Molecular Analysis in the Diagnosis and Management of Lesions of Uterine Cervix: The 95% solution. Mark H. Stoler, MD PSC Symposium USCAP 2008

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1 Molecular Analysis in the Diagnosis and Management of Lesions of Uterine Cervix: The 95% solution Mark H. Stoler, MD PSC Symposium USCAP 2008 Objectives: This presentation will briefly review the currently understood limitations of cyto- and histomorphology in the diagnosis of cervical neoplasia. Given the limitations are significant, then it will examine the question of whether molecular analysis using HPV or other markers can overcome some these limitations and how markers that are currently available can be used in daily practice. Do we need molecular help in cervical cytology and histology? Unfortunately, YES Management of intraepithelial neoplasia of the uterine cervix requires accurate diagnosis. If the Pap is normal, patients are told they have no risk of cancer for the near term. If it is abnormal they go to colposcopy and biopsy and often have surgical intervention with increasing recognized costs or trade-offs in term of fertility vs. cancer prevention. But how accurate is this system? Based on routine cytology, colposcopy and biopsy, how often do we get it right? Diagnostic Accuracy: The critical impact of sampling error and interpretive reproducibility. If a woman truly has CIN3, how often does the Pap- colposcopy-biopsy system, on a given pass find the CIN 3? If the cells aren t on the slide or in the vial, the sample is falsely negative. If the colposcopist can t see the lesion or sees it but fails to biopsy it, the tissue obtained is falsely negative. Sampling error is prominent in this system and may never be completely eradicated. The sensitivity of the Pap smear is widely cited as at best 80% with prominent reports as low as 50%. Randomized clinical trials have now shown that the latter (50-60%) is likely a more accurate estimate. Colposcopic biopsy is a poor standard, getting the lesion in only 50% of encounters. Even if a lesion is present on the slide, the variable skill of pathologists in the application of criteria causes significant interobserver variability in diagnosis. Many studies demonstrate that even experts exhibit significant interobserver variation in cervical specimen diagnosis. Remarkably, biopsy diagnosis is no more reproducible than Pap smear diagnosis. So the unfortunate answer to the above question is yes. Our prayer may be for the molecular marker to please make screening and diagnostic interpretations more accurate. But it will never be perfect. However 95% is a lot better than 50%. Diagnostic and Prognostic Markers By analogy to a prognostic factor, a diagnostic marker can be defined as an item of information concerning the pathology of interest that can increase the accuracy of diagnosis. Ideally such markers are highly correlated with biologic processes. However, the critical question then arises as to whether the biologic correlation is of clinical utility. The process of determining marker utility is a much more daunting problem then much of the literature suggests and explains why

2 Stoler, M.H. Page 2 of 6 many of the claims in the literature are confusing, conflicting or just plain examples of poor science. In partial response to the inappropriate and premature use of markers in many systems, guidelines for the evaluation of prognostic factors have been developed and refined. The American Joint Committee on Cancer (A.J.C.C.), C.A.P., Working Group on Ethical, Legal and Social Implications of the human genome research of the National Institutes of Health (N.I.H./E.L.S.I.) all have published position papers on this topic of prognostic markers. By analogy, many similar concerns can be applied to diagnostic markers as well. All proposals have certain common threads. To be useful a proposed new marker should be significant; meaning it is not a chance occurrence, independent and most importantly clinically relevant. Clinical relevance or validity is the most stringent standard since it requires that the additional information provided by the factor will affect patient management or outcome in a useful manner. All systems recognize that determination of analytic sensitivity and specificity is only a first step and factors like technical feasibility, assay codification, methodologic accuracy, precision, quality assurance, cost and turn around time all need determination before trials of clinical sensitivity, specificity predictive value and validity /utility can be fully established and a new marker can be considered ready for prime time general practice Within this framework, are there clinically valid molecular adjuncts that help our problems? Fortunately, YES! VALIDATED HPV TESTING: The 95% Solution for Cytology Cytology Screening: The answer is as simple as 50 vs. 95%. Do you want an algorithm that is 50% sensitive in each round or 95% sensitive? For screening, multiple studies and meta-analyses say cytology, even optimized cytology, is only 50-60% sensitive. In contrast HPV testing is always in the range of 90-95% sensitive. There is an important corollary in this concept. Sampling issues are not very large for cytology. There will always be some mis-sampling but the magnitude is on the order of only 5-10%. Thus the big issue is getting the abnormal cell on the slide (as opposed to in the sample), finding it and properly interpreting it. Cytology is first and foremost a screening test. Epidemiology 101 says screen with the most sensitive test possible. This is especially true if the prevalence of disease if low as it is the negative predictive value of the test that is the true value to most women. Cytology Triage: The same answer is available, 95 vs. 50%. Most abnormal Paps are ASCUS or LSIL not HSIL. Yet most CIN2/3 comes from patients who do not have an HSIL Pap. Ergo, there is a potential sampling or interpretive issue with abnormal cytology. For ASCUS, the answer is clear. HPV testing effectively segregates the 50% of ASC-US patients with true cancer risk from those that have essentially no risk. While not quiet as clear, HPV testing works well and is increasingly part of the algorithms for ASC-H and AGC. For LSIL, HPV testing may also be useful particularly in older cohorts. In all these setting the concerns of lack of specificity are in my opinion overstated or worth the trade-offs. Cytology Quality Control: The same sampling and interpretive issues that so greatly impact our primary interpretation of Pap smears plague our current QC routine. The one that is confounded the worst is cyto-histo 2

3 correlation. Call me a heretic, but given what we know, it is almost a complete waste of time as circular reasoning plagues it and high grade on anything always wins. What one needs for cytology is an objective measure of NIL (<10% HPV), equivocal (~50% HPV) or definite SIL /Cancer (~95%). Cyto-HPV correlation is superior quality control. Cytology Test of Cure: After a patient has been treated for proven precancer, traditionally they are placed in a serial follow-up program more intensive than routine screening. In the past this meant serial Pap smears every 3-4 months times three to four cycles. Remember though, that each of these cycles of Pap are still ~ 50% sensitive. In addition, early cycles may be confounded by repair or inflammation, later cycles by problems in getting to the area of interest. HPV is again better and current consensus guideline suggest a single test at 1 year is just as good as 2-3 interval Paps (both about 95% sensitive) at finding people who have a recurrence or persistence after therapy. What About Biopsy Histology? Histology Interpretation and Quality Control. For Cytology, validated HPV testing is the key to quality practice. Cervical biopsies are only obtained if there is an indication based on cytology (or rarely symptoms). Histology has classically been viewed as the gold standard, but it is now widely appreciated to be no better than brass. Why has there been this fall from grace? Colposcopy is now proven to be no more sensitive than cytology. Small lesions are hard to visually distinguish from benign mimics and even if they are seen, are hard to biopsy. Even if the biopsy is representative of a lesion, getting the lesion in the biopsy represented on the slides (~1/3 of the error) and interpretive variability (~2/3s of the error) are significant problems for biopsies, maybe even more so than for cytology. Remember that biologically, patients in this system exist in one of three states. First, they are normal or have infections, inflammations or mimics of cervical neoplasia, but are truly at no immediate risk and should be reassured. Second, they only have a low-grade lesion, the biologic manifestation of productive HPV infection, which in the vast majority of woman are transient and probably should not be treated. Third, they have a true precancer (or rarely cancer), more than 95% of which can be attributed to high risk HPV. Since essentially all the truly relevant pathology is caused by HPV and the mimics are not, segregating histo-morphology on the basis of HPV being present or not makes great sense. Unlike in cytology most truly normal biopsies are HPV negative. The problem is what assays are available and how good are they. Correlation with the validated HPV test done on the cytology specimen that led to the biopsy may be very valuable, but does induce a source of variation. On tissue, immunohistochemistry and in situ hybridization are most appealing. If a probe or set of probes for all the relevant HPVs that infect the cervix could be developed such that an easy, costeffective, single-slide ISH test with a dynamic range of 1- several thousand copies per cell could be performed in most labs, then we should absolutely do it. This would fix the biggest problem in histology, telling normal from LSIL. Unfortunately we do not have such a tool, and unlike the situation in cytology such a test would have to include low risk viruses to capture the 10-15% of low-grade lesions that would be morphologically obvious. An alternative would be to do broad spectrum HPV sandwich pcr on a slice of tissue between representative H&Es, the approach

4 taken by the vaccine trials, but not currently implementable in any practical way for most laboratories. Stoler, M.H. Page 4 of 6 Based on the reproducibility studies, the major problem is telling normal from CIN1. But almost as important is telling low grade from high grade with the implication that the former does not need treatment but the latter does. CIN2, our current treatment threshold, is the ASCUS of CIN. It is an equivocation most likely representing a mix of ~1/3 CIN1 and ~2/3s CIN3. HPV testing does not segregate these groups, as all are HPV positive. Furthermore, more than 80% of CIN1 are high risk HPV positive and more than 95% of CIN2 and CIN 3 are high risk HPV positive so HPV typing does not help. Ki-67, P16 and other Magic Bullets Could other HPV correlates be useful in correctly categorizing patient biopsies? Much is known about HPV's proposed molecular pathogenesis and selected markers in these pathways might prove to be discriminating. For example, expression of the E6 and E7 viral oncogenes is thought to be essential for cancer precursor development. The morphologic hallmark of precancer is clonal expansion and cell proliferation. For cervical neoplasia, the papillomavirus E6 and E7 proteins interactions with the retinoblastoma and p53 pathways respectively, place them squarely at the center of cellular regulation of both proliferation and apoptosis. Induction of cell proliferation either S-phase measurement or immunohistochemical indices like Ki-67 have been the widely reported correlate of cervical preneoplasia and the switch from a virally productive to cellular proliferative phenotype seems well correlated with the progression to high grade lesions. Thus, markers like Ki-67 may be useful in the problematic biopsy, where the differential diagnosis is between a reactive process like immature metaplasia or gland regeneration vs. a high-grade cancer precursor. As noted above, many experimental and epidemiological studies reveal that expression of the HR-HPV oncogenes E6 and E7 in cervical epithelia is required to induce and maintain neoplastic growth of cervical epithelia. There is increased expression of the E6 and E7 genes in high-grade precancer. However, attempts to monitor expression of viral oncogenes in routine clinical samples have been limited by the great variety of the HR-HPV types, the low expression level of the viral oncogene products and the lack of sufficiently specific technologies and reagents to detect them. To overcome these limitations a cellular surrogate marker, i.e., a gene expressed by the host cell in response to the expression of viral oncogenes, but not expressed in normal nontransformed cells could be a better test. Many reports now demonstrate that increased expression of the HR-HPV oncogenes in cervical dysplasia results in highly specific overexpression of p16 INK4a in cervical dysplasia and cervical cancer cells which can easily be detected by specific monoclonal antibodies. Essentially ~95% of high-grade cervical lesions and invasive cancers have been shown to express very high levels of p16 INK4a. Thus on histology p16 seems reasonably validated for distinguishing high-grade lesions from mimics. The problem is that it is yet to be defined as valid for distinguishing low grade from high grade. Part of the reason for this has to do with biology, the rest with study design in the published studies to date. However large-scale clinical validation trials of p16 are coming The literature is full of many reports describing numerous other genotypic and phenotypic associations with development of cervical neoplasia. Immunohistochemical studies looking for over or under expression of epidermal growth factor, her 2/neu, CEA, MN telomerase, ProEx C etc., etc., all have some biologic rationale, but at times the relationships are not well developed 4

5 or represent second-level correlates. Most of these are very similar to p16 in that they are best correlated with high-grade lesions. However, often the reports on these markers are directly conflicting and the reasons are in the details of study design. Sources of variability between these studies run the gamut from technical and reagent variability to differences in populations studied to differences in the type of statistical analysis and elements included in the multivariate model. While some data appear promising based on reasonable analyses, most are unconfirmed or not replicated and hence for practical purposes have to be considered not ready for prime time, i.e. not clinically validated. Marking high grade is the easy. But finding a marker or a small set of easy to implement markers that accurately segregate biopsies into normal vs. low grade vs. high grade at the 95% level not quite there yet, although in selected situations they can really help and my current favorites are p16 and Ki-67. TAKE HOME POINTS: Accurate diagnosis that directs appropriate management triage is the goal of the pap-colpo-biopsy sequence. Sampling issues and interpretive variability confound the system at most steps, making each step less sensitive than we think. Sensitivities of ~50% are common. Validated HPV testing can significantly improve the performance of cervical cytology screening, triage, QC and treatment, improving sensitivity to the 95% level. Similarly validated adjuncts for histology are needed and candidates like p16 and Ki-67 are ready for clinical trials. Routine daily practice ultimately requires tests that are clinically validated.

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