Original Article

Equivocal Cytology in Lung

Cancer Diagnosis
Improvement 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;
cking, MD, PhD1
Stefan Biesterfeld, MD, PhD1; and Alfred Bo

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 prospectively 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 conversion. Clinical and histological follow-up according to a reference standard, defined in advance, were available 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
C 2011 American Cancer
cancer would enhance sensitivity. Cancer (Cancer Cytopathol) 2011;119:177-92. V
Society.
KEY WORDS: lung cancer, cytology, FISH, DNA-image cytometry, promoter hypermethylation.

Corresponding author: Stefan Biesterfeld, MD, PhD, Institute of Cytopathology, Heinrich Heine University, Moorenstr.5, D-40225 Dusseldorf,
Germany; Fax: (011) 49-211-8118402; Stefan.Biesterfeld@med.uni-duesseldorf.de
1

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

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

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177

Original Article

Lung cancer is 1 of the most frequent causes of death

worldwide. It is estimated that in 2008, 161,840 people 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 disease
with metastases to lymph nodes and distant sites at diagnosis, 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 methods
include investigation of sputa, bronchial washings,
bronchial brushings, and transbronchial or transthoracic fine-needle aspiration biopsies.5 Because lung
cancer is often diagnosed in late stages, operative therapy is not always recommended, and final diagnosis is
not seldom solely based on cytology.6 Sometimes benign 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 specimens sometimes impede or even render a distinct cytological 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 9month 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) aneuploidy 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 speci178

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 identification
of prospective malignant lesions (ie, dysplasia) and prediction of prognosis in manifest cancers.29-35. Both, FISH
and DNA-image cytometry can be performed on the
same specimen subsequent to a cytological diagnosis.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 polymerase 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 methylated 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 predefined 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 chromosomal set =2n, which is because integrated-value multiples 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,
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Improving Equivocal Lung Cancer Cytology/Schramm et al

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

was sent to the Institute of Cytopathology during May

2008 and February 2009. These materials included bronchial washings, bronchial brushings, and transbronchial
fine-needle aspiration biopsies of peribronchial lesions
and intrapulmonary and mediastinal lymph nodes. A routine 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 bronchial 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 diagCancer Cytopathology

June 25, 2011

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

were applied to each group. The authors had no additional 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 potentially 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
179

Original Article

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 diagnostic 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)

smears. Residual material was stored at 4
C for subsequent

investigation. Bronchial brushings or transbronchial fineneedle aspiration biopsies were smeared on 2-10 glass
slides and immediately fixed with alcohol-spray (Merckofix; Merck KGaA, Darmstadt, Germany) by the bronchoscopist. For clinical routine cytology, all specimens were
Papanicolaou stained and interpreted by experienced cytopathologists. 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
180

cells present), or not sufficient (no cells from deeper airways 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, DNACancer Cytopathology

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Improving Equivocal Lung Cancer Cytology/Schramm et al

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 bronchoscopically
obtained 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 cytological 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 discrepancies in the adjuvant methods and the reference standard, 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 backside of a slide and processing as previously described.43
Briefly, the smears were uncovered, rehydrated, and
destained, then digested by using pepsin, washed in phosphate-buffered saline, and then fixed in formalin. After
dehydration, denaturation at 73
C, hybridization with
the FISH probe mix (7 lL LSI/WCP hybridization
buffer, 2 lL purified water, and 1 lL LAVysion multicolor 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 observers, 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
Cancer Cytopathology

June 25, 2011

DAPI staining) using the DAPI filter. Signals were

recorded from these cells. A cell was defined as chromosomally 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 not
considered abnormal (Fig. 3). A specimen was considered
positive for malignancy when 6 or more cells on a slide
exhibited chromosomal aneuploidy.18,21 When this number 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 consisting of a Motic BA400 microscope (Motic, Xiamen,
China) with a 40 objective, a 12-bit color CCD camera
with a resolution of 13601024 pixels (MoticamPro
285A; Motic, Xiamen, China), and the MotiCyte-DNAimage 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 interest 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 stemline 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 <1.80 c or >2.20
c and <3.60 c or >4.40 c. Single-cell aneuploidy was
181

Original Article

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

182

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Improving Equivocal Lung Cancer Cytology/Schramm et al

diagnosed when at least 1 cell per slide had a DNA content >9 c. Rarely, octaploid cells occur in noncancerous
epithelium of inflammatory affected lungs.44 Consequently, the threshold for the detection of rare aneuploid
cells had to be set at 9 c and not at 5 c. Examples of a diploid, 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 conducted after bisulfite treatment of DNA blinded to the
cytological diagnosis as follows: the genomic DNA of cells
from bronchial washings was isolated by using the Puregene DNA Isolation kit (Gentra Systems, Minneapolis,
Minnesota). Fully methylated DNA (CPGenome Universal 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 analysis 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. Correct size of amplified MYOD1 DNA and average samples
of APC, p16INK4A, and RASSF1A were controlled with
agarose gel electrophoresis. This indicates a sufficient
DNA extraction, sodium bisulfite treatment, and quantitative polymerase chain reaction (PCR). Sample DNA
sequencing was in accordance with published gene bank
sequences of APC, p16INK4A, and RASSF1A. 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 maCancer Cytopathology

June 25, 2011

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 pattern has the biggest stemline at 2.7 c and an additional fraction
of proliferating cells between 4 c and 6 c.

lignant tumor) provided by both reference standard

and tests. Sensitivity and specificity, both with 95%
confidence intervals, were calculated for cytological diagnosis, 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
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Original Article

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.

tables using Fleiss kappa statistics. The level of significance 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 Diag184

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 during the diagnostic procedure, 1 was treated for cancer after a suspicious cytological diagnosis with no histological
tumor confirmation, 2 patients chart reviews revealed
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Improving Equivocal Lung Cancer Cytology/Schramm et al

Table 1. Clinical Characteristics of Patient Population

Age, y

Benign
Lung
Diseasea
n563

Primary
Lung
Cancer
n5124

Othersb
n523

No. (%)
Median
[Range]

No. (%)
Median
[Range]

No. (%)
Median
[Range]

65 [26-88]

66 [34-88]

66 [26-83]

Sex
30 (48)
33 (52)

Female
Male

48 (39)
76 (61)

7 (30)
16 (70)

Smoking status
34
40
10
19

Smoker
Pack-years
Never smoker
No data

(54)
[6-150]
(16)
(30)

100 (81)
40 [10-150]
10 (8)
14 (11)

8 (35)
40 [30-60]
5 (22)
10 (43)

Stage

1
4
14
5
13
27
37
2
7
13

0
IA
IB
IIB
IIIA
IIIB
IV
d
Not determined
Limited disease
Extensive disease

(1)
(4)
(13.5)
(5)
(12.5)
(26)
(36)
(2)
(35)
(65)

84 (68)
38 (30.5)
2 (1.5)
f

Histology/Cytology
SCC
AC
NSCLC
SCLC
cSCLC
g
Miscellaneous

31
52
17
20
3
1

(25)
(42)
(14)
(16)
(2)
(1)

0 (0)
5 (22)
2 (8.5)
0 (0)
0 (0)
16 (69.5)

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

small cell lung cancer; NSCLC, non-SCLC; cSCLC, combined SCLC.
a
Benign lung disease: acute or chronic bronchitis (35), pneumonia (9), interstitial lung disease (7), scar (3), pulmonary embolism (3), pleuritis (1),
hemoptysis (1), atelectasis (1), gastroesophageal reflux (1), hamartoma (1),
mediastinal neurinoma (1).
b
Other cases include patients with metastasizing carcinoma to the lung:
poorly differentiated carcinoma of the breast (1), AC of the vulva (1), endometrioid AC of the cervix uteri (1), AC of the stomach (1), colorectal AC (3),
or miscellaneous conditions (refer to g).
c
Only primary lung carcinoma included. Percentages were calculated for
SCLC and NSCLC separately.
d
The exact tumour stage could not be determined for 2 patients. One
patient died, 1 patient left the hospital prior to complete staging.
e
Diffusely metastasizing SCLC (1), cervical and mediastinal lymph node
metastases of pulmonary AC with undetected primary (1).
f
Tumor classification was based on cytological diagnoses alone in 9 cases.
g
Metastatic melanoma (1), atypical carcinoid tumour (1), non-Hodgkin lymphoma (2), no reference standard (12).

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June 25, 2011

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 were
enrolled in this study, and FISH, DNA-image cytometry, and QMSP were performed.
FISH

Location
Central
Peripheral only
e
Miscellaneous

FIGURE 6. Specificity and sensitivity of cytology, FISH, DNAimage cytometry, and QMSP are shown in the whole cohort
of patients (n 198). Data are presented as percentages.
(No./total).

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 of
severe 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 chromosomal 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).
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Table 2. Diagnostic Accuracy of FISH, DNA Image Cytometry, and QMSP in Cytologically Equivocal Cases

Specimen Type

FISH
DNA image cytometry
QMSP

Sensitivity

Specificity

No./Total (%) [95% CI]

No./Total (%) [95% CI]

33/42 (79) [66-91]

29/35 (83) [70-95]
19/39 (49) [33-64]

13/14 (93) [79-100]

13/13 (100)
18/19 (95) [85-100]

<.001
<.001
.001

0.90
d
0.46
e
0.38

FISH indicates fluorescent in situ hybridization; QMSP, quantitative methylation specific polymerase chain reaction (PCR).
a
All specimens that were evaluable with either FISH, DNA image cytometry, or QMSP were included for calculation of diagnostic accuracy.
b
Kappa-values were calculated only in cases when both compared methods were evaluable.
c
Comparison of FISH and DNA image cytometry.
d
Comparison of DNA image cytometry and QMSP.
e
Comparison of FISH and QMSP.

One cytologically doubtful specimen showed chromosomal aneuploidy in a woman aged 72 years, but the
clinical follow-up was a chronic bronchitis. Careful rescreening of residual Papanicolaou-stained smears revealed a
misinterpretation of euploid-polyploidy as aneuploidy,
which was possibly caused by a tight signal constellation.
FISH is of special clinical value in the adjuvant application 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 subliminal amount of suspicious cells. In the whole cohort, the
specificity of DNA-image cytometry was 98.2% (P <
.001; Fig. 6). Subsequent to a negative cytological diagnosis, 2 cases were identified as false-negative by DNA
ploidy. Both cases were also identified by FISH, and histological follow-up revealed a malignant tumor in each
case. One patient with negative clinical follow-up was
diagnosed with DNA aneuploidy according to the applied
criteria for evaluation. A DNA histogram (Fig. 4B)
showed a euploid-polyploid pattern and 2 large, but normally configured, ciliated cells with a DNA content
greater than 9 c, the latter leading to the diagnosis of
DNA aneuploidy. All 67 cytologically positive diagnoses
evaluable with DNA-image cytometry were DNA aneuploid. Subsequent to an equivocal cytological diagnosis,
DNA-image cytometry was successfully applied to 75%
(48 of 64) of specimens. A malignant tumor was correctly
predicted in 82.9% (29 of 35) by DNA aneuploidy (Table 2). Particularly, subsequent to a highly suspicious
186

FIGURE 7. (Top) Papanicolaou-stained smear of a bronchial

washing contains only 1 group of singly lying cells with severe
abnormal changes. The sparse number of cells hampered a
clear-cut positive diagnosis of a nonsmall-cell carcinoma; cytology revealed a highly suspicious result only. (Bottom)
Same group is shown after hybridization with the LAVysion
probe set and computer-assisted relocalization. One cell at
the upper right corner was stripped off during hybridization
procedure. Chromosomal aneuploidy is shown with clusters
of EGFR gene amplification (7p12, red signals) in nonsmallcell carcinoma. (original magnification, 40; planar objective)

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Improving Equivocal Lung Cancer Cytology/Schramm et al

Table 3. Direct Comparison of FISH, DNA Image Cytometry, and QMSP for the Detection of
Malignant Lung Tumors in Cases Where All Methods Were Evaluable on the Same Specimen
(n 147)

Specimen Type

FISH
DNA image cytometry
QMSP

Sensitivity

Specificity

No./Total (%)
[95% CI]

No./Total (%)
[95% CI]

74/98 (76) [66-84]

75/98 (77) [68-85]
52/98 (53) [42-62]

48/49 (98) [94-100]

48/49 (98) [94-100]
49/49 (100)

ja

<.001
<.001
<.001

0.93b
0.52c
0.53d

FISH indicates fluorescent in situ hybridization; QMSP, quantitative methylation specific polymerase chain reaction (PCR).
a
Kappa-values were calculated only in cases when both compared methods were evaluable.
b
Comparison of FISH and DNA image cytometry.
c
Comparison of DNA image cytometry and QMSP.
d
Comparison of FISH and QMSP.

cytological diagnosis, malignancy was confirmed in

92.9% (26 of 28, P .014) of patients.
QMSP
Of the 198 specimens, 187 (94.4%) were evaluable,
including 18 cases in which the relevant cytological diagnoses for study inclusion were made on transbronchial
fine-needle aspiration biopsies (16) or bronchial brush
biopsies (2). In each case, an additional bronchial washing
specimen was evaluable for QMSP: 9 of the same pulmonary region and 9 of different or unknown pulmonary
regions evaluated for follow-up reference diagnosis. The
latter 9 patients were excluded from evaluation of QMSP
results. QMSP achieved 49.6% and 98.4% (P < .001)
sensitivity and specificity, respectively, in the whole
cohort (Fig. 6). Aberrant promoter methylation was correctly identified in 4 patients with false-negative cytological diagnoses. Follow-up revealed a malignant tumor in
each case. One patients malignant tumor was also
detected by FISH. Rescreening of Papanicolaou-stained
smears revealed sparse amounts of atypical cells in 2 cases.
The cytology of the other 2 specimens remained negative.
Aberrant promoter methylation was observed in 60.3% of
evaluable cytologically positive specimens. These patients
all had malignant lung tumors. After equivocal cytology,
application of aberrant promoter methylation detected
48.7% (19 of 39) of tumors (Table 2). One patient with
known rectosigmoid carcinoma and suspect lingula patches
on chest x-ray showed aberrant methylation of the APC
promoter. Postsurgical histological investigation of this
region yielded an inflammatory pseudotumor and no evidence of pulmonary malignancy or colorectal metastasis.
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June 25, 2011

Direct Comparison of Adjuvant Methods

In a subset of 147 patients, 59 with negative, 51 with
positive, and 37 with equivocal cytology, all additional
methods were performed and interpreted on the same
specimen, and a follow-up reference standard was available. This group comprised only bronchial washing specimens, which are often false-negative with reported overall
sensitivities of 43% for peripheral and 48% for central
tumors in a large meta-analysis.5 More powerful sampling
methods like bronchial brushings or transbronchial fineneedle aspiration biopsies were excluded because QMSP
was tested only on liquid material. Accordingly, diagnostic accuracy of FISH and DNA-image cytometry was
slightly lower than in the whole cohort (Fig. 6). Diagnostic accuracy of DNA-image cytometry and FISH was
nearly equal (j 0.93), whereas sensitivity of QMSP
remained lower (Table 3).

DISCUSSION
Equivocal diagnoses in pulmonary cytology are problematic for physicians, pathologists, and patients. Furthermore, diagnostic bronchial cytology suffers from high
rates of false-negative samples because the tumor cells are
often absent.5 In the present study, we compared diagnostic accuracy of FISH, DNA-image cytometry, and quantitative analysis of aberrant promoter methylation. An
algorithm is presented, defining the status of each adjuvant method in the diagnostic procedure.
Beyond that, we have developed a new scoring
algorithm for FISH analysis in pulmonary cytology with
the LAVysion set, taking the physiological phenomenon
of euploid polyploidization into account. With the
187

Original Article

advent of DNA-image cytometry techniques, euploid

polyploidization under benign conditions has been recognized in a large variety of human tissues.52 This
includes polyploidy associated with functional adaption
(ie, heart muscle cells),53 aging, or reactive conditions
(ie, pneumonia). It is known from DNA-image cytometry studies that single nuclei of inflamed bronchial epithelial cells contain a DNA content up to 8 c, which
corresponds with a 4-fold chromosomal set.44 Euploid
polyploidization, which resembles a more than 30-year
old diagnostic problem, gains new importance for the
detection of aneuploidy by chromosomal FISH because
it has to be differentiated from the latter.
The LAVysion set has been designed primarily to
detect lung cancer cells.18 This was confirmed by our
results, for 67 of 68 specimens with positive cytology
were also chromosomally aneuploid with FISH. FISH
was aneuploid in 79% of lung cancer patients subsequent
to a cytologically equivocal diagnosis. This is in good
agreement with Bubendorf and coworkers, who detected
chromosomal alterations in 45 cytological equivocal cases
with FISH after automated relocation and achieved 79%
sensitivity and 100% specificity.11 Taking euploid polyploidization into account and excluding these cells from
aneuploid values did not reduce sensitivity (78%) in the
whole series of patients compared with published
evidence. Bubendorf found enhanced sensitivity (73%)
for bronchial brushings compared with cytology alone
(49%) but no additional value in lung cancer diagnosis
for bronchial washings and transbronchial needle
aspirates in a group of 100 lung cancer patients and 71
controls.21 Halling et al performed cytology and FISH
on bronchoscopic brushing and washing specimens and
achieved 71% sensitivity on brushings and 49% sensitivity on washing specimens.22 Voss found an additional
32% of detected lung cancers by FISH compared with
cytology (41% and 20% for central and peripheral
tumors, respectively) in bronchoscopically collected cytology specimens in a cohort of 343 patients with indeterminate pulmonary lesions.26 The latter study defined
tetrasomy as nonmalignant for study purposes but did
not take euploid polyploidization beyond tetraploidy
into account. We observed chromosomal octaploid cells
in 8 of 56 cytologically negative or equivocal specimens
with negative follow-up. In part, marked euploid polyploidization was detectable in 24 of these specimens.
188

False-positive FISH diagnoses can easily be made when

octaploid cells are interpreted as aneuploid, and difficulties in signal counting (ie, tightly positioned or elongated
signals, degenerative changes) lead to false classification
of in-fact tetraploid cells as chromosomally aneuploid.
Nevertheless, 1 patient with chronic bronchitis
revealed a false-positive FISH result. Careful re-evaluation
showed, in part, very narrow to close contacting signal
positions, which hampered signal counting. With followup diagnosis in mind, a cytopathologist could have interpreted the suspicious cells as euploid-polyploid.
FISH was aneuploid in 3 of 65 patients after a negative cytological result; all proved to be malignant lung
tumors in follow-up. Two of their specimens were aneuploid on DNA-image cytometry, and 1 showed aberrant
promoter hypermethylation, too. In a group of 230
patients, Voss and coworkers detected a much higher
number of 37 FISH-positive specimens with confirmed
lung cancer.26 This could warrant use of FISH as a reflex
test on residual material after negative cytology, but a low
rate of FISH-positive specimens after negative cytology
findings, as observed in our study, makes this approach
appear too expensive.
Nuclear DNA content, measured with DNA-image
cytometry, increases with degree of cellular atypia up from
mildly dysplastic (metaplastic with mild atypia) changes
to invasive lung cancer.29,31 This has been used to facilitate the distinction between atypical squamous cells (precancer) and nonspecific cellular changes by measuring the
nuclear DNA content of dysplastic bronchial epithelial
cells.30,32 The morphological criteria of dysplasia and carcinoma were applied for both cytology and DNA-image
cytometry according to Saccomanno.54 We detected
82.9% aneuploid specimens with DNA-image cytometry
subsequent to an equivocal cytological diagnosis, confirmed as malignant by follow-up. This is in good agreement with Auffermann et al who detected 17 of 19
cancers with DNA-image cytometry subsequent to cytological dysplastic and suspicious cell findings.32 When
their specimens exhibit DNA aneuploidy with the criteria
applied in this study, our patients routinely receive the
diagnosis of a malignant tumor.
DNA-image cytometry revealed DNA aneuploidy
in 3 patients subsequent to a negative cytological diagnosis. Follow-up histological diagnosis of a malignant tumor
was obtained for 2 patients. One patient was diagnosed
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June 25, 2011

Improving Equivocal Lung Cancer Cytology/Schramm et al

with DNA aneuploidy because of 2 cells with a DNA content >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 configured, ciliated cells with a DNA content greater than 9
c. These had not reached the 16 c state yet and were obviously 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 suggested 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 cytopathic
effect).28 As proof that DNA aneuploidy is a reliable
marker of malignant lung tumors, all cytologically positive specimens evaluable with DNA-image cytometry
revealed stemline aneuploidy or single-cell aneuploidy.
We have recently suggested quantitative detection of
aberrant promoter hypermethylation of APC, p16INK4A,
and RASSF1A genes as a reflex test on bronchial cytologic
specimens in patients who are clinically suspected of having 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 by
49.6% 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 performance of the methods compared in this study. One
cytologically negative specimen of a peripheral adenocarcinoma was positive on FISH and on DNA-image cytometry, 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 displayed a false-positive QMSP assay with aberrant methylation of the APC promoter 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
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June 25, 2011

rectosigmoidal carcinoma with suspected lung metastasis

in the lingula. Surgical resection revealed an inflammatory
pseudotumor. It has been suggested that aberrant methylation of the APC promoter is associated with aging.58,59
This was 1 reason why Grote et al introduced a 35% cutoffcompared with the MYOD1 reference geneinto
investigation of aberrant APC promoter 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 and
FISH was reported in a screening approach by Belinsky et
al and Varella-Garcia et al in 2 studies on the same subgroup 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-specific 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% sensitivity
and 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 suggest 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.
189

Original Article

Conclusion
To a great extent, equivocal cytology in lung cancer diagnosis can be overcome by the use of additional methods
on the same specimen (ie, slide). Our diagnostic
algorithm recommends DNA-image cytometry on 1 of
the 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 Motic
Company, Xiamen, China, to develop instruments for DNAimage cytometry and multimodal cell analysis.

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