Shikha Bose

Protocol for the Examination of Specimens From Patients With Ductal Carcinoma In Situ of the Breast

Authors Susan C. Lester, MD, PhD, FCAP* Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts Shikha Bose, MD, FCAP Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, California Yunn-Yi Chen, MD, PhD, FCAP Department of Pathology, UCSF Medical Center, San Francisco, California James L. Connolly, MD, FCAP Department of Pathology, Beth Israel Deaconess Medical Center, Boston, Massachusetts Monica de Baca, MD, FCAP Physicians Laboratory, Sioux Falls, South Dakota Patrick L. Fitzgibbons, MD, FCAP Department of Pathology, St. Jude Medical Center, Fullerton, California Daniel F. Hayes, MD, Department of Medical Oncology, University of Michigan Medical Center, Ann Arbor, Michigan Celina Kleer, MD, FCAP Department of Pathology, University of Michigan Medical Center, Ann Arbor, Michigan Frances P. O’Malley, MD, FCAP Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, University of Toronto, Ontario, Canada David L. Page, MD, FCAP Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee Barbara L. Smith, MD, PhD Department of Surgical Oncology, Massachusetts General Hospital, Boston, Massachusetts Donald L. Weaver, MD, FCAP Department of Pathology, College of Medicine and Vermont Cancer Center, University of Vermont, Burlington, Vermont Eric Winer, MD Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts Jean F. Simpson, MD, FCAP† Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee For the Members of the Cancer Committee, College of American Pathologists

The Anal Pap smear : Cytomorphology of squamous intraepithelial lesions

Background Anal smears are increasingly being used as a screening test for anal squamous intraepithelial lesions (ASILs). This study was undertaken to assess the usefulness and limitations of anal smears in screening for ASILs. Methods The cytomorphological features of 200 consecutive anal smears collected in liquid medium from 198 patients were studied and findings were correlated with results of surgical biopsies and/or repeat smears that became available for 71 patients within six months. Results Adequate cellularity was defined as an average of 6 or more nucleated squamous cells/hpf. A glandular/transitional component was not required for adequacy. Dysplastic cells, atypical parakeratotic cells and bi/multinucleated cells were frequent findings in ASIL while koilocytes were infrequent. Smears from LSIL cases most frequently showed mildly dysplastic and bi/multinucleate squamous cells followed by parakeratotic cells (PK), atypical parakeratotic cells (APK), and koilocytes. HSIL smears contained squamous cells with features of moderate/severe dysplasia and many APKs. Features of LSIL were also found in most HSIL smears. Conclusions In this study liquid based anal smears had a high sensitivity (98%) for detection of ASIL but a low specificity (50%) for predicting the severity of the abnormality in subsequent biopsy. Patients with cytologic diagnoses of ASC-US and LSIL had a significant risk (46–56%) of HSIL at biopsy. We suggest that all patients with a diagnosis of ASC-US and above be recommended for high resolution anoscopy with biopsy.

Interobserver reproducibility in the diagnosis of flat epithelial atypia of the breast

Columnar cell lesions (CCLs) of the breast with low-grade/monomorphic-type cytologic atypia are being identified increasingly in biopsies performed owing to mammographic microcalcifications. The WHO Working Group on the Pathology and Genetics of Tumours of the Breast recently introduced the term ‘flat epithelial atypia’ (FEA) for these lesions. However, the ability of pathologists to reproducibly diagnose FEA and to distinguish it from CCLs without atypia has not been previously evaluated. Eight pathologists with an interest in breast pathology participated in a study to address this issue. The study reference pathologist provided the other seven study pathologists with a Powerpoint tutorial that included written criteria for, and representative images of, FEA and CCLs without atypia (ie, columnar cell change and columnar cell hyperplasia). Following review of the tutorial, the study pathologists examined images in Powerpoint format from 30 CCLs and were instructed to categorize each as either ‘FEA’ or ‘not atypical’. Overall agreement among the eight pathologists was 91.8% (95% CI, 84.0–96.9%), and the multi-rater kappa value was 0.83 (95% CI, 0.67–0.94), which is within the ‘excellent agreement’ range. Agreement was slightly better for determining absence of FEA (92.8%: 95% CI, 84.1–97.4%), than for determining its presence (90.4%: 95% CI, 79.9–96.7%). We conclude that the diagnosis of FEA and its distinction from CCLs without atypia is highly reproducible with the use of available diagnostic criteria.

Evidence-based evaluation of the risks of malignancy predicted by thyroid fine-needle aspiration biopsies

A National Cancer Institute (NCI) “Thyroid Fine‐Needle Aspiration (FNA) State of the Science Conference” recently proposed standardized nomenclature and “risks of malignancies” associated with various diagnostic categories. We evaluated the evidence levels of the data used by NCI to predict malignancy risks and whether those estimates had clinical validity in our patient population.

BD ProEx C : A Sensitive and Specific Marker of HPV-associated Squamous Lesions of the Cervix

BD ProEx C (ProEx C) is a recently developed immunocytochemical assay that targets the expression of topoisomerase II-alpha and minichromosome maintenance protein-2, 2 genes shown to be overexpressed in cervical cancers. Recent studies validated this reagent in liquid-based cytology specimens and suggested its usefulness as an adjunct in the diagnosis of challenging cases. Limited information is available on its expression in tissue sections. This study aims to assess ProEx C expression patterns in benign, atypical, and dysplastic lesions of the cervix and to compare these patterns with p16 and Ki67 expression and with the presence of human papilloma virus DNA as determined by in situ hybridization. ProEx C positivity was limited to the basal layers of the epithelium in 75% of benign cervices. In the remaining 25%, staining extended into the lower half of the epithelium particularly in areas of squamous metaplasia and immature squamous metaplasia. In 92% of high-grade dysplasias [cervical intraepithelial neoplasia (CIN) II and III] strong positive staining for ProEx C involved the lower and upper halves of the epithelium. Condylomas and CIN I showed greater variability in staining pattern with ProEx C positivity extending into the upper half of the epithelium in 48% of cases. Statistically significant correlations were noted with presence of human papilloma virus DNA and with p16/Ki67 expression. Atypical squamous metaplasia showed varied staining with 24% being positive. To summarize, ProEx C is a reliable marker for high-grade CIN that can be applied to tissue sections to confirm the diagnosis of high-grade CIN and to triage cases of atypical squamous metaplasia. ProEx C may also have a potential role in triaging patients with low-grade CIN. ProEx C expression patterns are similar to those for p16 and Ki67 with most discrepancies occurring in the CIN I category.

P16 and Ki67 immunostaining is a useful adjunct in the assessment of biopsies for HPV-associated anal intraepithelial neoplasia

P16 is a tumor suppressor gene product, shown to be overexpressed in most cervical carcinomas and dysplasias associated with high-risk human papilloma virus (HPV) infection. HPV is also associated with anal squamous dysplasias and carcinomas. Significant interobserver and intraobserver variation exists in the interpretation of biopsies for anal intraepithelial neoplasia (AIN). This study was undertaken to assess the potential role of p16 and Ki67 immunohistochemical expression in refining the diagnosis and grading of AIN.One-hundred and four anal biopsies from 74 patients were retrieved from the surgical pathology files of the department. After discrepancies were resolved and concurrence was achieved by at least 2 of 3 reviewing pathologists, the diagnoses were as follows: 37 negative, 12 condylomas without overt dysplasia, 14 AIN I, 25 AIN II, and 16 AIN III. p16 and Ki67 expression was evaluated by ABC immunoperoxidase staining whereas the presence of the high-risk subtypes of HPV virus was determined by in situ hybridization on a subset of the biopsies. Results were reviewed by 2 pathologists and positive and negative staining was correlated with H&E diagnoses. Nuclear and/or nuclear and cytoplasmic staining was considered as positive for p16 when present in >10% of squamous cells. Two patterns of positive p16 staining were observed: (1) “spotty” in which positive cells were scattered throughout the lesion and (2) “band” in which >90% of contiguous cells in the lesion stained positive. A band-like pattern of p16 immunoreactivity was seen in 21.4% AIN I, 80% AIN II, and 87.5% AIN III cases. None of the condylomas and only 1 of the negative cases showed a band of p16 positive staining. Spotty p16 immunoreactivity was observed in 8.1% negative, 8.3% condyloma, 14.3% AIN I, 12.0% AIN II, and 12.5% AIN III cases. More than 50% of nuclei stained positive for Ki67 in 28.6% AIN I, 48.0% AIN II, and 75.0% AIN III cases but in none of the negative or condyloma cases. On the basis of these results, a band-like pattern of p16 staining and Ki67 positivity in >50% of the squamous cell nuclei were strongly associated with high-grade AIN. Conversely, absence of a p16 band of positivity coupled with Ki67 positivity in <50% of nuclei was frequently associated with benign lesions. Band like p16 staining also correlated strongly with the presence of high-risk HPV. Most AIN I lesions stained similar to the nondysplastic cases. A small subset of biopsies studied did not conform to the pattern described above: 4 of 14 (28.6%) AIN I lesions showed a band-like pattern of p16 staining and/or >50% Ki67 positive nuclei. Two of these cases were positive for high-risk HPV DNA. 4 of 25 (16.0%) AIN II lesions comprising 9.8% of the 41 high-grade AINs (AIN II and III) showed spotty p16 positivity and <50% Ki67 positive nuclei. One was positive for high-risk HPV DNA. We conclude that when used together and evaluated in conjunction with H&E stained sections, p16 and Ki67 immunoexpression is a useful adjunct in the diagnosis and grading of AIN.

Epigenetic silencing of the tumor suppressor klotho in human breast cancer

Klotho is a single pass transmembrane protein, associated with premature aging. We identified tumor suppressor activities for klotho, associated with reduced expression in breast cancer. We now aimed to analyze klotho expression in early stages of breast tumorigenesis and elucidate mechanisms leading to klotho silencing in breast tumors. We studied klotho expression, using immunohistochemistry, and found high klotho expression in all normal and mild hyperplasia samples, whereas reduced expression was associated with moderate and atypical ductal hyperplasia. Promoter methylation and histone deacetylation were studied as possible mechanisms for klotho silencing. Using bisulfite sequencing, and methylation-specific PCR, we identified KLOTHO promoter methylation in five breast cancer cell lines and in hyperplastic MCF-12A cells, but not in the non-tumorous mammary cell line HB2. Importantly, methylation status inversely correlated with klotho mRNA levels, and treatment of breast caner cells with 5-aza-2-deoxycytidine elevated klotho expression by up to 150-fold. KLOTHO promoter methylation was detected in 8/23 of breast cancer samples but not in normal breast samples. Chromatin immunoprecipitation revealed that in HB2 KLOTHO promoter was enriched with AcH3K9; however, in breast cancer cells, H3K9 was deacetylated, and treatment with the histone deacetylase inhibitor suberoylanilide bishydroxamide (SAHA) restored H3K9 acetylation. Taken together, these data indicate loss of klotho expression as an early event in breast cancer development, and suggest a role for DNA methylation and histone deacetylation in klotho silencing. Klotho expression and methylation may, therefore, serve as early markers for breast tumorigenesis.

Overexpression of β1-chain-containing laminins in capillary basement membranes of human breast cancer and its metastases

IntroductionLaminins are the major components of vascular and parenchymal basement membranes. We previously documented a switch in the expression of vascular laminins containing the α4 chain from predominantly laminin-9 (α4β2γ1) to predominantly laminin-8 (α4β1γ1) during progression of human brain gliomas to high-grade glioblastoma multiforme. Here, differential expression of laminins was studied in blood vessels and ductal epithelium of the breast.MethodIn the present study the expressions of laminin isoforms α1–α5, β1–β3, γ1, and γ2 were examined during progression of breast cancer. Forty-five clinical samples of breast tissues including normal breast, ductal carcinomas in situ, invasive ductal carcinomas, and their metastases to the brain were compared using Western blot analysis and immunohistochemistry for various chains of laminin, in particular laminin-8 and laminin-9.ResultsLaminin α4 chain was observed in vascular basement membranes of most studied tissues, with the highest expression in metastases. At the same time, the expression of laminin β2 chain (a constituent of laminin-9) was mostly seen in normal breast and carcinomas in situ but not in invasive carcinomas or metastases. In contrast, laminin β1 chain (a constituent of laminin-8) was typically found in vessel walls of carcinomas and their metastases but not in those of normal breast. The expression of laminin-8 increased in a progression-dependent manner. A similar change was observed from laminin-11 (α5β2γ1) to laminin-10 (α5β1γ1) during breast tumor progression. Additionally, laminin-2 (α2β1γ1) appeared in vascular basement membranes of invasive carcinomas and metastases. Chains of laminin-5 (α3β3γ2) were expressed in the ductal epithelium basement membranes of the breast and diminished with tumor progression.ConclusionThese results suggest that laminin-2, laminin-8, and laminin-10 are important components of tumor microvessels and may associate with breast tumor progression. Angiogenic switch from laminin-9 and laminin-11 to laminin-8 and laminin-10 first occurs in carcinomas in situ and becomes more pronounced with progression of carcinomas to the invasive stage. Similar to high-grade brain gliomas, the expression of laminin-8 (and laminin-10) in breast cancer tissue may be a predictive factor for tumor neovascularization and invasion.

Human papillomavirus genotypes in anal intraepithelial neoplasia and anal carcinoma as detected in tissue biopsies.

Human papillomavirus (HPV) infection strongly correlates with the development of anal intraepithelial neoplasias and carcinomas; however, few studies have characterized the distribution of the specific subtypes of the virus in the varying grades of dysplasia. This report characterizes the distribution of HPV 16/18 in surgical specimens with anal intraepithelial neoplasia (AIN) I–III and histological variants of anal carcinoma. A total of 111 anal surgical specimens with no dysplasia (10), AIN I–III (53), and anal carcinomas (48) were evaluated for the presence of high-risk HPV infection and subtyped by nested PCR or the Invader Assay. High-risk virus types were detected in progressively greater number of anal intraepithelial lesions from 56% in low grade to 88% in high grade. Type 16 was the prevalent subtype and was noted in 28% of low grade and 68% of high-grade lesions. Moderate dysplasias showed type 16 in 20%, a prevalence similar to that in low-grade lesions. The non-16/18 subtypes of the virus predominated and were present in 50% of the cases. Most (89%) squamous carcinomas were associated with high-risk viruses, 68% with type 16, a prevalence similar to that noted in high-grade dysplasia. Non-16/18 subtypes were encountered more frequently in squamous carcinomas from immunodeficient individuals (57% cases) as compared with immunocompetent individuals (18% cases). The similarity in the prevalence of type 16 in high-grade dysplasia and squamous carcinomas suggests that anal intraepithelial lesion III is the true precursor of squamous carcinoma and warrants aggressive management. Anal intraepithelial lesions II showed a virus distribution that was similar to low-grade dysplasia. In addition, a subset of these that were associated with type 16 or 18 showed progression, whereas those associated with non-16/18 subtypes regressed, thereby raising the possibility of conservative management for these lesions.

p16, Ki-67, and BD ProEx™C immunostaining: a practical approach for diagnosis of cervical intraepithelial neoplasia

p16, Ki-67, and BD ProExC (TriPath Imaging, Inc., Burlington, NC) have each been shown to be helpful adjuncts in the diagnosis and grading of human papillomavirus-associated cervical intraepithelial neoplasia. However, no clear guidelines are available regarding their use in routine practice. This study was designed to evaluate the efficacy of the 3 stains alone and in combinations to determine the most useful strategy in the diagnosis of cervical intraepithelial neoplasia and provide guidance in instances with discordant staining patterns. Serial sections of 136 formalin-fixed paraffin-embedded cervical samples with consensus diagnoses of 39 benign-reactive, 46 low-grade cervical intraepithelial neoplasia (CIN I/human papillomavirus), and 51 high-grade cervical intraepithelial neoplasia (CINII/III) were immunostained using p16, Ki-67, and BD ProExC antibodies. Results of high-risk human papillomavirus testing were available in 70 cases. Immunostaining patterns were designated as negative, intermediate, and strongly positive based on the patterns observed most commonly in benign-reactive, low-grade cervical intraepithelial neoplasia, and high-grade cervical intraepithelial neoplasia lesions, respectively. A concordant staining pattern with all 3 stains correctly identified benign-reactive, low-grade cervical intraepithelial neoplasia, and high-grade cervical intraepithelial neoplasia cases. p16 was the most sensitive and specific individual stain (sensitivity, 33%; specificity, 93%; positive predictive value [PPV], 81%; negative predictive value [NPV], 58%). Performing all 3 immunostains and using concordant results of any 2 improved diagnostic accuracy (sensitivity, 35%; specificity, 98%; PPV, 93%; NPV, 67%). However, a significant proportion of low-grade cervical intraepithelial neoplasia cases showed aberrant staining patterns with 52% staining negative and 9% staining strongly positive. Low-grade cervical intraepithelial neoplasia cases with the negative staining pattern were more likely to be negative for high-risk human papillomavirus, whereas those showing the strongly positive staining pattern were high-risk human papillomavirus positive. Performing p16 and BD ProExC initially followed by Ki-67 only when p16 and BD ProExC yielded discordant results provided similar diagnostic accuracy at reduced cost because only one third of the cases required the additional stain.