CAP TODAY Pathology/Laboratory Medicine/Laboratory Management
Editors: Rouzan Karabakhtsian, MD, PhD, professor of pathology and director of the Women’s Health Pathology Fellowship, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, NY; Rachel Stewart, DO, PhD, molecular genetic pathology fellow, University of Utah/ARUP Laboratories, Salt Lake City; Nicole Panarelli, MD, associate professor of pathology, Albert Einstein College of Medicine, Montefiore Medical Center; and Shaomin Hu, MD, PhD, pathology resident, Albert Einstein College of Medicine, Montefiore Medical Center.
Analysis of ZC3H7B-BCOR high-grade endometrial stromal sarcomas
October 2018—High-grade endometrial stromal sarcoma likely encompasses underrecognized tumors harboring genetic abnormalities besides YWHAE–NUTM2 fusion. Triggered by three initial endometrial stromal sarcomas with ZC3H7B–BCOR fusion characterized by high-grade morphology and aggressive clinical behavior, the authors investigated the clinicopathologic features of this genetic subset by expanding the analysis to 17 such tumors. All of the tumors occurred in women who were a median age of 54 (range, 28–71) years. The tumors were predominantly based in the endomyometrium and demonstrated tongue-like or pushing myometrial invasion, or both. Most were uniformly cellular and displayed haphazard fascicles of spindle cells with mild to moderate nuclear atypia. Myxoid matrix was seen in 14 of 17 (82 percent) tumors, and collagen plaques were seen in eight (47 percent). The mitotic index was 10 or more mitotic figures per 10 high-power fields (HPFs) in 14 of 17 (82 percent) tumors, with a median of 14.5 mitotic figures per 10 HPFs. No foci of conventional or variant low-grade endometrial stromal sarcoma were seen. All tumors expressed CD10 with only limited or absent desmin, SMA, or h-caldesmon staining. Estrogen receptor and progesterone receptor expression in more than five percent of cells was seen in four of 12 (33 percent) tumors. Diffuse cyclin D1 and BCOR immunoreactivity were present in seven of eight (88 percent) and seven of 14 (50 percent) tumors, respectively. FISH or targeted RNA sequencing confirmed ZC3H7B–BCOR fusion in all tumors, including four and two previously diagnosed as myxoid leiomyosarcoma and undifferentiated uterine sarcoma, respectively. Limited clinical data suggest that patients present at higher stage and have worse prognosis compared with published outcomes in low-grade endometrial stromal sarcoma. Tumors with ZC3H7B–BCOR fusion constitute a distinct group of endometrial stromal sarcomas with high-grade morphology that should be distinguished from other uterine mesenchymal neoplasms that may demonstrate myxoid morphology.
Lewis N, Soslow RA, Delair DF, et al. ZC3H7B-BCOR high-grade endometrial stromal sarcomas: a report of 17 cases of a newly defined entity. Mod Pathol. 2018;31:674–684.
Correspondence: Dr. Sarah Chiang at chiangs@mskcc.org
Single-cell heterogeneity in ductal carcinoma in situ of the breast
Heterogeneous patterns of mutations and RNA expression have been well documented in invasive cancers. However, technological challenges have limited the ability to study the heterogeneity of protein expression. This is particularly true for pre-invasive lesions such as ductal carcinoma in situ of the breast. The authors conducted a study in which cell-level heterogeneity in ductal carcinoma in situ was analyzed in a single 5-μm tissue section using a multiplexed immunofluorescence analysis of the disease-related markers EGFR, HER2, HER4, S6, pmTOR, CD44v6, SLC7A5, and CD10, CD4, CD8, and CD20, plus pan-cytokeratin, pan-cadherin, DAPI, and Na+K+ATPase for cell segmentation. Expression was quantified at the cell level using a single-cell segmentation algorithm. K-means clustering was used to determine co-expression patterns of epithelial cell markers and immune markers. The authors documented the presence of epithelial cell heterogeneity within ducts, between ducts, and between patients with ductal carcinoma in situ. They found moderate heterogeneity in a distribution of eight clusters within each duct (average Shannon index, 0.76; range, 0–1.61). Within each patient, the average Shannon index across all ducts ranged from 0.33 to 1.02 (standard deviation, 0.09–0.38). Because the distribution of clusters within ducts was uneven, the analysis of eight ducts might be sufficient to represent all the clusters—that is, within- and between-duct heterogeneity. The pattern of epithelial cell clustering was associated with the presence and type of immune infiltrates, indicating a complex interaction between the epithelial tumor and immune system for each patient. This analysis provides evidence that simultaneous analysis of both the epithelial and immune/stromal components might be necessary to understand the complex milieu in ductal carcinoma in situ lesions.
Gerdes MJ, Gökmen-Polar Y, Sui Y, et al. Single-cell heterogeneity in ductal carcinoma in situ of breast. Mod Pathol. 2018;31:406–417.
Correspondence: Dr. M. J. Gerdes at gerdes@research.ge.com or S. S. Badve at sbadve@iupui.edu
Ultrasensitive automated RNA ISH for detecting B-cell clonality in tissue biopsies
Assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin-fixed, paraffin-embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluating plasma cells but are often insufficiently sensitive to detect the much lower abundance of light chains present in B cells. The authors described an ultrasensitive RNA in situ hybridization assay that has been adapted for use on an automated immunohistochemistry platform and compared the results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, RNA in situ hybridization identified light chain-restricted B cells in 85 (42 percent) of the 203 tissue biopsies versus 58 (29 percent) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified restricted B cells in 74 (89 percent) versus 56 (67 percent) by flow cytometry. B-cell clonality could be evaluated in only 23 (22 percent) of 104 bone marrow cases due to poor RNA preservation, but evaluable cases showed 91 percent concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry and highlighted unexpected findings, such as the co-expression of kappa and lambda RNA in two cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average κ, 0.92), and an automated image-analysis system showed high concordance (97 percent) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on common immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphological features in formalin-fixed, paraffin-embedded tissues with a clinical sensitivity similar or superior to flow cytometry.
Guo L, Wang Z, Anderson CM, et al. Ultrasensitive automated RNA in situ hybridization for kappa and lambda light chain mRNA detects B-cell clonality in tissue biopsies with performance comparable or superior to flow cytometry. Mod Pathol. 2018;31:385–394.
Correspondence: Dr. J. R. Cook at cookj2@ccf.org
Digital image analysis of Ki67 in hot spots: comparison to manual Ki67 and mitotic counts in breast cancer
During pathological examination of breast tumors, proliferative activity is routinely evaluated by a count of mitoses. Adding immunohistochemical stains of Ki67 provides extra prognostic and predictive information. However, the methods for these evaluations suffer from imperfect reproducibility. It is unclear whether Ki67 analysis should be performed in hot spots or the tumor periphery or as an average of the whole tumor section. The authors conducted a study to compare the clinical relevance of mitoses, Ki67, and phosphohistone H3 in two cohorts of primary breast cancer specimens (total n = 294). They evaluated manual and digital image-analysis scores for sensitivity and specificity for luminal B versus A subtype as defined by PAM50 gene-expression assays, high versus low transcriptomic grade, axillary lymph node status, and prognostic value in terms of predicting overall and relapse-free survival. Digital image analysis of Ki67 outperformed the other markers, especially in hot spots. Tumors with high Ki67 expression and high numbers of phosphohistone H3-positive cells had significantly increased hazard ratios for all-cause mortality within 10 years of diagnosis. Replacing manual mitotic counts with digital image analysis of Ki67 in hot spots increased the differences in overall survival between the highest and lowest histological grades and added significant prognostic information. The authors concluded that digital image analysis of Ki67 in hot spots is the marker of choice for routine analysis of proliferation in breast cancer.
Stålhammar G, Robertson S, Wedlund L, et al. Digital image analysis of Ki67 in hot spots is superior to both manual Ki67 and mitotic counts in breast cancer. Histopathol. 2018;72(6):974–989.