Validation of p16 immunocytochemical staining on alcohol-fixed aspirate smears: A single-institutional study with review of the literature.

BACKGROUND: Determining human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma can have a significant impact on treatment and clinical outcomes. Because fine-needle aspiration (FNA) is typically an initial diagnostic modality in a patient workup for primary or suspected metastatic disease, immunostaining for p16 on FNA material is a promising option to determine HPV tumor status, possibly avoiding biopsies or excisions. In this study, the authors investigated the possibility of using alcohol-fixed smears as a reliable alternative for reporting p16 status. METHODS: Twenty HPV-associated tumors and 20 non-HPV-associated tumors were identified using the gold-standard histologic cutoff for positivity of ≥70% strong nuclear and cytoplasmic staining. Matched FNA specimens were identified for comparison staining, and a positive p16 result was rendered on a single aspirate smear using the same cutoff of ≥70%. RESULTS: On alcohol-fixed cytology smears, 16 of 20 (80%) HPV-associated tumors showed positive p16 staining in ≥70% tumor cells. Four cases showed lower level (30%-60%) nuclear and cytoplasmic staining. Nineteen of 20 (95%) non-HPV-associated tumors showed no or minimal p16 staining (0%-10%), and 1 case had a p16-equivocal cytology result. CONCLUSIONS: The authors performed immunocytochemical validation for p16 using alcohol-fixed smears and observed promising results, offering this technique as a potential alternative to formalin-fixed tissue in the appropriate clinical context. By using a positive staining cutoff of ≥70%, this technique offers 80% sensitivity and 95% specificity for detecting HPV-associated tumors. Although it was not performed in the current study, HPV-specific testing on available formalin-fixed, paraffin-embedded tissue should be considered in cases with equivocal or negative p16 staining.

NKX2.2, PDX-1 and CDX-2 as potential biomarkers to differentiate well-differentiated neuroendocrine tumors.

BACKGROUND: Well-differentiated neuroendocrine tumors (NET) most frequently arise from the gastrointestinal tract (GI), pancreas, and lung. Patients often present as metastasis with an unknown primary, and the clinical management and outcome depend on multiple factors, including the accurate diagnosis with the tumor primary site. Determining the site of the NET with unknown primary remains challenging. Many biomarkers have been investigated in primary NETs and metastatic NETs, with heterogeneous sensitivity and specificity observed. METHODS: We used high-throughput tissue microarray (TMA) and immunohistochemistry (IHC) with antibodies against a panel of transcriptional factors including NKX2.2, PDX-1, PTF1A, and CDX-2 on archived formalin-fixed paraffin-embedded NETs, and investigated the protein expression pattern of these transcription factors in 109 primary GI (N = 81), pancreatic (N = 17), and lung (N = 11) NETs. RESULTS: Differential expression pattern of these markers was observed. In the GI and pancreatic NETs (N = 98), NKX2.2, PDX-1, and CDX-2 were immunoreactive in 82 (84%), 14 (14%), and 52 (52%) cases, respectively. PDX-1 was expressed mainly in the small intestinal and appendiceal NETs, occasionally in the pancreatic NETs, and not in the colorectal NETs. All three biomarkers including NKX2.2, PDX-1, and CDX-2 were completely negative in lung NETs. PTF1A was expressed in all normal and neuroendocrine tumor cells. CONCLUSIONS: Our findings suggest that NKX2.2 was a sensitive and specific biomarker for the GI and pancreatic neuroendocrine tumors. We proposed that a panel of immunostains including NKX2.2, PDX-1, and CDX-2 may show diagnostic utility for the most common NETs.

Significance of positive and inhibitory regulators in the TGF-ß signaling pathway in colorectal cancers.

Inactivation of genes in the transforming growth factor (TGF)-ß/SMAD signaling pathway is a well-known step for the progression of colorectal cancers (CRCs). Genetic mutations can occur in the precursors, and the combined prevalence of SMAD4, SMAD2, and SMAD3 mutations was seen in up to 50% of CRCs. High levels of serum TGF-ß1 were reported in patients with CRC and were associated with poor clinical outcome. PPM1A is an important inhibitory regulator in the TGF-ß signaling pathway and contributes to terminating the TGF-ß/SMAD signaling activity. We recently showed that PPM1A expression was lost in approximately 45% of pancreatic ductal adenocarcinomas and loss of PPM1A was associated with worse overall survival. Genome-wide analyses from The Cancer Genome Atlas revealed that abnormal TGF-ß signaling pathway is among the most common molecular changes in CRC. The complexity of the TGF-ß signaling pathway is its dual function as a tumor suppressor and tumor-promoting factor, depending on the cellular and molecular context. In this study, we simultaneously investigated the protein expression pattern of 3 regulators in the TGF-ß/SMAD signaling pathway, including SMAD4, PPM1A, and TGF-ß1, and their clinicopathological correlations in CRCs by immunohistochemistry. We observed that loss of SMAD4 and PPM1A was seen in 37.8% and 7.3% of CRCs, respectively. Loss of SMAD4, lymphovascular invasion, and distant metastasis were independently associated with worse overall survival in multivariate analysis. However, loss of PPM1A was associated with worse overall survival with less statistical strength. Our findings would provide new insights into the pathophysiological function of different components in the TGF-ß signaling pathway in CRC.

Immunohistochemical analysis of ER, PR, Her2 and CK5/6 in infiltrative breast carcinomas in Indian patients.

AIM: Breast cancer is biologically a heterogeneous disease. Patients with the same diagnostic profile have markedly different clinical outcomes. Gene expression studies identified distinct breast cancer subtypes that differ in prognosis. Aim is to identify the immunohistochemical subtypes of breast carcinoma and correlate the results with pathological features associated with adverse prognosis in our study population. METHOD: We included 107 consecutive cases of invasive breast carcinoma and sub classified using immunohistochemical staining for ER, PR, Her2, and CK5/6 into the following subtypes: luminal A, luminal B, basal-like, Her2(+) and unclassified. Associations between tumor subtypes and tumor characteristics were examined. RESULTS: The proportion of each subtype in our patient population was: luminal A 37.4%, luminal B 11.1%, Her2(+) 29% and basal-like 7.5%. The following variables were significantly associated with IHC breast cancer subtypes: patient age (p<0.05), overall histopathology grade (p<0.001), nuclear grade (p<0.005) and mitotic index (p<0.001). Her2(+) and basal like subtypes were associated with poor differentiation (p<0.01), higher nuclear grade (p<0.05) and high mitotic index (p<0.05). CONCLUSIONS: Our data show a higher proportion of patients in the study population undergo total mastectomy and harbor poorly differentiated, node positive tumors than reported. There was also a relatively high percentage of the Her2(+) subtype (29%).

The binding of thyroid transcription factor-1 and hepatocyte paraffin 1 to mitochondrial proteins in hepatocytes: a molecular and immunoelectron microscopic study.

We studied the antibodies hepatocyte paraffin 1 (Hep Par 1) and thyroid transcription factor-1 (TTF-1; clone 8G7G3/1) in normal human liver tissue with immunoelectron microscopy using renal tubules as control samples. TTF-1 (8G7G3/1) and Hep Par 1 bound exclusively to the mitochondria of hepatocytes but not to those of the renal tubular epithelium. Both antibodies labeled mitochondria in a similar pattern and density. These findings confirm that the binding site of Hep Par 1 in hepatocytes is mitochondrial. The specific binding of TTF-1 (8G7G3/1) in hepatocyte mitochondria suggests its potential usefulness for identifying hepatocellular carcinoma. Western blot analysis with cellular proteins extracted from normal human liver and thyroid tissue demonstrated that Hep Par 1 and TTF-1 (8G7G3/1) bound to a protein band of approximately 150 kd in liver cells, with TTF-1 (8G7G3/1) showing less affinity. It is likely that different epitopes to Hep Par 1 and TTF-1 (8G7G3/1) share the same protein molecule in hepatocyte mitochondria.