HPV RNA in situ hybridization can inform cervical cytology-histology correlation.

BACKGROUND: In situ hybridization for human papillomavirus (HPV) messenger RNA (HPV RNA ISH) recently was introduced as an ancillary tool in the diagnosis of cervical squamous intraepithelial lesions, and can aid in the distinction between low-grade squamous intraepithelial lesions (LSILs) versus reactive/negative biopsies. Prior work has shown that up to one-half of cases originally diagnosed as LSIL are reclassified as negative/reactive by expert consensus review of morphology, and negative HPV RNA ISH results most often correlate with an expert diagnosis of negative/reactive. Given that LSIL overdiagnoses on biopsy may result in the erroneous clinical impression that a cervical lesion has been sampled appropriately, the authors proposed that HPV RNA ISH can inform cytology-histology correlation for challenging LSIL biopsies. METHODS: A total of 92 cervical biopsies originally diagnosed as LSIL were reviewed by 3 gynecologic pathologists and reclassified based on consensus opinion of morphology. ISH was performed for high-risk and low-risk HPV E6/E7 mRNA. Prior/concurrent cytology results were collected. RESULTS: Based on expert consensus morphologic review, 49% of biopsies (45 of 92 biopsies) originally diagnosed as LSIL were reclassified as negative, 6.5% (6 of 92 biopsies) were reclassified as high-grade squamous intraepithelial lesion, and 44.5% (41 of 92 biopsies) were maintained as LSIL. The majority of LSIL biopsies reclassified as negative (80%; 36 of 45 biopsies) were HPV RNA negative, whereas 93% of LSIL biopsies (39 of 41 biopsies) and 100% of high-grade squamous intraepithelial lesion biopsies were HPV RNA positive. CONCLUSIONS: LSIL often is overdiagnosed by morphology on biopsy, potentially leading to the false impression that a lesion identified on cytology has been sampled. Performing RNA ISH on biopsies decreases histologic LSIL overdiagnosis, and potentially can prompt further sampling when there is cytology-histology discordance. Cancer (Cancer Cytopathol) 2018. © 2018 American Cancer Society.

HPV E6/E7 mRNA In Situ Hybridization in the Diagnosis of Cervical Low-grade Squamous Intraepithelial Lesions (LSIL).

Cervical low-grade squamous intraepithelial lesions (LSIL) (aka cervical intraepithelial neoplasia, grade 1 [CIN1]) can present considerable diagnostic challenges and are associated with poor interobserver reproducibility and overdiagnosis. Furthermore, ancillary studies such as p16 immunohistochemistry have shown little utility in resolving the LSIL versus negative/reactive differential. Human papillomavirus (HPV) RNA in situ hybridization (ISH) has shown promise as a diagnostic aid in this setting, but has not been studied in a large case series. We herein investigate high-risk and low-risk HPV RNA ISH in 126 cervical biopsies originally diagnosed as LSIL/CIN1 and compare HPV RNA ISH results to expert-adjudicated morphologic diagnosis to assess whether this assay can help routine cases attain the existing "gold standard" of morphologic consensus diagnosis. We also assess whether this criterion standard can be further improved by integration of HPV RNA ISH results. A consensus diagnosis of intraepithelial lesion (CIN1) was confirmed in 61% of cases, whereas 57% were HPV RNA. HPV-RNA positivity was 84% sensitive and 86% specific for an expert-adjudicated diagnosis of CIN1. Conversely, consensus diagnosis was 90% sensitive and 78% specific for the presence of HPV RNA. Integrating RNA ISH into morphologic review led to further reclassification of 10% of cases, resulting in 95% sensitivity and 98% specificity of HPV RNA ISH for a CIN1 diagnosis and 98% sensitivity and 92% specificity of CIN1 for the presence of HPV RNA. These findings suggest that judicious use of HPV RNA ISH can improve the accuracy of LSIL/CIN1 diagnosis for morphologically ambiguous cases.

Mismatch repair status and PD-L1 expression in clear cell carcinomas of the ovary and endometrium.

Clear cell carcinoma represents a distinct histologic type of müllerian carcinoma that is resistant to conventional chemotherapy. Expression of programmed cell death ligand (PD-L1) has been associated with immune evasion in numerous tumor types and may be used to identify patients who will benefit from targeted immunotherapy, particularly in the setting of mismatch repair defects. We evaluated PD-L1 expression in 23 ovarian clear cell carcinomas and 21 endometrial clear cell carcinomas, and correlated expression with mismatch repair status. Tumor PD-L1 staining was seen in 43% of ovarian tumors and 76% of endometrial tumors, including 71% of cases (67% of ovarian and 75% of endometrial) with mismatch repair defects. Extensive tumoral staining (>50%) was seen in only one case (an endometrial case with MSH6 loss). However, tumoral PD-L1 expression remained common in mismatch repair-intact tumors and mismatch repair status was not significantly correlated with PD-L1 expression. The increased incidence of PD-L1 positivity in tumor cells (P=0.04) in endometrial vs ovarian clear cell carcinomas suggests differences in the tumor microenvironment of these histologically and molecularly similar tumors that may inform treatment options. These results suggest that clear cell histology may be a useful susceptibility marker for immunotherapy targeting the PD-1/PD-L1 axis irrespective of mismatch repair status, particularly in endometrial carcinomas.

PD-L1 Expression in Mismatch Repair-deficient Endometrial Carcinomas, Including Lynch Syndrome-associated and MLH1 Promoter Hypermethylated Tumors.

Mismatch repair (MMR)-deficient endometrial carcinomas (ECs) bearing Lynch syndrome (LS)-associated germline mutations or sporadic MLH1 promoter hypermethylation (MLH1hm) are highly immunogenic and may represent excellent candidates for therapies targeting the programmed cell death (PD)/programmed cell death ligand-1 (PD-L1) immune checkpoint pathway. This study evaluates PD-L1 expression in MMR-deficient ECs including LS-associated and MLH1hm cases, in comparison with MMR-intact tumors. Immunohistochemistry for PD-L1/CD274 was performed on 38 MMR-deficient and 29 MMR-intact ECs. Staining was scored in the tumor and the peritumoral immune compartment. The majority of MMR-deficient tumors were PD-L1 positive (53%) in at least a subset of tumor cells. LS-associated tumors were more likely to be PD-L1 positive relative to MLH1hm tumors (70% vs. 33%, P=0.05). Only 10% of MMR-intact ECs demonstrated any tumoral PD-L1 expression; this was significantly lower than was observed in MMR-deficient tumors (P=0.0005). When reviewed by histologic grade, PD-L1 expression remained highest in LS-associated ECs followed by MLH1hm and MMR-intact carcinomas, respectively. The MMR immunohistochemical pattern most uniformly associated with PD-L1 expression was MSH6 loss. Immune PD-L1 expression was seen in 100% of MMR-deficient and 66% of MMR-intact cases. This study represents the first to characterize differences in PD-L1 expression between LS-associated and MLH1hm endometrial cancers. It demonstrates that tumoral PD-L1 expression is more common in LS-associated endometrial cancers relative to MLH1hm and MMR-intact tumors, although sporadic cancers often show PD-L1 positive immune staining. These data suggest that MMR deficiency may be a better predictor of response to PD-1/PD-L1 inhibitor therapy than tumor grade in EC, and that potential benefit may vary based on the molecular mechanism of MMR defects.

Parafibromin, APC, and MIB-1 Are Useful Markers for Distinguishing Parathyroid Carcinomas From Adenomas.

BACKGROUND: Differentiation of parathyroid carcinoma (PC) from parathyroid adenoma (PA) relies solely on the pathologic determination of invasion of surrounding structures and/or distant metastasis. Parathyroid lesions with atypical histologic features with no demonstration of invasion or metastasis present a diagnostic dilemma. Different authors report a parafibromin and adenomatous polyposis coli (APC) loss or reduction in PC cases. High proliferative activity of MIB-1 and increased galectin 3 expression are reported in PC. There is no clear cutoff for the sensitivity, specificity, or predictive value for all these markers. METHODS: The immunohistochemical expression of parafibromin, APC, MIB-1, and galectin 3 was studied in 73 adenomas, 21 PCs, and 3 atypical adenomas. The presence or absence of each marker was identified through the use of a comprehensive scoring system based on multiplying the percentage of tumor cells stained (0 to 100) and the staining intensity (0 to 3) on each biopsy. The highest score that any slide could reach was 300. A cutoff of >100 was used to consider the specimen positive for parafibromin, APC, or galectin 3 staining. MIB-1 proliferation indices were calculated using image cytometry; proliferation indices >5% were considered positive. RESULTS: We identified parafibromin loss in 7/21 (33%) carcinomas and 1/73 (1%) adenomas. Loss of APC was seen in 20/21 (95%) carcinomas and 38/73 (52%) adenomas. MIB-1 indices were elevated in 18/21 (86%) carcinomas. MIB-1 indices were <5% in all (100%) adenomas. MIB-1 indices were elevated in 2/3 (67%) atypical adenomas. CONCLUSIONS: Our study presents a clear cutoff to determine the practicality of using parafibromin, APC, and MIB-1 as immunohistochemical markers to differentiate between PCs and PAs. Loss of parafibromin and a high MIB-1 index are both independently sensitive and specific markers for the diagnosis of PC. Loss of APC was only specific for PC. This panel of markers provides a novel, useful approach in the diagnosis and differentiation of PCs from PAs.

Two Case Reports of Nodular Fasciitis: A Newly Recognized Placental Lesion.

We describe two occurrences of nontrophoblastic mesenchymal tumors of the placenta. The first placental tumor was found along the placental margin, and the second was identified close to the insertion of the fetal membranes along the placental disc. Microscopically both lesions demonstrated bland fibroblastic cells with intricate vasculature and inflammatory cells. Both lesions were negative for estrogen receptor (ER), progesterone receptor (PR), beta-HCG, PLAP, CD34, desmin, h-caldesmin, and smooth muscle actin by immunohistochemistry. Some cells were weakly positive for CD10, a nonspecific finding. The morphologic and immunohistochemical characteristics of these lesions were most consistent with nodular fasciitis, a tumor most commonly found in the soft tissues. FISH positive for USP6 gene rearrangement in our two patients confirmed the molecular similarity of these lesions to nodular fasciitis of soft tissue. Such lesions can be easily dismissed on gross placental examination as infarcts or thrombi, thus these rare entities are likely underreported.

SOX10: a useful marker for identifying metastatic melanoma in sentinel lymph nodes.

SOX10 (Sry-related HMg-Box gene 10) is a key nuclear transcription factor in the differentiation of neural crest progenitor cells to melanocytes. It has been shown to be a sensitive and specific marker of malignant melanoma of multiple histologic types. We evaluated the immunohistochemical profile of SOX10 in 107 sentinel lymph nodes and compared it to S100 protein, HMB-45, and Melan-A. Seventy-seven lymph nodes originally reported as positive for metastatic melanoma, and 30 reported as negative were reviewed. SOX10 identified metastatic melanoma in 58 of 58 (100%) positive cases included in the final analysis. SOX10 staining showed a statistically significant increase in staining intensity when compared with S100 protein, HMB-45, and Melan-A (P = 0.000, 0.000, and 0.003, respectively). In addition, there was a statistically significant increase in percentage of tumor cells stained by SOX10, compared with S100 protein, HMB-45, and Melan-A (P = 0.015, 0.000, and 0.001, respectively). SOX10 stained nodal nevi in a similar manner to S100 protein and Melan-A in 4 cases. Interpretation of nodal nevus staining (negative by HMB-45) was significantly aided by the crisp nuclear staining produced by SOX10 as compared with the obscuring cytoplasmic staining produced by S100 protein and Melan-A in 3 cases. Identification of micrometastases was facilitated by the nuclear staining pattern of SOX10 and lack of background dendritic cell staining seen in S100 protein. We conclude that given the sensitivity and specificity of SOX10 for detecting metastatic melanoma in sentinel lymph nodes, it is a reliable marker for supplementing and potentially replacing traditionally utilized immunohistochemical stains.