Introductory slide. Took 3 hours to come up with this very witty title.

Transcription

1 1 Urine Cytology De Bait in FISHing for the Best Approach to Diagnosing and Managing Patients with Urothelial Cancer USCAP Genitourinary Evening Session, March 25, 2010 Thomas J. Sebo, M.D., Ph.D. Consultant, Department of Anatomic Pathology Divisions of Surgical Pathology & Cytopathology Mayo Clinic, Rochester, MN Professor of Laboratory Medicine and Pathology, Mayo Medical School Introductory slide. Took 3 hours to come up with this very witty title. To view small print, go to zoom drop down and click on 200%. That will allow you to see in greater detail the finer details of the inserted PPT images. 2 Sebo Case 4a with Accompanying Virtual Voided Urine Cytology 60 YOM; History of Renal Insufficiency Sebo Case 4a with Accompanying Virtual Voided Urine Cytology 60 YOM; History of Renal Insufficiency. 1

2 3 Case 4a Follow-Up: Renal Transplant Patient With Allograft Kidney Tubules Infected with Polyoma Virus (Left to Right Decoy Cells in Urine, Renal Tubule H&E, and in Situ BK Hybridization) Follow-up of Case 4a Renal transplant patient with allograft kidney. Tubules infected with polyoma virus. These decoy cells can often mimic urothelial carcinoma but typically will have some suggestion of ground glass chromatin appearance. Renal transplant patients are at risk for BK virus reactivation while on immunosuppressive therapy. 1 10% - 30% of Renal Transplant Patients on Immunosuppressive Therapy Have BKV Reactivation (Nickeleit et al. N Engl J Med 2000;342: ) Of note, and to briefly introduce one of the main themes of tonight s discussion is the fact that genetic alterations are well-documented to occur in polyoma virus-infected urothelial/ renal tubular cells. Thus, clinical tests for DNA alterations (by digital image analysis [DIA] or fluorescence in situ hybridization [FISH]) can be falsely positive. 2 0 Association Between Urinary BK Viral (Decoy) Cell Titer and DNA Content by Image Analysis (IA) and Fluorescence in Situ Hybridization (FISH) Nondiploid Cells by IA (%) YOM, 9p21del 52 YOM, trisomy 7 (EGFR) FISH negative FISH positive 75 YOM, polysomic 70 YOM, 9p21del Cells with Abnormal Nuclei but Without Ground-Glass Change Kipp et al. Am J Clin Pathol 2005 Dec; 124(6): BK Viral cells/ Ten High Power Fields 4 Sebo Case 4b Without Virtual Voided Urine Cytology 75 YOM; Hematuria and History of Prostate Cancer Sebo Case 4b Without Virtual Voided Urine Cytology 75 YOM; Hematuria and History of Prostate Cancer. 2

3 5 Case 4b Follow-Up Patient s prostate cancer had been treated with radiation Cystoscopy identified changes of radiation cystitis Clinical follow-up, to date (12 months) negative for cancer This urine cytology reflects radiation cystitis in men who have undergone either interstitial brachytherapy or external beam radiation for prostate cancer. To date, we have evaluated about a dozen patients in this setting with no evidence of urothelial carcinoma on follow-up. Most of these cases have also had FISH performed on the urine samples and, when available, the bladder biopsies without evidence of polysomy. 6 Sebo Case 4c Without Virtual Right (Top) and Left (Bottom) Ureteral Brushing Cytologies 68 YOM; Hematuria with Current Cystoscopic Evidence of Bladder Mass and History of BCG-Treated Bladder Cancer Sebo case 4c without virtual slide. This shows the bilateral ureteral brushings urine cytologies in a 68-yearold gentleman with hematuria. At the time of the brushings, cystoscopic examination identified a bladder mass. The patient has a history of BCG treated bladder cancer. 3

4 7 Case 4c Follow-Up Diagnosis Positive for malignancy. Urothelial carcinoma. The findings could reflect ureteral contamination from bladder cancer primary. Discussion with clinician Further work-up identified synchronous right renal pelvic urothelial cancer The urine cytology was reported as Positive for malignancy. Urothelial carcinoma. The findings could reflect ureteral contamination from the bladder cancer primary. After discussion with the clinicians involved in the care of this patient, further evaluation identified a synchronous right renal pelvic urothelial cancer. 8 Controversies in Urine Cytology Each case highlights challenging aspects of urine cytology interpretation Discussion points My perspective after 17 years of practice at Mayo Clinic»Viewing the landscape»the role of urine cytology»the impact of equivocal cytology findings»fish as a paradigm for novel biomarker use»the role of clinicopathologic discourse These cases highlight some of the challenging aspects of urine cytology interpretation. In the allotted time, it is, of course, impossible to discuss in detail all the potential controversial issues that urine cytology brings to the table, so my goal for this evening is to give you my perspective on urine cytology relying heavily on my own experience of evaluating many thousands of urine cytology specimens over a 17 year period of time while on staff at Mayo Clinic. For a detailed review of urine cytology and the role biomarkers play, I direct your attention to two wonderful reviews. 3,4 I will focus on 5 main discussion points (1) Viewing the landscape of urothelial cancer and why, perhaps, it has been difficult for any test whether routine urine cytology or a novel biomarker to make its mark in the field; (2) The role routine urine cytology plays in the diagnosis and management of urothelial cancer; (3) The impact equivocal diagnoses in urine cytology have had on the diagnosis and management of urothelial cancer; (4) A discussion of FISH using it as a paradigm for novel, ancillary biomarker analysis in other aspects of clinical practice; and (5) The crucial role communication and teamwork between pathologist and clinician have on the efficient management of patients with symptoms concerning for or biopsy proven urothelial cancer. 4

5 9 My Mentors Two of the Giants Farrow Goellner Me a Little Giant from Canton, Illinois I trained under two of the greats in urine cytopathology George Farrow and John Goellner. While they taught me essential morphologic keys to diagnosis, they also taught me something, perhaps, more important a humble perspective that while we often would like to view cytology whether urine or otherwise as a gold standard, it is often more important to communicate our findings whatever they may be in a timely and articulate manner to our clinical colleagues who can then correlate their observations with our cytologic examination and formulate the best game plan for managing their patients. Dr. Farrow was fond of saying that pathology is as much art as it is science and I think no where does that mantra apply more fittingly than in the interpretation of urine cytology. I view these two individuals as true giants. Being from Canton, Illinois (near Peoria), where our high school mascot was the Little Giant (eerily reminiscent of the Purdue Boilermaker), that makes me a little giant in more ways than one. 10 The Landscape: ; Incidence of Urothelial Carcinoma (Top) Has Risen but Mortality (Bottom) Has Remained Unchanged Urothelial carcinoma represents the fourth most common malignancy in the United States and ninth leading cause of cancer death. The etiology is multifactorial and is rarely diagnosed before the age of 50 particular in patients who have not smoked or been exposed to known carcinogens. In 2009, there were 71,000 new cases with 14,000 deaths. 5 The incidence of urothelial cancer has increased over the past 30 years, but mortality has remained essentially unchanged. 3 Mitra and Cote. Nat Rev Urol. 7(1):11-20,

6 11 The Landscape: ; Stage-Stratified 5-Year Overall Survival of Urothelial Carcinoma If caught early and, where appropriate, treated aggressively, patient survival is high Easier Said Than Done! Mitra and Cote. Nat Rev Urol. 7(1):11-20, 2010 Most bladder cancers are diagnosed at the superficial stage with a small percentage progressing to muscle invasive cancer. 6 In some respects, the increased incidence of tumor detection without a concomitant increase in death rates is encouraging for it suggests that more urothelial cancers are being detected early in their life span. However, what is also true is that the lack of survival improvement with advanced stage disease reflects a lack of success with adjuvant therapies subsequent to surgical removal of the bladder and cancer debulking. Furthermore, while a vast majority of patients present with hematuria, 7 most patients with hematuria do not have cancer unless they are in a high risk category including male gender over the age of 50, smoking history, and/ or exposure to known urinary tract carcinogens such as aromatic hydrocarbons. 8 This slide indicates that five year overall survival has also not changed much. However, if the urothelial carcinoma is caught early and, where appropriate, treated aggressively, patient survival is high. The problem, of course, is that this is easier said than done given the above-stated nuances of urothelial cancer detection and the lack of generally accepted algorithms for evaluating patients for the possibility of identifying clinically occult urinary tract malignancy. 12 The Role of Urine Cytology Specimens Evaluated at Mayo Clinic, Total Cases Year # of cases 75% of Urine Samples Collected as Part of Managing Patients with Urothelial Cancer Urine cytology evaluation varies considerably throughout the world. 9 At Mayo Clinic, however, we have been very active in evaluating urine cytologically for a very long time (more than 20 years). This slide shows the number of urine samples evaluated on a yearly basis between 2004 and 2009 at our institution. It is estimated that roughly 75% of these samples are collected as part of a standardized management plan of urine cytology and urinary tract evaluation (cystoscopy and upper urinary tract studies) subsequent to a biopsyproven diagnosis of urothelial cancer. 6

7 13 Two Main Challenges in Conventional Urine Cytology Scattered Atypical Cells and Mildly Atypical Clusters I view the two main challenges in urine cytology interpretation as (1) scattered urothelial cells with degenerative type atypia; and (2) clusters of urothelial cells with mild atypia in which the differential diagnosis is the so-called big three instrumentation effect, the effects of urolithiasis, and low grade papillary neoplasm. TURP Radiation Catheter In general, any etiology that causes urinary stasis can generate cytologic atypia. This includes benign prostatic hypertrophy, catheterization, and renal insufficiency, which often manifests as urinary casts with scattered degenerating urothelial cells as well urothelial cell clusters with mild atypia. Renal Insufficiency Urothelial Clusters Degenerating Cancer Cells The challenge is to distinguish a degenerating cell from a benign process from that of a cancer. The photomicrograph in the lower right hand corner highlights the fact that sometimes degenerating cells sloughing from a high grade carcinoma may be subtly altered as to render an unequivocal diagnosis of urothelial cancer difficult. 14 Processing Urine for Cytology ThinPrep Fluid Conservative; Reflex to Biomarker In the old days, when I trained and then became a staff pathologist, we evaluated urine cytology by collecting cells onto Millipore filters via vacuum filtration. 10 The current approach using ThinPrep is more fluid conservative in the event ancillary biomarker studies are necessary. In my opinion, we have lost some cellular nuclear detail with this process possibly making urine cytology interpretation more problematic. The red arrow points to an example of a urothelial carcinoma with nice nuclear detail from a slide generated from the Millipore filtration method. 7

8 15 Voided Urine Cytology Diagnoses, 2004 March 2007 ( Equivocal Diagnoses: Big 3*, Atypical, and Suspicious ) Year (Jan-March) N Negative (%) Filtration ThinPrep Equivocal (%) Equivocal (N) Positive (%) Voss et al. Am J Clin Pathol Sep;130(3): * - Urothelial Cell Clusters. Differential Diagnosis Includes the Effects of Instrumentation or Urolithiasis and Low Grade Papillary Urothelial Neoplasm To highlight this concern, a recent study from our group showed that when we made the switch from the vacuum filtration method to the ThinPrep method, our equivocal urine cytology diagnosis rate essentially doubled. 11 An equivocal diagnosis is defined as either (a) big 3 (clusters of urothelial cells with mild atypia; the differential diagnosis includes the effects of instrumentation and urolithiasis or a low grade papillary neoplasm); (b) scattered atypical cells with features favoring reactive/ degenerative changes; or (3) urothelial cells with cytologic features worrisome for carcinoma (related either to insufficient numbers of cells with qualitative features of malignancy or sufficient numbers of cells with borderline cytologic features of malignancy). 16 Percentage Patients With Cancer on Follow-Up (Problem Undefined Patient Population With Different Risks) 0.5 YEARS 1.0 YEARS Negative Big 3 Atypical Suspicious Positive Overall Before ThinPrep 4.0% 9/ % 56/ % 45/ % 91/ % 162/ % 363/1376 After ThinPrep 5.1% 9/ % 18/ % 39/ % 78/ % 198/ % 342/1392 Before ThinPrep 5.3% 12/ % 58/ % 58/ % 104/ % 172/ % 404/1376 After ThinPrep 6.3% 11/ % 24/ % 47/ % 81/ % 210/ % 373/1392 Conversely, the detection rate of malignancy up to one year subsequent to the increased number of equivocal urine cytologies decreased. While the data are not clean (i.e., this was not a prospective study and patients entered the follow-up phase at different times in their cancers lifetime ), this information is still intriguing and suggests that by enhancing our sensitivity (increasing the number of equivocal urine cytologies), we have decreased our specificity in detecting malignancy subsequent to the urine cytology evaluation. 11 8

9 17 Percent of Cases in Category Urine Cytology Classification Per Mayo Clinic Pathologist, 2009 (Overall 16% Equivocal ) 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 2% 2% 2% 1% 3% 1% 0% 2% 2% 3% 2% 2% 4% 5% 6% 5% 3% 3% 2% 2% 5% 3% 4% 2% 1% 3% 6% 2% 1% 7% 7% 7% 13% 11% 2% 13% 13% 4% 6% 5% 6% 75% 5% 77% 87% 6% 9% 74% 71% 94% 82% 92% 81% 92% 82% Positive Suspicious Atypical Big 3 Negative The data on this slide show the equivocal rate of urine cytology diagnoses per pathologist evaluating urine specimens at Mayo Clinic for The overall equivocal rate was 16%, which, as you will note from slide 15 has not significantly changed in the last three years (unpublished data). 0% B (252) C (783) E (1003) G (734) J (1233) M (258) Q (1583) W (1125) X (1318) Z (945) Lab Total (9234) 18 Lack of Relationship Between Percent Equivocal Urine Cytology Classification and Number of Cases Examined by Pathologist % Equivocal (Atypical, Big 3, Suspicious) Number of Urine Cytology Cases Reviewed Per Pathologist There is a rather significant difference in the equivocal rates amongst our group of pathologists ranging from 5% to about 27%. This figure, however, suggests that these differences are not due to a lack of experience viewing urine specimens over the course of a year. Rather, there are likely to be philosophic differences amongst the group in which some pathologists are either more or less inclined to flag a urine as equivocal regardless of other clinical factors that may help solidify a diagnosis of either benign or malignant (unpublished data). 9

10 19 What Impact Does Equivocal Cytology Have on Practice? (Assume Urothelial Carcinoma Prevalence = 3% in General Population [N=1000]) Cancer Test Yes No Positive Negative Cytology Sensitivity = 34%, Cytology Specificity = 99%*; All Patients With Positive Urine Test Result Undergo Evaluation of Urinary Tract; Positive Predictive Value (PPV) = 50.0% Negative Predictive Value (NPV) = 98.0% (Clinically Acceptable Since Persistent Hematuria Requires Further Evaluation) * - Lotan and Roehrborn. Urology. 2003;61: However, it s important to remember that the prevalence of a specific disease you are trying to detect by a test with a certain degree of sensitivity and specificity will impact on the clinically important positive and negative predictive values of the test result. To show this with some hard numbers, this slide combines the reported sensitivity and specificity of urine cytology garnered from a meta-analysis of several studies 12 with the purported risk or prevalence of urothelial cancer in a general population of patients ( ) 13 Under these circumstances, the positive predictive value (PPV) is only 50%, but the negative predictive value (NPV) is very high (98%). Our urologist colleagues at Mayo indicate that under most clinical circumstances, this combination of low PPV and high NPV is acceptable. 20 Impact of Urine Test Predictive Value (General Population Cancer Prevalence of 3%) if All Patients With Equivocal Test Undergo Evaluation Predictive Values Positive Negative Predictive Values for Urine Cytology With Low (Left) and High (Right) Sensitivity 34%/ 99% 75%/ 80% 50.0% 10.2% 98.0% 99.0% In Low Prevalence Disease, Increasing Test Sensitivity at Expense of Specificity Increases Likelihood More Patients Will Undergo Surgical Evaluation for False Positive Test 12 More Patients Detected with Cancer by Evaluating 196 More Patients (6.1%) The rationale behind this reasoning is highlighted in this slide. Equivocal urine cytology diagnoses have a greater tendency to force urologists to pursue a urinary tract source for the atypia. Thus, if one makes the assumption that any non-negative urine cytology finding leads to further investigation (endoscopy, upper urinary tract studies) particularly in the setting of unexplained hematuria increasing your sensitivity of urine cytology (by statistically classifying the non-negative urines as potential positive samples) will capture in this particular scenario 12 more patients but potentially at the expense of evaluating 196 more patients. This represents a true positive rate of 6.1%. 10

11 21 Low Sensitivity and High Equivocal Rate of Cytology Paved Way for Ancillary Biomarker Analysis Why FISH? Assay Sensitivity BTA-Stat FISH Hb Dipstick 5/12 (42%) 15/25 (60%) 34/36 (94%) 54/73 (74%) Telomerase 3/10 (30%) 12/25 (48%) 17/35 (49%) Cytology Grade 1 Grade 2 Grade 3 6/12 (50%) 18/25 (72%) 32/35 (91%) 4/11 (36%) 19/25 (76%) 36/37 (97%) 3/11 (27%) 13/24 (54%) 24/34 (71%) Grade 1973 W.H.O. Classification Halling et al. J Urol May;167(5): Overall 56/72 (78%) 59/73 (81%) 32/70 (46%) 40/69 (58%) Due, in part, to the low reported sensitivity and increasingly higher equivocal rate of urine cytology at Mayo Clinic and other institutions, we embarked upon a comparative analysis looking at various assays/ tests including BTA Stat, hemoglobin dipstick, telomerase, and fluorescence in situ hybridization (FISH). 14 The results of the study, which kick-started the use of FISH for urine evaluation at Mayo, showed that, overall, FISH was the most sensitive in detecting the so-called clinically significant urothelial cancers including high grade papillary urothelial carcinoma and urothelial carcinoma-in-situ. 22 Fluorescence in Situ Hybridization (FISH) Chromosome 3 Centromere.... P16 Gene (LSI 9p21).. Chromosome 7 Centromere.. UroVysion Probe Set FISH for urine specimens uses the Vysis UroVysion probe set looking for various colored fluorophores to specific DNA sequences matching the centromeres of chromosomes 3, 7, and It also includes a locus specific gold-colored fluoroprobe to 9p21 where several tumor suppressor genes, including p16, reside. 16,17 Chromosome 17 centromere 11

12 23 Fluoresence in Situ Hybridization (FISH) Signal Patterns Disomy (Negative) Polysomy ( 5 Cells; Positive) Trisomy 7 ( 10 Cells; Equivocal) CEP 17 CEP 3 CEP 7 LSI 9p21 ( 10 Cells; Positive) 9p21 (First, Most Common Deletion) Trisomy Polysomy Loss of signal to 9p21 is thought to be the first and most common deletion, which occurs in low grade papillary neoplasms. 18 Significant percentages of polysomic cells in the urine typically correspond to high grade urothelial cancers. 19 Initial studies with this probe set established criteria by which to classify a urine specimen as either negative or positive. 17 In general, the more subtle the genetic alterations (loss of signal to 9p21, trisomy 7), the greater the number of cells required to view the FISH result as reflective of tumor. Conversely, it takes fewer polysomic cells (defined as 5 or more cells with gains of 2 or more chromosomes) to establish a sample as positive and polysomic. 24 Under What Circumstances Can Biomarker (FISH) Help? (Meta-Analysis of Test) 14 studies involving 2477 FISH tests The overall prevalence of urothelial cancers was 35% FISH Cytology Sensitivity 72% 42% Specificity 83% 96% In a meta-analysis performed by Hajdinjak, overall sensitivity for FISH and cytology was 72% and 42%, respectively, and overall specificity for FISH and cytology was 83% and 96% for FISH and cytology, respectively. In the collection of studies reviewed, the prevalence of urothelial cancers was 35%. 20 Hajdinjak T. Urol Oncol Nov-Dec;26(6):

13 25 Impact of Urine Test Predictive Value (High Risk Cancer Prevalence of 35%) If All Patients With Positive Test Undergo Evaluation Predictive Values Positive Negative Predictive Values for Cytology (Left) and FISH (Right) 42%/ 96% 72%/ 83% 85.0% 69.6% 75.5% 84.6% In High Risk Population, Increasing Test Sensitivity at Expense of Specificity Increases by Almost 10-Fold the Identification of Cancer in Patients Undergoing Surgical Evaluation 105 More Patients Detected with Cancer By Evaluating 189 More Patients (55.6%) Applying the same statistical analysis as in slide 20, we can compare the PPV and NPV of routine cytology and FISH when the likelihood of detecting urothelial cancer is high in reality, similar prevalence to the risk of cancer progression in patients with high grade carcinoma (papillary or flat). Under these circumstances, increased test sensitivity at the expense of specificity increases by almost tenfold the likelihood of identifying patients with cancer who undergo surgical evaluation because of a positive FISH test. Specifically, 105 more patients will be detected with cancer by evaluating the urinary tract of 189 more patients. This improves the detection rate from 6.1% (see slide 20) to 55.6%. To play devil s advocate, this still means that about half of patients with a positive FISH test will not have clinicopathologic evidence of recurrent tumor upon further evaluation. Thus, it becomes a matter of which side of the ratio one likes in the cost/ benefit ratio scheme. 26 What Constitutes a Positive FISH Test? Relationship Between % Abnormal Cells and Recurrence Patient Histories of pta, pcis, or pt1 Bladder Cancer What Impacts on the % of Abnormal Cells? Grade, Tumor Burden, Background CIS, Location Kipp BR, et al. J Mol Diagn Mar;11(2): Furthermore, as we gain more experience with clinical FISH testing, it becomes clear that how one defines a positive FISH test will determine the likelihood of urothelial cancer detection in follow-up. In fact, whenever I am told that a patient s FISH test was positive, my first response is to ask the question, What do you mean by positive? This can play out in several different ways. For example, a patient with 15% of their urothelial cells marking as 9p21 deletions with no cystoscopic or ureteroscopic evidence of a papillary neoplasm should be viewed with caution. Conversely, it is hard for me to imagine a truly negative urine cytology when the FISH test is 40% polysomic. A Mayo study showed that quantitating the degree of FISH polysomy could predict bladder recurrence in patients originally diagnosed with urothelial cancer. 19 Potential problems with the study include the lack of grading information in the pta group, the lack of standardized treatment in the patients, and no data on urine cytology. 13

14 27 Phenotype/ Genotype Interplay (Refer to #16 Not Clean Database) Polysomic FISH <5% Polysomic FISH 5% - 30% Polysomic FISH >30% Negative Big 3 Atypical Suspicious Positive In fact, if we present the data from slide 16 in a similar Kaplan-Meier assessment, there is rough similarity between the percent polysomic FISH findings from the study mentioned in the previous slide and the risk of cancer progression in equivocal and positive urine cytology results. The approach to most of the studies on FISH at Mayo have been to take the urine cytology diagnosis gleaned from the pathology reports and compare that with, perhaps, a more rigorously defined collection of FISH results. Furthermore, by not normalizing the data to tumor grade, stage, volume, and location and the presence or absence of CIS in the background, it is somewhat speculative to make dogmatic statements regarding the strength of quantitative FISH in predicting cancer progression. 28 Clinical Urine FISH Tests Performed At Mayo Clinic (Top) and Percent of Tests Abnormal (Bottom), Volume of Tests % Abnormal (Trisomy, Deletion, Tetrasomy, Polysomy) Year Another way of looking at this is to track total clinical urine FISH test volume per year with the percent of cases classified as abnormal. The abnormal category includes 9p21 deletions, trisomy 7, tetrasomy, and polysomy. Furthermore, there is no information on the percent of cells quantified as abnormal in each category. Regardless, one can see from this figure that there is at least a suggestion of an inverse relationship between the number of FISH tests performed and the likelihood of the test being classified as abnormal (unpublished data). 14

15 29 30 Full Circle Statistics in Urine Evaluation There Are Three Kinds of Lies: Lies, Damned Lies, and Statistics Attributed to Benjamin Disraeli in Mark Twain autobiography, 1924, volume 1, p. 246 Cytology diagnoses Negative Atypical Suspicious Positive FISH diagnoses Negative Equivocal Positive + +» (deletion, trisomy, tetrasomy, polysomy; percentage thereof) A Unified Field Theory of Cytology Phenotype And Genotype All of this reminds me of the famous quote attributed to Benjamin Disraeli (former British Prime Minister in the 1870 s) in Mark Twain s autobiography (Chapters From My Autobiography) There are three kinds of lies: Lies, damned lies, and statistics. There are so many clinicopathologic permutations to most published reports on the relative values of routine cytology and FISH or for that matter, any ancillary biomarker that it behooves all of us as medical scientists to maintain a critical eye when it comes to assessing the clinical utility of any biomarker that has the potential to increase the cost of patient care without fully understanding its PPV and NPV. As we have discussed before, from the pathology side of the equation, we can certainly boost sensitivity by lumping, for statistical purposes, any non-negative urine cytologic interpretation. However, this comes at a cost of decreased specificity. Even our FISH lab has begun reporting equivocal FISH findings e.g., tetrasomy, particularly from upper urinary tract sources, polysomy in which the number of polysomic cells is borderline. On the clinical side of the equation, accurate and reproducible classification of urinary tract biopsy material, adjuvant therapy after debulking of tumor, and anticipatory positive results (positive pathologic test result which precedes the identification of urothelial cancer) will also impact on the PPV and NPV of a particular test. I also have taken the approach that just because a study is published which shows that a biomarker is superior to routine cytology does not mean we should stop trying to improve our cytomorphometric skills. Albert Einstein searched his whole life in vain for the Holy Grail of physics: the unified field theory. He also thought that much of scientific discovery was serendipitous. I view molecular pathology as contributing along two great fronts towards a better understanding of pathogenesis of disease and allowing us an opportunity to merge old concepts with new ones and, in so doing, becoming generally the better for it. 15

16 31 DAPI Stain for FISH Evaluation of Urine Specimen Look for the Large Cells to Identify FISH Abnormalities As a first example, consider a Mayo study that showed polysomic urothelial cells have significantly weaker DAPI nuclear counterstaining, nuclear enlargement, more irregular nuclear shape, and more chromatin clumping when compared to disomic cells. 21 This begs the question of why FISH routinely appears to outperform routine cytologic examination of urine. Kipp et al. Anal Quant Cytol Histol Dec;27(6): Quantitating Nuclear Size Relationship Between DNA Content and Nuclear Area FISH/ DIA Positive Cell Nuclear Area (Microns 2 ) FISH/ DIA Negative Diploid Mean = 64 53% Increase Aneuploid Mean = 98 Very early studies at Mayo when we first began merging our routine cytology work with digital image analysis (DIA) and FISH showed these nuclear features which were polysomic by FISH could be compared with quantifiable nuclear parameters collected by DIA. The figure shows that evaluating nuclear area in cells that were classified as positive by routine cytology, aneuploid by DIA, and polysomic by FISH had a 53% increase in their mean nuclear areas compared to the nuclei from urine specimens categorized as negative by cytology, diploid by DIA, and disomy by FISH (unpublished data). 16

17 33 Quantifiable Nuclear Morphometry Combined into Visual Nuclear Morphometric Score (VNMS) Feature Definition Value Area There are several quantifiable nuclear features which can be viewed as reflective of nuclear cytologic findings by routine microscopic examination. 22 This allows one to generate a visual nuclear morphometric score (VNMS), which can be compared with routine cytology findings and FISH results. 23 Perimeter Feret x Feret y Max. Diameter Min. Diameter Elongation VNMS Bile Duct Nuclear Morphometry Versus Cytology Result Visual Nuclear Morphometric Score 30 Barr-Fritcher et al. Am J Clin Pathol 2007;128: P< Negative Atypical Suspicious Positive Cytology Result In a more organized study looking at the relationship among routine cytology, DIA, and FISH in bile duct brushing specimens, we showed that there was a gradual alteration in quantifiable nuclear parameters as the cytology specimen progressed from negative to atypical to suspicious and, finally, positive for malignancy

18 35 36 Bile Duct Nuclear Morphometry Versus FISH Result Visual Nuclear Morphometric Score 30 Barr-Fritcher et al. Am J Clin Pathol 2007;128: Disomy Gain of Single Chromosome; Slight Increase in Nuclear Size/ Score Trisomy 7 FISH Result Polysomy Ratios of Quantifiable Nuclear Features Between Benign and Malignant Cells from Three Different Organ Systems Feature Area Perimeter Shape Elongation Urothelial Pancreatobiliary Benign Cancer Ratio Benign Cancer Ratio Benign Cancer Ratio Barr Fritcher et al. Am J Clin Pathol 2007;128(2):272-9 Gastroesophageal Pending Area microns 2 ; Perimeter microns; Shape Perimeter 2 / Area; Elongation Maximum Diameter/ Minimum Diameter Our data suggested that, on average, routine cytology and quantifiable nuclear morphometry (VNMS) could discern FISH alterations as subtle as a single chromosome gain specifically, trisomy 7. These data, similar to patients with high grade urothelial cancer, reflect a patient population with a high prevalence of developing pancreatobiliary carcinoma (45% in our database). 24 As the cytologic interpretation became more atypical, the likelihood of detecting FISH abnormalities increased significantly Relationship Between FISH and Cytology In Bile Duct Brushings (Cancer Prevalence 45%) Percent of Cases (Barr-Fritcher et al. Am J Clin Pathol 2007;128:272-9) 5% 30% Negative Atypical Suspicious Positive Cytology Result 70% 90% Results with FISH and Cytology Likely to Be Similar in Urine Samples? Negative Trisomy 7 Polysomy While generating VNMS is academically intriguing, it appears that the one feature that is most strongly correlated with FISH abnormalities is nuclear area. Although our cytology/ DIA/ FISH work was rejected for publication, as mentioned in the previous slides, we have shown in publication that nuclear area increases significantly in cells as they proceed from cytologically negative to malignant. 25 Similar data have been collected in gastroesophageal brushings from patients with Barrett s specialized esophageal mucosa and a high risk of progressing to cancer (unpublished data). Perhaps, no unified field theory can be established in the sense that the mean nuclear enlargement in each organ system tested to date with this multi-modality approach is different. However, the results confirm the very logical assumption that as the nucleus adds more genetic material, its nuclear area increases concordantly. 18

19 37 Urothelial Cancer Large and Hyperchromatic (Left); Benign Atypia Small and Hyperchromatic (Right) What does this all mean? Well, with more experience evaluating urine cytology and with the knowledge gained with these sorts of studies, my approach to urine cytology classification has increasingly become more black and white, i.e., negative or positive. I do the best I can in reducing my diagnoses of big 3 and atypical urothelial cells by focusing on nuclear size and degree of hyperchromasia. The general rule of thumb that I typically now follow is that small and hyperchromatic nuclei are most likely to be reactive in nature whereas large and hyperchromatic nuclei are the cells which portend a more ominous clinical picture. We all recognize this as a very hyperchromatic appearance to a urine specimen on low magnification. This will manifest as the typical sort of nuclear enlargement one expects from a polysomic urothelial cancer cell. 38 Clinicopathologic Discourse Management of Mayo Patients With Biopsy- Proven Urothelial Cancer Urine Cytology, Cystoscopy, Upper Urinary Tract Studies Correlate Cytology With Cystoscopy Don t Allow Clinical to Drive Cytology Interpretation The last discussion point to consider is that, in the long run, good clinicopathologic correlation will often either trump or at least equal the predictive power of the latest, hottest biomarker. Many of my urine cytology consultations historically have often been generated due to a lack of good communication between pathologist and urologist. When we do our urine cytology sign out, we sometimes have at our disposal the urinary tract findings from the urologist during the routine follow-up examination of patients with treated urothelial cancer. Some people may think this is cheating, which I have always thought is a strange way of describing the process. More accurately, it is true that we do not want clinical observation to drive our cytologic interpretation. To avoid that, I always review the urine samples in a clinical vacuum, rendering a diagnosis or differential diagnosis prior to reviewing the cystoscopic findings of the patient. This helps eliminate a significant number of equivocal diagnoses ( big 3 and atypical ) and reduces the number of patients who require further evaluation with a low cancer detection rate. 19

20 39 Urine Clinical FISH Tests Performed, Jan 08 Jan 10 (Extramural Vs. Mayo Patients) # Cases Jan Mar-08 May-08 Clinical History Unknown Jul-08 Sep-08 Nov-08 Jan-09 Mar-09 Month May-09 Jul-09 Sep-09 Nov-09 MML Mayo Total FUROs Clinical History Known; Interaction with Clinicians Jan-10 This collaborative effort plays out in our clinical FISH test volumes. This slide shows that the vast majority of our test requests come through our reference lab (Mayo Medical Labs MML). I think it highlights a very important observation about the critical role pathologists must play in the era of molecular pathology. We cannot forget our past completely if the future means more patient expense in the coming era of cost containment. While no one can know it all, those pathologists who understand the clinical landscape, are excellent microscopists, and can navigate through the increasingly complex molecular universe should be the role models by which we base future training in pathology. This becomes an issue for us as we have reduced the number of years for trainees to become board-eligible and, at the same time, drawn them away from basic surgical pathology and cytopathology exposure so they can learn the latest techniques in molecular pathology. The approach I am most comfortable with is to have a highly skilled anatomic pathologist who knows clinical practice well apply a new technology where it makes the most sense rather than having the practice driven by technocrats searching for a clinical niche to apply their molecular technology. 40 Sensitivity, Specificity, and Positive and Negative Predictive Values of Cystoscopy and FISH for Detection of Superficial and Muscle-Invasive Bladder Cancer When Urine Cytology is Equivocal Test Cystoscopy FISH Cystoscopy + FISH p-value 3 Sensitivity 1 Sensitivity 2 Specificity 57/85 (67) 53/85 (62) 74/85 (87) < /17 (56) 15/17 (88) 16/16 (94) /39 (85) 57/63 (90) 33/61 (54) 34/39 (87) 53/58 (91) 34/66 (52) 31/39 (78) 74/82 (90) 31/42 (74) PPV NA NPV Compare to #25 1 Sensitivity Detecting Superficial Or Muscle-Invasive Bladder Cancer 2 Sensitivity Detecting Only Muscle-Invasive Bladder Cancer 3 p-value Comparing Cystoscopy Alone to Cystoscopy + FISH Kipp et al. J Urol 2008;179(4): NA A recently published study from our group shows the potential utility in applying routine cytopathology skills, routine urologic clinical skills, and FISH testing in a structured manner. Using FISH as a reflex test when the urine cytology interpretation is equivocal may allow patients to be managed in a more standardized fashion depending upon the combination of findings. In particular, the NPV in managing a patient with a history of urothelial cancer and an equivocal urine cytology is enhanced when both the cystoscopic findings and the FISH test are negative. Of course, while this may be a statistically significant improvement, it is by no means an indication that repeat cystoscopy is not necessary or that the PPV is improved beyond the typical 50% level

21 41 Some Final Thoughts Simpson s paradox Aggregated data can appear to reverse trends in numbers being combined (weighted averages); My favorite example My final thought on this subject comes from an article I recently read in the Wall Street Journal regarding an anomaly in statistics termed Simpson s Paradox. In text form, it simply means that aggregated data can appear to reverse trends in numbers being combined due to the effect of weighted averages. Being a former baseball player, I find the best explanation of this phenomenon comes from showing why Derek Jeter s two year batting average (BA) is higher than David Justice s BA over the same period of time despite the fact that for each year, Mr. Justice s BA was actually higher. Player Total Justice BA (H/ AB).253 (103/ 411).321 (45/ 140).269 (148/ 551) Jeter BA (H/ AB).250 (12/ 48).314 (183/ 582).310 (195/ 630) 42 Simpson s Paradox (My Second Favorite Example): Urine Cytology and FISH Parameter No./ Total No. (%) P-Value Sensitivity Stage pta ptis pt1 - pt4 Sensitivity Grade Cytology FISH 17/ 36 (47) 14/ 18 (78) 9/ 15 (60) 3/ 11 (27) 13/ 24 (54) 24/ 34 (71) 24/ 37 (65) 17/ 17 (100) 18/ 19 (95) 4/ 11(36) 19/ 25 (76) 36/ 37 (97) Overall Sensitivity 40/ 69 (58) 59/ 73 (81) Specificity 48/ 49 (98) 75/ 78 (96) This segues into my second most favorite example of Simpson s Paradox one that has likely escaped the attention of most experts in the field of urine cytology and ancillary biomarker utilization. In the first major study from our group comparing the sensitivities and specificities of routine cytology and FISH, it seemed very clear that FISH markedly enhanced the sensitivity of urothelial cancer detection without compromising specificity. 17 Halling et al. J of Urol 2000;164:

22 43 Simpson s Paradox (My Second Favorite Example): Urine Cytology and FISH Study % Sensitivity (#) % Specificity (#) Grade 1 Grade 2 Grade 3 Before (150) 75 (421) 94 (426) 95.9 (10,421) 1990 and After 11 (265) 31 (452) 60 (388) 97.5 (1,096) All 21 (415) 53 (873) 78 (814) 96.0 (11,517) However, further evaluation showed that there wasn t a clinically relevant difference between the two methods sensitivities if comparing the published sensitivities of urine cytology prior to The reasons may be two-fold: First, there were ten times as many urine samples evaluated prior to 1990; and second, all of the data came mainly from two studies and 90% of that data came from one individual Dr. Farrow at Mayo Clinic. 27,28 FISH Overall Sensitivity = 81% (59/ 73) Cytology Overall Sensitivity (before 1990) = 77% (772/ 997) Halling et al. J of Urol 2000;164: That s OK, Tom. It s the first 100,000 cases that are the most difficult. - George M. Farrow, M.D. This is a nice way to both end my discussion and introduce or remind people of Dr. George Farrow s famous quote whenever I or one of his colleagues misinterpreted a urine cytology. Diagnosis? 22

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