Abstract. Anatomic Pathology / DETECTING PANCREATOBILIARY TRACT MALIGNANCY

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1 Anatomic Pathology / DETECTING PANCREATOBILIARY TRACT MALIGNANCY Correlating Routine Cytology, Quantitative Nuclear Morphometry by Digital Image Analysis, and Genetic Alterations by Fluorescence In Situ Hybridization to Assess the Sensitivity of Cytology for Detecting Pancreatobiliary Tract Malignancy Emily G. Barr Fritcher, CT(ASCP), 1 Benjamin R. Kipp, MS, CT, MP(ASCP), 1 Jeffrey M. Slezak, MS, 2 Laura E. Moreno-Luna, MD, 3 Gregory J. Gores, MD, 3 Michael J. Levy, MD, 3 Lewis R. Roberts, MBChB, PhD, 3 Kevin C. Halling, MD, PhD, 1,* and Thomas J. Sebo, MD, PhD 1 Key Words: Pancreatobiliary; Image analysis; Fluorescence in situ hybridization; FISH; Quantitative nuclear morphometric features Abstract Routine cytologic (RC), fluorescence in situ hybridization (FISH), digital image analysis (DIA), and quantifiable morphometric results from 284 pancreatobiliary stricture brushings were compared. We chose specific DIA nuclear features assessed by pathologists in evaluating RC specimens, such as area and shape. A visual nuclear morphometric score (VNMS) was calculated. There was a difference (P <.1) in the mean VNMS when RC results were classified as negative (11.5), atypical (12.5), suspicious (13.8), and positive (16.5). The mean VNMS of specimens diagnosed as disomy (11.3), trisomy 7 (12.1), and polysomy (14.7) by FISH was also different (P <.1). There was no difference in the VNMS of false-negative and true-negative cytologic specimens (P =.225). Our findings substantiate the relationship between cell nuclear visual alterations and genetic FISH abnormalities. The low sensitivity of cytologic examination for pancreatobiliary carcinoma is due to an absence of tumor cells or the presence of welldifferentiated tumor lacking recognizable nuclear atypia. Biliary brush cytologic examination is commonly used to assess for malignancy in patients with pancreatobiliary tract strictures identified by endoscopic retrograde cholangiopancreatography (ERCP). Pathologic and clinical interpretation can be difficult because strictures can be caused by benign conditions, including choledocholithiasis, chronic pancreatitis, and ischemia, and malignancies, such as cholangiocarcinoma and pancreatic cancer. Furthermore, cells isolated from strictures may not show the same cytologic findings as those isolated from tumor masses because of cohesion and lack of necrosis. In this context, cytopathologic interpretation of pancreatobiliary tract brushing specimens usually demonstrates high specificity but relatively poor sensitivity for the detection of malignant strictures. Previous studies have shown that the sensitivity of cytologic examination for detecting pancreatobiliary tract malignancy ranges from as low as 8% to as high as 68%. 1-3 The low sensitivity of cytologic examination for detecting pancreatobiliary tract malignancy is often attributed to inadequate sampling, paucicellular cytologic specimens, and/or pathologic misinterpretation caused by subtle changes between malignant and nonmalignant cells. 2,4,5 Brushing specimens from patients with primary sclerosing cholangitis (PSC), an inflammatory disease of the bile ducts that predisposes patients to the development of cholangiocarcinoma, can be particularly difficult to interpret cytopathologically because reactive cells from the patients can mimic cancer cells. 4,6,7 Recently, the poor sensitivity of cytologic examination for pancreatobiliary malignancy with ERCP-guided brushings has been improved with new ancillary cytologic testing methods, such as fluorescence in situ hybridization (FISH), digital image analysis (DIA), and immunohistochemical labeling of fine-needle aspiration specimens. 1,4,8 FISH uses 272 Am J Clin Pathol 7;128: Downloaded 272 from

2 Anatomic Pathology / ORIGINAL ARTICLE fluorescently labeled DNA probes targeting specific chromosomes or chromosomal loci in interphase cells. Because cells from most solid tumors contain structural and numeric chromosomal abnormalities, FISH is able to diagnose a specimen as positive for malignancy with as few as 5 chromosomally abnormal cells present in the sample. 9 Previous studies conducted at our institution indicate that FISH significantly increases the sensitivity for detecting pancreatobiliary tract malignancy over routine cytologic (RC) examination while maintaining acceptable specificity. 1,4 DIA has also been shown to distinguish malignant from benign strictures with a higher sensitivity than cytologic examination with comparable specificity DIA uses a camera interfaced with a microscope to digitize the images of Feulgen-stained cells. DNA ploidy software can then produce a histogram of DNA content plotted against cell number. A nondiploid cell by DIA could represent nonclonal DNA synthesis or true aneuploidy. Thus, DNA ploidy of a cell population is an interpretation of the DNA histogram generated by analyzing the cells of interest in a given specimen. Although this may be viewed by some as a weakness of DIA, one of the strengths of this method is that each time a nucleus is collected for DNA content, the instrument automatically collects and stores quantifiable nuclear morphometric features that can be retrieved and analyzed by the instrument s morphometry software. Many different nuclear morphometric features such as size, shape, and chromatin texture can be evaluated. With tissue specimens containing a known diagnostic entity, the morphometric data from a given neoplasm can be used for prognostication of tumor biology 14,15 and for providing insight into the role of specific nuclear proteins in tumorigenesis. 16 For example, quantitative nuclear morphometry has been used to differentiate between biliary and pancreatic adenocarcinoma in paraffin tissue sections, 17 and with cytologic specimens, quantitative morphometry has been used for diagnostic purposes. 18,19 We recently published data from a subset of patients dating from October 3 to August 4, which correlated the results of this 3-pronged test with clinical follow-up. 4 In the present study, we expanded the database to include more patients. Furthermore, rather than focus on clinical follow-up, we wanted to address the interplay between genetic alterations and nuclear morphometry in bile duct epithelial cells. To our knowledge, our study represents a unique look at the relationship among RC examination, DIA, and FISH. No other study has assessed quantitative nuclear features of pancreatobiliary tract cells and correlated these findings with the results of RC and FISH genetic alteration studies. In an effort to objectively compare features that are recognized and used by cytopathologists in their practice of evaluating pancreatobiliary tract cells and to obtain a better understanding of the interaction between genotypic and phenotypic alterations of cells during biliary tract cancer pathogenesis, this study quantitatively assessed the cells based on morphologic features that can be visualized by cytopathologists. Materials and Methods Patient Demographics As approved by the Mayo Clinic Institutional Review Board (Rochester, MN), this retrospective study evaluated 284 patients pancreatobiliary tract RC and FISH results, quantifiable nuclear morphometry by DIA, and clinical follow-up information. All patients included in this study were clinically viewed as high risk for the presence of pancreatobiliary tract malignancy, underwent ERCP between September 3 and September 5, and met the following study requirements: (1) a pancreatobiliary stricture diagnosed by ERCP; (2) a definitive diagnosis of the stricture as benign or malignant by surgery, surgical pathology, or sufficient follow-up (at least 6 months for patients without clinical or pathologic evidence of malignancy; mean, 14 months) to ensure a benign or malignant course (ie, obvious progression of a malignant disease with metastases, the presence of a progressive tumor mass on cross-sectional imaging studies, and/or death from cancer); and (3) an available DIA file for nuclear morphometric analysis. There were 33 patients who fulfilled study requirements. Specimens were excluded from analysis if any of the following occurred: (1) an inadequate number of cells to perform cytologic and/or FISH studies (n = 7); (2) presence of a malignancy outside the liver, bile ducts, or pancreas (eg, colorectal carcinoma) (n = 7); or (3) therapy prevented a definitive diagnosis of carcinoma (n = 5). Only specimens from the initial ERCP were included in the analysis if a patient had multiple visits during the time evaluated. A total of 284 patients were assessed in this study Table 1. Table 1 Demographics for 284 Patients * Characteristic Result Mean age (range), y 59.7 (9-91) Males 166 (58.5) Females 118 (41.5) Cancer 13 (45.8) No cancer 154 (54.2) Cholangiocarcinoma 82 (63.1) Pancreatic adenocarcinoma 41 (31.5) Gallbladder carcinoma 4 (3.1) Ampullary carcinoma 3 (2.3) Primary sclerosing cholangitis 17 (37.7) Cancer 33 (3.8) No cancer 74 (69.2) Non primary sclerosing cholangitis 177 (62.3) Cancer 97 (54.8) No cancer 8 (45.2) * Data are given as number (percentage) unless otherwise indicated. Downloaded from Am J Clin Pathol 7;128:

3 Barr Fritcher et al / DETECTING PANCREATOBILIARY TRACT MALIGNANCY Sample Acquisition and Preparation Each brushing sample was collected using a standard DLB or DLB brush (Wilson-Cook, Winston- Salem, NC). The brush was advanced through the stricture with at least 5 to 8 to-and-fro movements. To optimize the cellular yield, the brush was pushed from the end of the sheath, as opposed to pulling the brush from the sheath, and the cut brush was placed in a vial containing ml of PreservCyt solution (Cytyc, Marlborough, MA) and immediately transferred to the Mayo Clinic Cytopathology Laboratory. On arrival in the laboratory, vials collected during ERCP were gently shaken and split equally for cytologic, DIA, and FISH testing. Cytologic Examination A ThinPrep slide (Cytyc) was made with the cytologic portion of the collected specimen and Papanicolaou-stained for cytologic examination. Each specimen was classified as negative, atypical, suspicious, or positive for malignancy by cytopathologists as part of routine clinical practice using established criteria published previously. 2,6,7 In the atypical cases, epithelial cells usually had more cytoplasm and nuclear features possibly reflective of reactive changes. In suspicious cases, the cells may have quantitatively not been sufficient to classify as carcinoma or did not exhibit sufficient nuclear changes to be viewed as clearly malignant. For this study, the cytologic (and FISH and DIA) results were not rereviewed for consensus. The interpretation was made by 1 group of 5 cytopathologists who routinely evaluate cytology specimens as part of their clinical practice. The same criteria used in our previously published work were used by all cytopathologists. 4,6 Fluorescence In Situ Hybridization The FISH slide was prepared as previously described 1 with the following exceptions: (1) The cell pellet was resuspended in 15 ml of phosphate-buffered saline solution instead of potassium chloride. (2) The pellet was washed with fixative a total of 2 times. (3) The cell suspension was placed in one 1- cm well until the desired cell density was attained. The slide was hybridized with the UroVysion probe set (Abbott Molecular, Des Plaines, IL), which targets the pericentric regions of chromosomes 3, 7, and 17 and chromosomal locus 9p21. The specimens were evaluated by cytotechnologists trained to evaluate FISH cases, and they assessed the cells for abnormal signal patterns and diagnosed the samples as follows: (1) negative (disomy); (2) trisomy 7 ( 1 cells with 3 copies of chromosome 7 and 2 of the other 3 probes); or (3) polysomy ( 5 cells with 3 signals in 2 or more of the 4 probes). 4 Digital Image Analysis DIA slides were prepared using a ThinPrep processor (Cytyc), Feulgen stained, and evaluated using a CAS image analyzer (Bacus Laboratories, Lombard, IL) as previously described. We collected 5 of the most abnormalappearing nuclei for each specimen. A case was considered positive if the peak DNA index was aneuploid (DNA index, or >2.1) or tetraploid (DNA index, ). Specimens were classified as negative if the peak DNA index was diploid (DNA index, ) or near diploid (DNA index, ) without a well-formed ( clonal ) peak of cells with DNA indices in the aneuploid or tetraploid range. Quantitative Nuclear Morphometric Analysis Cells collected for ploidy interpretation by DIA were evaluated using CellSheet software (Bacus Laboratories), which quantifies nuclear morphometric features. Nine nuclear features were selected for quantitation based on the premise that they represent morphologic characteristics visualized by pathologists during routine cytologic examination of Papanicolaou-stained slides. Some such as area, perimeter, and shape are self-explanatory. Others require more explanation and are defined in the legend to Figure 1. Picograms of DNA and DNA index were also selected for quantitation. These features are not necessarily visual assessments, but they indirectly reflect the degree of nuclear hyperchromasia, which can be visualized by light microscopy and as such were included in this evaluation. The CellSheet software calculates the raw data for these nuclear features from preexisting.ilm files, which represent the compilation of all morphometric features collected for each nucleus per patient. The mean value of each nuclear feature per patient was determined for all 11 morphometric features. These values were added to each other and divided by 11 (total number of features captured per nucleus) to generate a visual nuclear morphometric score (VNMS) for each specimen. Statistical Analysis Statistics were calculated using SPSS 11.5 software (SPSS, Chicago, IL). A 2-tailed t test was used to identify the significance of VNMS in patients with and without cancer. The McNemar test was used to compare the sensitivity and specificity of cytologic with DIA and FISH results, whereas a 1-way analysis of variance and Tamhane post hoc test were performed to compare the mean VNMS of specimens based on cytologic, FISH, and DIA results. P values of.5 or less were considered statistically significant. Results The relative sensitivities and specificities for detecting pancreatobiliary tract cancer in our study based on RC, DIA, and FISH results are shown in Figure 2. The sensitivity of RC results was 15% if only a positive diagnosis was considered positive for statistical purposes, 38% if suspicious and positive diagnoses were considered positive, and 48% if atypical, 274 Am J Clin Pathol 7;128: Downloaded 274 from

4 Anatomic Pathology / ORIGINAL ARTICLE Area Perimeter Percentage Shape Mass DNA index Density Feret x perimeter 2 area pg DNA pg tumor DNA pg benign DNA mass area Sensitivity Specificity Cytology 1 Cytology 2 Cytology 2 DIA FISH 1 FISH Figure 2 Relative sensitivities and specificities of cytology, digital image analysis (DIA), and fluorescence in situ (FISH) in detection of pancreatobiliary carcinoma. Cytology 1, positive cytologic results were considered positive diagnoses; cytology 2, positive and suspicious cytologic results were considered positive diagnoses; cytology 3, positive, suspicious, and atypical cytologic results were considered positive diagnoses; FISH 1, polysomy FISH results were considered positive diagnoses; FISH 2, polysomy and trisomy 7 FISH results were considered positive diagnoses Feret y Max diameter Min diameter Elongation Figure 1 Visual nuclear morphometric features. Area equals π nuclear radius squared, perimeter is 2π the nuclear radius, and shape is quantified as the perimeter squared divided by the area. Mass is DNA content in picograms, and DNA index is the ratio of the peak DNA content of tumor normalized to the peak DNA content of normal cells. Density is defined as DNA mass divided by nuclear area and, therefore, reflects nuclear hyperchromasia. Feret x and feret y are defined as the width and height (in micrometers), respectively, of a bounding rectangular box around the nucleus. Thus, these features reflect nuclear size or area. The maximum (Max) diameter is the length (in micrometers) of the greatest distance when measuring all possible diameters of the cell. The minimum (Min) diameter is the length (in micrometers) of the least distance when measuring all possible diameters of the cell. Elongation is calculated by dividing the maximum diameter by the minimum diameter and is, therefore, associated with nuclear contour irregularity or shape. suspicious, and positive diagnoses were considered positive. However, the specificity of cytologic results dropped from 1% (if only positive cases were considered positive) to 98% (if suspicious cases were included) to 91% (if atypical and suspicious cases were included). When aneuploid/tetraploid DIA cases and polysomy FISH cases were considered positive, the sensitivities of DIA and FISH results were significantly higher (P <.1) than cytologic results (43% vs 15% and 44% vs 15%, respectively), without a significant difference in specificity between FISH and cytologic results (98% vs 1%; P =.25). However, the specificity of DIA results was significantly lower than cytologic results (92% vs 1%; P <.1). Nuclear morphometric data are summarized in Table 2. As these data demonstrate, there were significant (P <.1) differences in area, perimeter, shape, picograms of DNA, DNA index, feret x, feret y, maximum and minimum diameters, and VNMS between patients with and without cancer. When we evaluated patients based on cytologic results and cancer status, the data demonstrated no significant differences in noncancer patients for any feature based on the cytologic results. However, in patients with cancer, there was a significant increase in morphometric scores as the cytologic diagnosis increased in severity for all features except density and elongation. In an effort to elucidate reasons for the relatively low sensitivity of cytologic examination in detecting pancreatobiliary malignancy, we assessed for correlations between the VNMS and cytologic and FISH results Figure 3A and Figure 3B, respectively. As these Figures illustrate, there was a significant (P <.1) increase in the VNMS scores as one moves from Downloaded from Am J Clin Pathol 7;128:

5 Barr Fritcher et al / DETECTING PANCREATOBILIARY TRACT MALIGNANCY Table 2 Morphometric Nuclear Feature Scores * Cytologic Diagnosis Nuclear Feature Score/ Presence of Cancer All Cases P Negative Atypical Suspicious Positive P Area <.1 No 43.8 ± ± ± ± 6.3 NA.45 Yes 54. ± ± ± ± ± 27.6 <.1 Perimeter <.1 No 25.4 ± ± ± ± 1.9 NA.514 Yes 28. ± ± ± ± ± 6. <.1 Shape <.1 No 14. ± ± ± ±.1 NA.51 Yes 14.1 ± ± ± ± ±.3 <.1 DNA (pg) <.1 No 7.7 ± ± ± ±.8 NA.832 Yes 9.3 ± ± ± ± ± 4.3 <.1 DNA index <.1 No 1.1 ± ± ± ±.1 NA.911 Yes 1.3 ± ± ± ± ±.6 <.1 Density.374 No.2 ±..2 ±.9.2 ±..2 ±. NA.366 Yes.2 ±..2 ±..2 ±..2 ±.1.2 ±..163 Feret X <.1 No 8.5 ± ± ± ±.5 NA.393 Yes 9.3 ± ± ± ± ± 1.8 <.1 Feret Y <.1 No 8.2 ± ± ± ±.6 NA.597 Yes 8.9 ± ± ± ± ± 1.7 <.1 Maximum diameter <.1 No 9.2 ± ± ± ±.6 NA.543 Yes 1.1 ± ± ± ± ± 1.9 <.1 Minimum diameter <.1 No 6.5 ± ± ± ±.5 NA.254 Yes 7.1 ± ± ± ± ± 1.4 <.1 Elongation.731 No 1.4 ± ± ± ±. NA.18 Yes 1.4 ± ± ± ± ± Average score <.1 No 11.4 ± ± ± ± 1. NA.48 Yes 13.1 ± ± ± ± ± 4.1 <.1 NA, no cases without cancer were diagnosed as positive by routine cytology on follow-up. * Data are given as mean ± SD. The t test was used to compare individual nuclear feature scores based on cancer status. One-way analysis of variance comparing means based on cytologic diagnoses. negative to equivocal to positive cytologic diagnoses and from negative to trisomy to polysomy FISH diagnoses. The same trend was true for the results of DNA ploidy by DIA. The VNMS was significantly higher for aneuploid and tetraploid specimens than for diploid specimens (mean VNMS: diploid specimens, 11.3; near-diploid specimens, 12.1; aneuploid specimens, 14.4; and tetraploid specimens, 15.3). However, there was not a significant difference in the VNMS between patients with cholangiocarcinoma and patients with pancreatic adenocarcinoma (mean, 12.7 vs 13.5, respectively; P =.195) and between patients who did and did not have PSC (mean, 12.9 vs 13.1, respectively; P =.756). To identify morphometric differences, if any, between cells from specimens that were cytologically negative with cancer on follow-up (false-negatives) and cells from specimens that were cytologically negative without cancer on follow-up (true-negatives), the mean VNMSs of the following 4 groups were compared: (1) noncancer patients with cytologically negative results (mean, 11.4); (2) patients with cancer with cytologically negative and ancillary test negative results (mean, 11.4); (3) patients with cancer with cytologically negative and ancillary test positive results (mean, 12.3); and (4) patients with cancer with cytologically positive and ancillary test positive or negative results (mean, 15.) Figure 3C. This comparison showed a significant difference (P <.1) in the VNMS for each of the groups when comparing all 4 categories. However, a post hoc test revealed that there was no significant difference (P >.5) between any 2 groups with negative cytologic results (aforementioned groups 1, 2, or 3). There was a significant difference (P <.1) between the VNMS of specimens with a positive cytologic result compared with the other 3 groups (aforementioned group 4 vs groups 1, 2, and 3). 276 Am J Clin Pathol 7;128: Downloaded 276 from

6 Anatomic Pathology / ORIGINAL ARTICLE A B 3 Visual Nuclear Morphometric Score 1 Negative Atypical Suspicious Positive Visual Nuclear Morphometric Score 3 1 Negative Trisomy 7 * Polysomy Cytology Result FISH Result C Visual Nuclear Morphometric Score * Figure 3 Box and whisker plots demonstrating visual nuclear morphometric score (VNMS) based on cytologic (A), fluorescence in situ hybridization (FISH) (B), and cytologic true-negative and false-negative results (C). Ancillary tests include DNA ploidy by digital image analysis (DIA) and genetic alterations by FISH. The median is marked by a horizontal line in the box. The lower and upper hinges of the box represent scores at the 25th and 75th percentile, respectively. The box length is defined as the interquartile range (IQR). The whiskers show the range of values that fall within 1.5 IQR of the box, while circles and asterisks represent values that are more than 1.5 IQR (outlier) and 3. IQR (extreme outlier) outside the box, respectively. A and B, P <.1. C, No significant difference between VNMS of 1 vs 2, 1 vs 3, and 2 vs 3. P <.1 between VNMS of 1 vs 4, 2 vs 4, and 3 vs 4. FU, follow-up. Cytology Negative Negative Negative Positive Ancillary tests Positive/Negative Negative Positive Positive/Negative Cancer on FU No Yes Yes Yes Finally, the relationship between each cytologic category and the respective FISH results is illustrated in Figure 4. The percentage of cases that were polysomic by FISH increased with each incremental diagnosis by RC examination (negative, 6%; atypical, 29%; suspicious, 69%; and positive, 9%). Trisomy 7 cases by FISH were more likely to be considered atypical in RC examination than any other cytologic diagnosis. Discussion This study is a unique evaluation of pancreatobiliary stricture pathology using 3 modalities (RC examination, DIA, and FISH) that, to the best of our knowledge, has not previously been reported. First, our study shows a logical progression of more quantifiable nuclear atypia as RC results progress from benign or negative to atypical, suspicious, and positive for malignancy (Figure 3A). Second, we showed that there are no significant differences in the quantifiable visual nuclear morphometric features of cells for true-negative and false-negative cytologic cases (Figure 3C). This suggests that false-negative cytologic results are not due to an inability of cytopathologists to recognize the cells as morphologically abnormal but rather to other causes. The most likely explanations for false-negative cytologic results are that the cancer cells are not present in the cytologic Downloaded from Am J Clin Pathol 7;128:

7 Barr Fritcher et al / DETECTING PANCREATOBILIARY TRACT MALIGNANCY specimen (inadequate sampling) or that the cancer cells do not exhibit morphologic features that differ significantly from normal cells. However, data from FISH results link disomic, trisomic, and polysomic cells to quantifiable nuclear morphometric features that steadily progress to a higher degree of atypia. Therefore, it seems more likely that inadequate sampling is the main cause of false-negative results. Possible explanations for inadequate sampling of the tumor include poor visibility of the lesion during ERCP, challenging anatomic position of the lesion for sampling, a malignancy that is externally compressing the duct rather than arising from within it, or a desmoplastic tumor such as cholangiocarcinoma that does not shed cells readily. 2 The most likely explanation for false-negative cytologic results in cases in which the tumor has been adequately sampled is that the tumor is well differentiated. The cells do not display significant morphologic changes and, therefore, are difficult to categorize as malignant. 5,21 Although it is logical to assume that changes in nuclear architecture reflect structural and numeric chromosomal aberrations during carcinogenesis, 22 our study, to the best of our knowledge, is the first to quantify the nuclear changes by DIA and correlate with FISH. In this study, we assessed for differences in the morphometric features of cells from patients with and without cancer in 2 ways (Table 2). First, the mean value for each nuclear feature was compared in patients with and without cancer. This comparison revealed a statistically significant difference in the values for each nuclear feature for patients with cancer and patients without except for density and elongation. The second way in which differences between the morphologic features of cells from patients with and without cancer was examined was to determine the differences in a particular nuclear feature based on the cytologic diagnosis (Table 2). In patients with cancer, each of the nuclear features (except density and elongation) changed in tandem with the degree of severity identified by RC examination. For example, the mean nuclear areas for cancer cases diagnosed as negative, atypical, suspicious, and positive by cytologic examination were 45.4, 53.1, 59.1, and 76.2, respectively. This reflects a mean change in nuclear size of 68% between benign and carcinoma cells. These results indicate that among patients who are found to have cancer, the atypical, suspicious, and positive cytologic categories reflect true differences in nuclear structure. However, this same trend is not observed among these cytologic categories for patients who are not found to have cancer on clinical follow-up. This suggests that although pathologists appropriately categorize cytologic specimens in patients who are identified as having cancer, the nuclear features used for classifying RC specimens as abnormal are apparently not as striking in patients whose clinical follow-up did not identify cancer. Previous studies have suggested that the inflammatory changes that occur in patients with PSC can induce reactive changes in cells that reduce the specificity of cytologic examination in these patients. 6,23 A reduced specificity in patients with Percentage of Cases Negative FISH Result Negative Trisomy 7 Polysomy Atypical Suspicious Cytology Result Positive Figure 4 Comparison of fluorescence in situ hybridization (FISH) and cytologic results. PSC has been reported by other authors 6,24,25 ; however, in our study, there was no significant difference in the specificity of cytologic examination for patients with and without PSC. Our findings are consistent with those of Lindberg et al, 23 who reported 1% specificity in patients with PSC. In addition, there was not a difference in the VNMS between patients with and without PSC (in the cancer and noncancer populations). The trends in ancillary tests parallel the trend seen in RC examination. As the severity of the diagnosis increases, so does the VNMS. There is a significant difference in VNMS between specimens with diploid, near-diploid, aneuploid, and tetraploid DIA results. Furthermore, Figure 3B highlighted the gradual increase in VNMS among negative, trisomy 7, and polysomy FISH results. Trisomy 7 has been reported in benign and malignant conditions as an isolated FISH abnormality. 4 In the present study, 54% of patients with trisomy 7 were found to have cancer on follow-up. This compares with 25% for disomy and 95% for polysomy (data not shown). Our study also indicates a slight (mean, 11.3 vs 12.1) but significant increase (P =.1) in the VNMSs of cells from cases diagnosed as trisomy 7 compared with cells from cases diagnosed as negative (Figure 3B). This seems to be reflected mainly in nuclear area. The mean nuclear area of disomic cells was 42.9 µm 2, and the mean nuclear area of trisomic cells was 48.1 µm 2 an increase of 12% (data not shown). This may explain the finding that a higher fraction of trisomy 7 specimens by FISH were categorized as atypical (29%) than other cytologic categories indicating that the cells in such specimens display at least some degree of nuclear atypia (Figure 4). Therefore, although trisomy 7 is not unequivocally associated with the presence of malignancy, it increases the relative risk of having cancer and may represent an early genetic alteration that occurs in pancreatobiliary neoplasia. As such, our recommendation to clinicians is to closely observe these patients with reevaluation at a later date Am J Clin Pathol 7;128: Downloaded 278 from

8 Anatomic Pathology / ORIGINAL ARTICLE For this study, the cytologic, FISH, and DIA results were not rereviewed for consensus. We thought that this approach more accurately reflected clinical practice. An argument can certainly be made that reviewing all findings for consensus may be superior. Despite this potential limitation to our study, our data show a strong correlation between genetic alterations at the level of FISH and nuclear morphometric features identified by the naked eye (in RC examination) or with a computer (in DIA). The present study quantified nuclear morphologic features that can be visually identified by cytopathologists in cells from pancreatobiliary strictures. Our findings suggest that cytopathologists accurately recognize abnormal nuclear features in cells from patients with cancer. The absence of a difference in the quantifiable morphologic findings between false-negative and true-negative cytologic cases suggests that the low sensitivity of cytologic examination is generally not due to pathologic misinterpretation, but rather inadequate sampling of the tumor or, less likely, the presence of a well-differentiated tumor that has nuclear features similar to those of normal cells. This study also indicates that cells from cases diagnosed as trisomy 7 by FISH have morphologic features that differ subtly from normal cells, possibly representing an early change in carcinogenesis of the pancreatobiliary tract. Our study represents a unique look at pancreatobiliary epithelial cells harvested by ERCP and analyzed by 3 separate modalities RC examination, DIA, and FISH. The results show a statistically significant and logical trend highlighting the strong link between the visual assessment of cells by RC examination and quantifiable nuclear morphometric features. From the Departments of 1 Laboratory Medicine and Pathology, 2 Biostatistics, and 3 Gastroenterology and Hepatology, Mayo Clinic and Foundation, Rochester, MN. * Dr. Halling has a patent on the use of the UroVysion probe set used in this study and receives royalties from its sale. Address reprint requests to Dr Sebo: Dept of Anatomic Pathology, Hilton 11, Mayo Clinic, First St, SW, Rochester, MN References 1. Kipp BR, Stadheim LM, Halling SA, et al. A comparison of routine cytology and fluorescence in situ hybridization for the detection of malignant bile duct strictures. Am J Gastroenterol. 4;99: Logrono R, Kurtycz DF, Molina CP, et al. Analysis of falsenegative diagnoses on endoscopic brush cytology of biliary and pancreatic duct strictures: the experience at 2 university hospitals. Arch Pathol Lab Med. ;124: Govil H, Reddy V, Kluskens L, et al. Brush cytology of the biliary tract: retrospective study of 278 cases with histopathologic correlation. 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