploid cells occur in noncancerous

epithelium of inflammatory affected lungs.44 Conse-

quently, the threshold for the detection of rare aneuploid

cells had to be set at 9 c and not at 5 c. Examples of a dip-loid, euploid-polyploid, and aneuploid histogram are

shown in Figure 4. All technical instruments, all software

used, and guidelines for diagnostic interpretation and

quality assurance met the standard requirements of the

consensus reports of the European Society for Analytical

Cellular Pathology.45-48

QMSP

Analysis of gene promoter hypermethylation was con-

ducted after bisulfite treatment of DNA blinded to thecytological diagnosis as follows: the genomic DNA of cells

from bronchial washings was isolated by using the Pure-

gene DNA Isolation kit (Gentra Systems, Minneapolis,

Minnesota). Fully methylated DNA (CPGenome Univer-

sal Methylated DNA; Millipore, Billerica, Massachusetts)

served as positive control. One microgram of DNA per

sample was modified by sodium bisulfite treatment

according to Herman et al.49 Promoter methylation anal-

ysis of adenomatous polyposis coli promoter 1A (APC),

cyclin-dependent kinase inhibitor-2A (p16INK4A), and

RAS association domain family protein 1 (RASSF1A)was made by using a LightCycler (Roche Diagnostics

GmbH, Mannheim, Germany) as previously described

(Fig. 5).40,50 Myogenic differentiation antigen (MYOD1)

was used as internal reference to control for input DNA.

A sample without DNA served as a negative control. Cor-

rect size of amplifiedMYOD1DNA and average samples

of APC, p16INK4A, and RASSF1A were controlled with

agarose gel electrophoresis. This indicates a sufficient

DNA extraction, sodium bisulfite treatment, and quanti-

tative polymerase chain reaction (PCR). Sample DNA

sequencing was in accordance with published gene bank

sequences ofAPC,p16INK4A, andRASSF1A. A specimen

was assigned as positive when at least 1 tumor suppressor

gene exhibited promoter hypermethylation.

Statistical Analysis

The Fisher exact test was used for contingency-table

analysis of categorical data (positive or negative for ma-

lignant tumor) provided by both reference standard

and tests. Sensitivity and specificity, both with 95%

confidence intervals, were calculated for cytological di-

agnosis, FISH, DNA-image cytometry, and QMSP.

Cytologically suspicious and equivocal diagnoses were

set as positive for statistical evaluation. Two methods

each of FISH, DNA-image cytometry, and QMSP

were directly correlated by construction of contingency

FIGURE 4. DNA-content (x-axis) is plotted against number of

cells (y-axis). (A) Diploid DNA pattern is shown with stemline

at 2 c. Euploid polyploid DNA pattern has typical stemlines at

2 c, 4 c and approximately 8 c. (B) This euploid polyploid DNA

pattern has typical stemlines at 2 c, 4 c and approximately 8 c.

Two cells with a DNA-content greater than 9 c led to a false

positive diagnosis. (C) This example of an aneuploid DNA pat-

tern has the biggest stemline at 2.7 c and an additional fraction

of proliferating cells between 4 c and 6 c.




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tables using Fleiss kappa statistics. The level of signifi-

cance was set to P.05.

RESULTS

Participants

The flow of the patients (210 in total) through the study

is shown in a modified Standards for Reporting of Diag-

nostic Accuracy (STARD) diagram (Fig. 1).51 Follow-up

reference standard was met in 68, 64, and 66 patients

with positive, uncertain, or negative cytological tumor

diagnosis, respectively. Twelve patients were not eligible

for evaluation: 5 patients had missing charts, 2 died dur-

ing the diagnostic procedure, 1 was treated for cancer af-

ter a suspicious cytological diagnosis with no histological

tumor confirmation, 2 patients chart reviews revealed

FIGURE 5. Quantitative polymerase chain reaction (PCR) runs are shown. Cycle number (x-axis) is plotted against fluorescence

intensity (y-axis). Examples are of target genes (A) p16INK4A, (B) RASSF1A, and (C) APC. APC promoter methylation level was

calculated as the ratio of the endpoint fluorescence intensity values (APC/MYOD1 [reference] x 100). The cutoff (bar) was 35%.

p16INK4A or RASSF1A promoter hypermethylation is shown at crossing points >0. AC indicates adenocarcinoma; SCC, squamous

cell carcinoma; SCLC, small-cell carcinoma; NSCLC, nonsmall-cell carcinoma.

Original Article



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known lung cancer, and 2 patients refused further

diagnostic confirmation of their highly suspected lung

cancers. Refer to Table 1 for detailed characteristics of

the patient population. The remaining 198 patients wereenrolled in this study, and FISH, DNA-image cytome-

try, and QMSP were performed.

FISH

A newly developed scoring algorithm for evaluation of

chromosomal aneuploidy with multicolor FISH was

applied to exclude false-positive results caused by euploid

polyploidization, for example, in tissue repair during

chronic bronchitis. Of 198 specimens, 189 (95.5%) were

evaluable, and 9 specimens were excluded because ofsevere degenerative changes of the bronchial material or

insufficient hybridization of the DNA probe. FISH

yielded 98.2% (P< .001) specificity in the whole cohort

of patients (Fig. 6). After a negative cytological diagnosis,

FISH correctly identified malignancy in 3 patients, thus

suggesting a false-negative cytology due to screening

errors. Careful rescreening of Papanicolaou-stained slides

revealed sparse amounts of atypical cells in 2 cases and

misinterpretation of tumor cells as benign caused by

severe air-drying artifacts in 1 case. All 68 patients with a

certain cytological diagnosis of malignant tumor were

evaluable with FISH. Malignancy was correctly identified

in 67 cases. One small-cell lung carcinoma (SCLC) with

well-recognized typical nuclear molding and crowding

pattern on DAPI counter-stained slides showed no chro-

mosomal aneuploidy with the 4 FISH probes used. In the

group of patients with equivocal cytological diagnoses,

78.6% (33 of 42) of evaluable specimens were positive for

FISH in patients with a malignant lung tumor (Table 2).

Table 1.Clinical Characteristics of Patient Population

BenignLungDiseasea

n563

PrimaryLungCancern5124

Othersb

n523

No. (%)

Median

[Range]

No. (%)

Median

[Range]

No. (%)

Median

[Range]

Age, y 65 [26-88] 66 [34-88] 66 [26-83]

Sex

Female 30 (48) 48 (39) 7 (30)

Male 33 (52) 76 (61) 16 (70)

Smoking status

Smoker 34 (54) 100 (81) 8 (35)

Pack-years 40 [6-150] 40 [ 10-150] 40 [ 30-60]

Never smoker 10 (16) 10 (8) 5 (22)

No data 19 (30) 14 (11) 10 (43)

Stagec

0 1 (1)

IA 4 (4)

IB 14 (13.5)

IIB 5 (5)

IIIA 13 (12.5)

IIIB 27 (26)

IV 37 (36)

Not determinedd

2 (2)

Limited disease 7 (35)

Extensive disease 13 (65)

Location

Central 84 (68)

Peripheral only 38 (30.5)

Miscellaneouse

2 (1.5)

Histology/Cytologyf

SCC 31 (25) 0 (0)

AC 52 (42) 5 (22)

NSCLC 17 (14) 2 (8.5)

SCLC 20 (16) 0 (0)

cSCLC 3 (2) 0 (0)

Miscellaneousg

1 (1) 16 (69.5)

SCC indicates squamous cell carcinoma; AC, adenocarcinoma; SCLC,

small cell lung cancer; NSCLC, non-SCLC; cSCLC, combined SCLC.aBenign lung disease: acute or chronic bronchitis (35), pneumonia (9), inter-

stitial lung disease (7), scar (3), pulmonary embolism (3), pleuritis (1),

hemoptysis (1), atelectasis (1), gastroesophageal reflux (1), hamartoma (1),

mediastinal neurinoma (1).

bOther cases include patients with metastasizing carcinoma to the lung:poorly differentiated carcinoma of the breast (1), AC of the vulva (1), endo-

metrioid AC of the cervix uteri (1), AC of the stomach (1), colorectal AC (3),

or miscellaneous conditions (refer to g).cOnly primary lung carcinoma included. Percentages were calculated for

SCLC and NSCLC separately.dThe exact tumour stage could not be determined for 2 patients. One

patient died, 1 patient left the hospital prior to complete staging.eDiffusely metastasizing SCLC (1), cervical and mediastinal lymph node

metastases of pulmonary AC with undetected primary (1).fTumor classification was based on cytological diagnoses alone in 9 cases.gMetastatic melanoma (1), atypical carcinoid tumour (1), non-Hodgkin lym-

phoma (2), no reference standard (12).

FIGURE 6. Specificity and sensitivity of cytology, FISH, DNA-

image cytometry, and QMSP are shown in the whole cohort

of patients (n 198). Data are presented as percentages.

(No./total).




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One cytologically doubtful specimen showed chromo-

somal aneuploidy in a woman aged 72 years, but the

clinical follow-up was a chronic bronchitis. Careful rescre-

ening of residual Papanicolaou-stained smears revealed amisinterpretation of euploid-polyploidy as aneuploidy,

which was possibly caused by a tight signal constellation.

FISH is of special clinical value in the adjuvant applica-

tion after highly suspicious cytology (Fig. 7). Under this

condition, it confirmed a malignant tumor in 87.9% (29

of 33, P .147) of patients.

DNA-Image Cytometry

Of the 198 specimens, 179 (90.4%) were evaluable, and

19 were excluded because of cell degeneration or sublimi-nal amount of suspicious cells. In the whole cohort, the

specificity of DNA-image cytometry was 98.2% (P 9 c.28 Refer to Figure 4B for the DNA histogram,

which displays a typical euploid polyploid pattern with

the exception of 2 large, but cytologically normally con-

figured, ciliated cells with a DNA content greater than 9c. These had not reached the 16 c state yet and were obvi-

ously octaploid cells in the S-phase of the cell cycle.

Because follow-up did not show any sign of malignancy,

we prefer to handle single DNA values up to 16 c as not

aneuploid in the case of euploid polyploidization as sug-

gested for bronchial epithelial cells of the human lung by

measurement of nuclear area and volume and as reported

for other organs with inflammation and reactive

changes.55-57 In addition, we strongly recommend strict

rules for ploidy interpretation as have been summarized

by Bocking, to avoid potential pitfalls (ie, viral cytopathiceffect).28 As proof that DNA aneuploidy is a reliable

marker of malignant lung tumors, all cytologically posi-

tive specimens evaluable with DNA-image cytometry

revealed stemline aneuploidy or single-cell aneuploidy.

We have recently suggested quantitative detection of

aberrant promoter hypermethylation ofAPC, p16INK4A,

andRASSF1Agenes as a reflex test on bronchial cytologic

specimens in patients who are clinically suspected of hav-

ing lung cancer but do not display a final cytological or

histological diagnosis of malignancy.40 QMSP had 53%

overall sensitivity in this study, which is confirmed by49.6% sensitivity here. The 98.4% specificity is in good

agreement with >99% detected in our previous study.

Aberrant promoter methylation was detected in 4 out of

22 patients with proven lung carcinoma subsequent to a

negative cytology finding and, therefore, had the best per-

formance of the methods compared in this study. One

cytologically negative specimen of a peripheral adenocar-

cinoma was positive on FISH and on DNA-image cytom-

etry, too. Bronchial washings of 1 central small-cell

carcinoma, 1 central adenocarcinoma, and 1 peripheral,

multifocal adenocarcinoma were exclusively positive with

QMSP. Rescreening of residual Papanicolaou-stained

slides displayed suspicious cells in 2 cases; the others

remained negative. In this study, 1 nontumor patient dis-

played a false-positive QMSP assay with aberrant methyl-

ation of the APCpromoter similar to that described by

Schmiemann et al.40 The 68-year-old patient observed in

our study with a 90% APC promoter methylation

compared with MYOD1 had a 3-month history of

rectosigmoidal carcinoma with suspected lung metastasis

in the lingula. Surgical resection revealed an inflammatory

pseudotumor. It has been suggested that aberrant methyl-

ation of the APCpromoter is associated with aging.58,59

This was 1 reason why Grote et al introduced a 35% cut-offcompared with the MYOD1 reference geneinto

investigation of aberrantAPCpromoter methylation with

QMSP.50 One could speculate whether this cutoff is not

high enough in some rare cases.

This is the first study, to our knowledge, that

directly compared the diagnostic power of 3 different

approaches in addition to cytological investigation of lung

cancer. FISH and DNA-image cytometry achieved very

similar rates of aneuploidy detection, whereas sensitivity

of QMSP was significantly lower. The prediction of lung

cancer incidence with aberrant promoter methylation andFISH was reported in a screening approach by Belinsky et

al and Varella-Garcia et al in 2 studies on the same sub-

group of patients, all enrolled in the Colorado High-Risk

Cohort Study from 1993 to 2003. Belinsky reported

increasing prevalence of promoter hypermethylation of

multiple genes, diagnosed with nested methylation-spe-

cific PCR, in sputum samples with decreasing time to

lung cancer diagnosis.42 Methylation of 3 or more genes

in sputum, collected within 18 months before manifest

cancer, predicted lung cancer with 64% sensitivity and

specificity. Varella-Garcia reported 76% sensitivityand 88% specificity for a positive FISH assay with the

LAVysion probes within 18 months before cancer

diagnosis.20

Subsequent to an equivocal cytological result in the

current report, the diagnostic accuracy of FISH and

DNA-image cytometry were nearly equal (j 0.9), but

more smears were evaluable with FISH (56 of 64) than

with DNA-image cytometry (48 of 64), in most cases,

because of a subliminal amount of atypical cells. We sug-

gest performing DNA-image cytometry when there are

enough cells (more than approximately 70 cells) in the

specimen, because this method is cheaper than FISH

($120 US dollars for DNA cytometry, $595 US dollars

for multicolor FISH according to a German medical-fee

schedule) and not as time consuming.60 When there is a

sparse amount of cells, which often occurs, hampering a

clear-cut cytological cancer diagnosis, we recommend

chromosomal FISH because only 6 aneuploid cells are

needed for a positive diagnosis.




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Conclusion

To a great extent, equivocal cytology in lung cancer diag-

nosis can be overcome by the use of additional methods

on the same specimen (ie, slide). Our diagnostic

algorithm recommends DNA-image cytometry on 1 ofthe cytological smears when there are enough atypical

cells, otherwise FISH should be performed. In most cases,

unequivocal cancer diagnoses are possible. Subsequent to

a negative cytology finding, QMSP can be performed as a

reflex test on the residual liquid specimen in the case of

persisting lung cancer suspicion.

CONFLICT OF INTEREST DISCLOSURES

Professor A. Bocking is receiving grant support from MoticCompany, Xiamen, China, to develop instruments for DNA-

image cytometry and multimodal cell analysis.

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articulo pulmon metodos auxiliars de diagnóstico

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