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Squamous cell carcinoma (SqCC) is the most common malignancy of the anal canal, where it is strongly associated with HPV infection. Characteristic genomic alterations have been identified in anal SqCC, but their clinical significance and correlation with HPV status, pathologic features, and immunohistochemical markers are not well established. We examined the molecular and clinicopathologic features of 96 HPV-positive and 20 HPV-negative anal SqCC. HPV types included 89 with HPV16, 2 combined HPV16/HPV18, and 5 HPV33. HPV-positive cases demonstrated frequent mutations or amplifications in PIK3CA (30%; p = 0.027) or FBXW7 mutations (10%). HPV-negativity was associated with frequent TP53 (53%; p = 0.00001) and CDKN2A (21%; p = 0.0045) mutations. P16 immunohistochemistry was positive in all HPV-positive cases and 3/20 HPV-negative cases (p < 0.0001; sensitivity: 100%; specificity: 85%) and was associated with basaloid morphology (p = 0.0031). Aberrant p53 immunohistochemical staining was 100% sensitive and specific for TP53 mutation (p < 0.0001). By the Kaplan-Meier method, HPV-negativity, aberrant p53 staining, and TP53 mutation were associated with inferior overall survival (OS) (p < 0.0001, p = 0.0103, p = 0.0103, respectively) and inferior recurrence-free survival (p = 0.133, p = 0.0064, and p = 0.0064, respectively). TP53/p53 status stratified survival probability by HPV status (p = 0.013), with HPV-negative/aberrant p53 staining associated with the worst OS, HPV-positive/wild-type p53 with best OS, and HPV-positive/aberrant p53 or HPV-negative/wild-type p53 with intermediate OS. On multivariate analysis HPV status (p = 0.0063), patient age (p = 0.0054), T stage (p = 0.039), and lymph node involvement (p = 0.044) were independently associated with OS. PD-L1 expression (CPS ≥ 1) was seen in 30% of HPV-positive and 40% of HPV-negative cases, and PD-L1 positivity was associated with a trend toward inferior OS within the HPV-negative group (p = 0.064). Our findings suggest that anal SqCC can be subclassified into clinically, pathologically, and molecularly distinct groups based on HPV and TP53 mutation status, and p16 and p53 immunohistochemistry represent a clinically useful method of predicting these prognostic groups.

DNA methylation is the most well-studied epigenetic modification in cancer biology. 5-hydroxymethylcytosine is an epigenetic mark that can be converted from 5-methylcytosine by the ten-eleven translocation gene family. We recently reported the loss of 5-hydroxymethylcytosine in melanoma compared with benign nevi and suggested that loss of this epigenetic marker is correlated with tumor virulence based on its association with a worse prognosis. In this study, we further characterize the immunoreactivity patterns of 5-hydroxymethylcytosine in the full spectrum of melanocytic lesions to further validate the potential practical application of this epigenetic marker. One hundred and seventy-five cases were evaluated: 18 benign nevi, 20 dysplastic nevi (10 low-grade and 10 high-grade lesions), 10 atypical Spitz nevi, 20 borderline tumors, 5 melanomas arising within nevi, and 102 primary melanomas. Progressive loss of 5-hydroxymethylcytosine from benign dermal nevi to high-grade dysplastic nevi to borderline melanocytic neoplasms to melanoma was observed. In addition, an analysis of the relationship of nuclear diameter with 5-hydroxymethylcytosine staining intensity within lesional cells revealed a significant correlation between larger nuclear diameter and decreased levels of 5-hydroxymethylcytosine. Furthermore, borderline lesions uniquely exhibited a diverse spectrum of staining of each individual case. This study further substantiates the association of 5-hydroxymethylcytosine loss with dysplastic cytomorphologic features and tumor progression and supports the classification of borderline lesions as a biologically distinct category of melanocytic lesions.

The diagnosis of gastric epithelial dysplasia, a precursor lesion of gastric adenocarcinoma, is hindered by interobserver variability and by its resemblance to regenerative changes. Loss of cell polarity, a histological feature of gastric epithelial dysplasia, may be difficult to ascertain, especially in the setting of inflammation or injury. A biomarker of cell polarity could be useful in diagnosis of dysplasia, but has not been reported. The Lethal giant larvae (lgl) gene controls apical-basal polarity of epithelial cells in Drosophila, and has properties of a tumor-suppressor gene. Two homologs, lgl1 and lgl2, are present in mammals and lgl2 mRNA is highly expressed in the stomach. The goal of our study was to test the hypothesis that Lgl2 protein expression and/or localization are disrupted in gastric epithelial dysplasia and adenocarcinoma. Routinely processed pathology specimens including 94 benign mucosae of digestive organs, in addition to 36 reactive gastropathy, 57 gastric epithelial dysplasia, and 77 gastric adenocarcinomas, were immunostained for Lgl2 protein. All normal, reactive, and chronically inflamed gastric epithelia showed basolateral Lgl2 staining. Normal esophageal, duodenal, colonic, biliary, and pancreatic duct mucosae, as well as gastric intestinal metaplasia, did not express Lgl2. All but one case each of gastric epithelial dysplasia and adenocarcinoma showed either complete loss of anti-Lgl2 immunoreactivity or diffuse, mostly weak, cytoplasmic staining. Complete loss of immunoreactivity was significantly more often observed in diffuse-type than in intestinal-type adenocarcinomas (79 vs 48%, respectively). Our data suggest that Lgl2 expression is either aberrantly localized or lost in gastric epithelial dysplasia and adenocarcinoma, whereas it is maintained in reactive gastric mucosa. We propose that Lgl2 may be a potential marker to rule out gastric epithelial dysplasia and adenocarcinoma in diagnostic specimens. However, the consistently negative anti-Lgl2 immunoreactivity seen in intestinal metaplasia does not allow differentiation of dysplasia from intestinal metaplasia with reactive change.

The subtype of Muir-Torre syndrome, allelic to hereditary nonpolyposis colorectal cancer is typically associated with germline mutations in the mismatch repair proteins MSH-2 and/or MLH-1. More recently, mutation in an additional mismatch repair protein MSH-6 has been documented in a patient with Muir-Torre syndrome. Given this, the aim of the present study was to ascertain the frequency of the same in unselected sebaceous gland neoplasms. Overall, we found that 59% of sebaceous neoplasms exhibited a mutation in at least one mismatch repair protein gene -- a prevalence rate similar to that reported previously by others. Of interest, we found MSH-6 to be the mismatch repair protein most commonly lost 17/41 (41%), followed by MSH-2 14/41 (34%) and MLH-18/41 (20%) and the positive predictive value of each were as follows: MLH-1 88%, MSH-6 67% and MSH-2 55%. The frequency of a MSH-6 germline mutation in our cohort indicates that it is not a rare finding. Evidence indicating microsatellite stability in three of 17 patients with a clinical history indicative of Muir-Torre syndrome and a mutation in only MSH-6 suggests that the phenotype of a germline MSH-6 mutation differs from that of MLH-1 and MSH-2 mutations and further supports the use of immunohistochemistry as a screening tool in patients with Muir-Torre syndrome with an extended panel that includes MSH-6.

Even though immunohistochemical comparisons of microcystic adnexal carcinoma vs infiltrative basal cell carcinoma and desmoplastic trichoepithelioma exist, they are mostly restricted to the use of a single stain. In addition, a comparison with squamous cell carcinoma has not been reported previously. In this study, we compare the expression of cytokeratin (CK) 15, CK7, CK20, CK903, carcinoembryonic antigen (CEA), CD10, CD15 and BerEP4 in 13 microcystic adnexal carcinoma, eight desmoplastic trichoepithelioma, 10 infiltrative basal cell carcinoma, and eight squamous cell carcinoma of which five exhibited ductal differentiation. We found that the majority of microcystic adnexal carcinoma (92%) and desmoplastic trichoepithelioma (100%) cases expressed CK15 while the infiltrative basal cell carcinoma and squamous cell carcinoma cases were all negative. Forty percent of infiltrative basal cell carcinoma expressed CK7; while only two microcystic adnexal carcinoma cases (15%) and one squamous cell carcinoma with ductal differentiation (12%) expressed CK7 in the remaining three tumor categories. None of the desmoplastic trichoepithelioma expressed CK7. All tumors were strongly positive for CK903. While the neoplastic cells were negative, luminal staining of ductal structures was noted for CK7, CD15 and CEA in some of the microcystic adnexal carcinoma, desmoplastic trichoepithelioma and squamous cell carcinoma with ductal differentiation cases. Sixty percent of infiltrative basal cell carcinoma, 31% of microcystic adnexal carcinoma, and 25% of squamous cell carcinoma express CD10. BerEP4 expression was noted in 38% of microcystic adnexal carcinoma, 57% of desmoplastic trichoepithelioma, 100% of infiltrative basal cell carcinoma, and 38% of squamous cell carcinoma. In conclusion, we found CK15 to be a useful marker in distinguishing microcystic adnexal carcinoma from infiltrative basal cell carcinoma and squamous cell carcinoma with ductal differentiation. Our experience indicates that microcystic adnexal carcinoma and desmoplastic trichoepithelioma have a similar immunohistochemical profile that is, CK15+ and BerEP4+/-; thus, additional studies are needed to separate these two entities.

More recent techniques to characterize the genetic profile of soft-tissue tumors include the use of gene arrays. Using this technique, Apolipoprotein D (Apo D), a 33-kDa glycoprotein component of high-density lipoprotein, has been found to be highly expressed in dermatofibrosarcoma protuberans. To corroborate these results, we sought to ascertain the utility of Apo D by investigating its sensitivity and specificity in a variety of CD34-positive and CD34-negative cutaneous neoplasms including superficial acral fibromyxoma, sclerotic fibromas, and cellular dermatofibromas. Of interest, we found absence of Apo D expression in all four cases of superficial acral fibromyxoma. Of the remaining CD34-positive lesions, Apo D expression was noted in 35/36 (97%) cases of dermatofibrosarcoma protuberans, 3/5 (60%) giant-cell fibroblastomas, 4/4 (100%) sclerotic fibromas, 8/8 (100%) neurofibromas, and 1/1 (100%) solitary fibrous tumor. Of the CD34-negative lesions, Apo D expression was noted in 2/22 (9%) regular dermatofibroma, 23/45 (51%) cellular dermatofibroma, 10/10 (100%) malignant fibrous histiocytoma, 9/10 (90%) atypical fibroxanthoma, 7/8 (86%) cellular neurothekeoma, 9/9 (100%) malignant melanoma, 8/8 (100%) melanocytic nevi (100%), 0/2 superficial angiomyxoma, 0/15 fibromatosis, 0/1 nodular fasciitis, and 1/2 (50%) desmoplastic fibroblastomas. In summary, our findings indicate that Apo D expression is not specific to dermatofibrosarcoma protuberans. Its principal use as an immunohistochemical adjunct lies in its utility in differentiating superficial acral fibromyxoma from dermatofibrosarcoma protuberans. Although strong positive staining of Apo D in a markedly atypical fibrohistiocytic lesion is suggestive of atypical fibroxanthoma and/or malignant fibrous histiocytoma, further studies with the inclusion of other atypical spindled cell neoplasms are required to conclusively prove the same.

Insulin-like growth factor-II mRNA-binding protein 3 (IMP3) is a newly identified oncofetal mRNA-binding protein that is involved in embryogenesis and carcinogenesis of some malignant neoplasms. To investigate the diagnostic and clinicopathologic significance of this protein in endometrial carcinomas, we evaluated immunohistochemical expression of IMP3 in the two most common forms of endometrial malignancies, endometrioid adenocarcinoma and serous carcinoma. We selected 167 endometrial adenocarcinoma cases including 122 cases of endometrioid adenocarcinoma and 45 cases of serous carcinoma. Twenty samples of benign endometrium obtained from 20 patients with nonmalignant uterine lesions were used as controls. Positive immunohistochemical stain for IMP3 was identified in all serous carcinoma cases, among which, 39 (86%) and 3 (7%) cases showed IMP3 immunoreactivity in >50%, and 21-50, or 6-20% of tumor cells, respectively. Immunohistochemical reaction intensity for IMP3 was identified to be strong in 38 (84%) and intermediate in 7 (16%) cases of serous carcinoma. Fifty-four (44%) cases of endometrioid adenocarcinoma were negative for IMP3. Thirty (25%), 20 (16%), 10 (8%), and 8 (7%) cases of endometrioid adenocarcinoma demonstrated positive immunoreactivity for IMP3 in 1-5, 6-20, 21-50, and >50% of the tumor cells. Strong IMP3-staining intensity was noted in 34 (28%), intermediate in 26 (21%), and weak in 8 (7%) cases of endometrioid adenocarcinoma. All 20 control cases were negative for IMP3. To compare p53 with IMP3 expressions, we found that 35 (78%) of the serous carcinoma cases showed strong p53 immunohistochemical activity in >50% of the tumor cell nuclei. In contrast, 11 of 112 (10%) endometrioid adenocarcinoma cases demonstrated strong p53 positivity in >50% of the tumor cell nuclei. In conclusion, our findings demonstrate significant expression of IMP3 in serous carcinoma as compared to endometrioid adenocarcinoma (P<0.0001). Expression of IMP3 and p53 may be helpful biomarkers in the distinction of endometrial serous carcinoma from endometrioid adenocarcinoma. In addition, expression of IMP3 in endometrioid adenocarcinoma correlates with higher nuclear and architecture grades of the tumor (P=0.0000 and P=0.0002, respectively).

Adenocarcinoma in situ of the uterine cervix remains a diagnostic challenge in a small proportion of cases. This suggests a need for biomarker that may be of help in establishing the diagnosis. The aim of this study was to evaluate the potential of insulin-like growth factor-II mRNA-binding protein 3 and cyclin-dependent kinase inhibitor p16(INK4a) as biomarkers for adenocarcinoma in situ. Forty-four samples of adenocarcinoma in situ from 40 patients and 23 control cases of benign uterine cervix were included in this study. In addition to benign endocervical epithelium, 19 of these 23 control cases also showed focal tubal metaplasia. Cytoplasmic immunoreactivity for insulin-like growth factor-II mRNA-binding protein 3 was identified in 41 (93%) adenocarcinoma in situ samples, among which, 29 (71%), 10 (24%), and 2 (5%) samples showed insulin-like growth factor-II mRNA-binding protein 3 positive staining in 50% or more, >5 to <50 and <5% of adenocarcinoma in situ lesional cells, respectively. Immunohistochemical reaction intensity for insulin-like growth factor-II mRNA-binding protein 3 was found to be strong in 34 adenocarcinoma in situ samples, intermediate in five, and weak in two. All 23 control cases were negative for insulin-like growth factor-II mRNA-binding protein 3. p16(INK4a) expression was identified in all of the adenocarcinoma in situ samples with intermediate staining intensity seen in seven samples and strong in the remainder. Fourteen of 19 (74%) tubal metaplasia cases showed p16(INK4a) immunoreactivity in >50% of the tubal metaplastic epithelium with staining intensity ranging from weak to strong. Our findings demonstrate significant expression of insulin-like growth factor-II mRNA-binding protein 3 and p16(INK4a) in adenocarcinoma in situ as compared to benign endocervical glands, suggesting that expression of these biomarkers may be helpful in the distinction of adenocarcinoma in situ from benign endocervical glands, particularly in difficult borderline cases.