p16 expression in cutaneous squamous cell carcinoma of the head and neck is not associated with integration of high risk HPV DNA or prognosis

Head and neck cutaneous squamous cell carcinoma (HNcSCC) can present with cervical metastases without an obvious primary. Immunohistochemistry for p16 is established as a surrogate marker of human papillomavirus (HPV) in oropharyngeal cancer. p16 expression in HNcSCC needs to be elucidated to determine its utility in predicting the primary site. The aim of this study was to evaluate the rate of p16 expression in HNcSCC and its association with prognostic factors and survival. p16 immunohistochemistry was performed on 166 patients with high risk HNcSCC (2000-2013) following histopathology review. Chromogenic in situ hybridisation (CISH) for HPV was performed. Fifty-three (31.9%) cases showed strong, diffuse nuclear and cytoplasmic p16 expression including 14 (41%) non-metastatic and 39 (29.5%) metastatic tumours (p = 0.21). HPV CISH was negative in all cases. p16 expression significantly increased with poorer differentiation (p = 0.033), but was not associated with size (p = 0.30), depth of invasion (p = 0.94), lymphovascular invasion (p = 0.31), perineural invasion (p = 0.69), keratinisation (p = 0.99), number of involved nodes (p = 0.64), extranodal extension (p = 0.59) or survival. Nearly 32% of HNcSCCs, particularly poorly differentiated HNcSCCs, show p16 expression. A primary HNcSCC should be considered in p16 positive neck node metastases in regions with high prevalence of HNcSCC. p16 expression is not associated with improved survival in HNcSCC

ASCT2 regulates glutamine uptake and cell growth in endometrial carcinoma

Glutamine commonly becomes a conditionally essential amino acid in cancer. Glutamine is supplied to the cell by transporters such as ASCT2 (SLC1A5), which is frequently upregulated in multiple cancers. Here we investigated the expression of ASCT2 in endometrial carcinoma, and evaluated the contribution of ASCT2 to glutamine uptake and endometrial cancer cell growth. Analysis of human gene expression data showed that ASCT2 was significantly upregulated in both endometrioid and serous subtypes of endometrial carcinoma, compared to normal, age-matched endometrium. Furthermore, immunohistochemical staining of primary human endometrioid adenocarcinomas showed that tumours stain positive for ASCT2 in either a uniform or mosaic expression pattern, while normal adjacent glands appeared predominantly negative for ASCT2 staining. Chemical inhibition of glutamine transport by benzylserine or GPNA led to a significant decrease in endometrial cancer cell growth and spheroid cross-sectional area. ASCT2 knockdown recapitulated the decrease of cell growth and spheroid cross-sectional area in HEC1A cells, suggesting a reliance on ASCT2-mediated glutamine uptake. ASCT2 knockdown in Ishikawa cells led to lower glutamine uptake and cell growth, but did not affect spheroid area. Ishikawa cells express higher levels of the glutamine transporter SNAT1 compared to HEC1A cells, suggesting these cells may rely on both ASCT2 and SNAT1 for glutamine uptake. Since SNAT1 is also significantly upregulated in the endometrioid and serous subtypes, these data indicate that ASCT2 and SNAT1 could be used as markers of malignancy, and/or potential therapeutic targets in patients with endometrial carcinoma

PD-L1 expression predicts longer disease free survival in high risk head and neck cutaneous squamous cell carcinoma

Programmed cell death (PD-1) and its ligand (PD-L1) inhibitors have shown clinical response in many tumours. PD-L1 data are limited in head and neck cutaneous squamous cell carcinoma (HNcSCC) and no clinical trials of PD-1/PD-L1 inhibitors are published. We performed PD-L1 immunohistochemistry on 74 cases of high risk HNcSCC with 38 matched metastases and evaluated clinicopathological associations, prognostic significance and heterogeneity in matched metastases. We observed PD-L1 expression in \u3e5% of primary tumour cells in 29 cases (39.2%), primary tumour infiltrating lymphocytes (TILs) in 40 cases (70.2%), metastatic tumour cells in 15 cases (39.5%), and metastatic TILs in 18 cases (47.4%). PD-L1 expression in \u3e5% of primary tumour cells was associated with an inflammatory phenotype (p = 0.04), and in primary TILs with clear margins (p = 0.05). PD-L1 expression in \u3e5% of primary tumour cells (p = 0.01), primary TILs (p = 0.001), and metastatic TILs (p = 0.02) was associated with improved disease free survival. PD-L1 expression in \u3e5% of tumour cells was heterogeneous between primary and metastatic tumours in 13 cases (34.2%). PD-L1 expression is common in HNcSCC supporting the rationale for a clinical trial of PD-1/PD-L1 inhibitors. PD-L1 expression in tumour cells or TILs predicts longer disease free survival and demonstrates temperospatial heterogeneity

Real-world programmed death-ligand 1 prevalence rates in non-small cell lung cancer : correlation with clinicopathological features and tumour mutation status

Aims: The detection of programmed death-ligand 1 (PD-L1) protein expression on tumour cells by immunohistochemistry (IHC) is a predictor of response to immune checkpoint inhibitors. New immunotherapeutic options are changing the treatment paradigm for patients with advanced non-small cell lung cancer (NSCLC). The aim of this retrospective study was to investigate real-world prevalence of PD-L1 expression in NSCLC and any correlations with clinicopathological features. Methods: We reviewed 425 NSCLC cases at a Sydney metropolitan hospital that had PD-L1 IHC (SP263 clone) expression estimated as part of routine diagnostic assessment during a 30-month period. Results: Overall, 32.2% of cases were negative for PD-L1 expression (<1%), 40.3% demonstrated low expression (1%-49%) and 27.5% exhibited high protein expression (≥50%). High PD-L1 expression rates were more likely in non-lung resection cases and in KRAS mutant NSCLC as opposed to KRAS wildtype, while lower expression rates were more commonly found in EGFR mutant NSCLC compared with EGFR wildtype tumours. Conclusions: Ongoing observation and comparison of PD-L1 expression rates is an important practice for ensuring its validity as a predictive biomarker. The results from our study align with and contribute to the growing field of published real-world PD-L1 prevalence rates in NSCLC

Molecular assays in breast cancer pathology

Recent advances in understanding the molecularpathology of breast cancer offer significantpotential to identifypatients who may benefit from adjuvant therapies. To date, few of these advances are utilised in a routine setting. We review molecular assays that are currently in use or are in the advanced stages of development, which may be used aspredictive orprognostic biomarkers in breast cancer. The only widely used breast cancer molecular assay is in situ hybridisation (ISH) for human epidermal growth factor receptor 2 (HER2) gene amplification and we highlight key issues with the interpretation of this assay, withparticular attention to the difficulties of the equivocal category. New molecular assays such as ISH for the topoisomerase II alpha (TOP2A) gene and for the aberrations in the copy number of the centromeric region of chromosome 17 are readilyperformed in a standard histopathology laboratory, but to date there are insufficient data to support their routine use.We also review the current data on two commercially available multigene expression assays, Oncotype DX and MammaPrint and discuss theirpotential use. Overall, while new molecular assays have significant potential to improve patient selection for therapy, well-performed histopathology with reliable interpretation of standard hormone and HER2 assays provides the most important predictive and prognostic information in early breast cancer

Telomerase expression as a predictive marker of radiotherapy response in rectal cancer : in vitro and in vivo study

AIMS: To investigate telomerase as a predictive marker of radiotherapy response in rectal cancer. METHODS: Expression of the telomerase catalytic subunit hTERT was quantified with reverse transcription PCR in the radioresistant colorectal cancer cell line SW620 following exposure to 5Gy of radiation. Additionally, 52 rectal cancer cases were pre-operatively treated with either the short (n = 19) or long (n = 33) course radiotherapy (SCR, LCR, respectively) regimes, before and after which their hTERT expressions were semi-quantified with immunohistochemistry (IHC). This was correlated with the histological tumour regression in the resected bowel, dichotomised into good and poor responses. RESULTS: SW620 cells expressed gradually increasing levels of hTERT after radiation. hTERT IHC positivity of ≤75% tumour cells in pre-radiotherapied cancer was the optimal negative cut-off level [sensitivity 63.2%, specificity 45.8%, area under curve (AUC) 0.5362] in predicting good tumour response. As significantly more LCR cases showed good tumour response (p<0.0001), the SCR cases were excluded and AUC re-analysed, which still remained low (0.5357). CONCLUSIONS: While our in vitro results suggest that hTERT up-regulation may contribute to radiation resistance of colorectal cancer cells, our in vivo results demonstrated poor ability of hTERT IHC in predicting histological tumour regression in rectal cancer

Concordant BRAFV600E mutation status in primary melanomas and associated naevi : Implications for mutation testing of primary melanomas

There is concern that BRAF mutant naevus cells admixed with melanoma cells could cause false positive mutation tests in BRAF wild-type melanomas. We sought to assess the frequency of BRAFV600E mutations in primary melanomas arising with/without associated naevi and determine BRAFV600E concordance between melanomas and associated naevi. Formalin fixed, paraffin embedded (FFPE) tissue from 57 patients with primary melanomas with/without associated naevi was immunohistochemically stained to detect BRAF V600E mutation. In a subset of patients (n=29), molecular mutation testing was also carried out using a panel of 238 known genetic variants. Of the primary melanomas with an associated naevus (n=29), 55% were BRAF V600E mutant with 100% concordance between the melanoma and associated naevus. In contrast, only 21% of the primary melanomas unassociated with naevi were BRAFV600E mutant (p=0.009). Our results suggest that melanomas with associated naevi have a higher frequency of BRAFV600E mutations than melanomas unassociated with naevi. Furthermore, melanomas and their associated naevi were concordant in BRAFV600E status, which suggests that false positive mutation tests occurring as a consequence of admixed BRAF mutant naevus cells in BRAF wild-type primary melanomas are unlikely to be a problem in clinical practice. The findings have important implications for adjuvant clinical trials of targeted therapies.6 page(s

Intrapatient homogeneity of BRAFV600E expression in Melanoma

Concern regarding the presence of intertumoral heterogeneity of BRAF mutation status in patients with metastatic melanoma has led to uncertainty surrounding which specimens should preferentially undergo BRAF testing. We sought to examine the extent of intrapatient heterogeneity of BRAF protein expression in patients with multiple tumors. Sixty-four patients with 171 tumors at various stages of disease progression had tumor BRAF protein expression immunohistochemically (IHC) assessed using the BRAF mutant-specific antibody VE1. Melanoma sections were examined for staining intensity (score 0 to 3), the presence of intratumoral heterogeneity, and concordance with molecular BRAF genotype. Intrapatient, intertumoral heterogeneity of BRAF expression was also assessed by comparing VE1 staining on different tumors within the same patient. All specimens from 64 patients displayed complete intertumoral homogeneity of BRAF expression status, and all tumors had concordant molecular and IHC BRAF status. Only 1 patient demonstrated >1 level of staining intensity heterogeneity between specimens. Intratumoral heterogeneity of staining intensity was not observed in any specimen. IHC-measured BRAF protein expression displays complete intertumoral homogeneity, minimal intertumoral intensity heterogeneity, and no intratumoral heterogeneity in metastatic melanoma patients in various stages of disease progression. Our results suggest that, provided there is adequate quantity of viable tumor cells and minimal admixture of nontumor cells, testing any melanoma sample from a patient with metastatic disease will accurately determine BRAF status for treatment planning.6 page(s

BRAF mutations in non-small cell lung cancer

Background: BRAF is a proto-oncogene encoding a serine/threonine protein kinase which promotes cell proliferation and survival. BRAF mutations are commonly seen in melanoma and papillary thyroid carcinoma. We aimed to investigate the prevalence and clinicopathological features of BRAF mutations in non-small cell lung cancer (NSCLC) cases submitted for routine mutation testing at our institution. Methods: Mutation analysis for BRAF, EGFR and KRAS was performed using Sequenom MassARRAY platform with OncoCarta panel v1.0. Pathological features were reviewed and immunohistochemistry for BRAF V600E was also performed. Results: Seven out of 273 cases (2.6%) had BRAF mutations (three males and four females, median age 70 years, all smokers), with six adenocarcinomas and one NSCLC, not otherwise specified (NOS). All had wild-type EGFR and KRAS. The identified BRAF mutations were V600E (4/7, 58%), K601N, L597Q and G469V. BRAF V600E immunohistochemistry was positive in two cases with V600E and negative in one case with K601N (tissue available in three cases only). No significant difference in age or gender was found (BRAF mutant vs. wild-type). Conclusions: BRAF mutations occur in a small proportion of NSCLC that lack other driver mutations. The clinicopathological profile differs from that of EGFR mutant tumours. The potential benefits of BRAF-inhibitors should be investigated

Diagnostic and prognostic utility of Mastermind-like 2 (MAML2) gene rearrangement detection by fluorescent in situ hybridization (FISH) in mucoepidermoid carcinoma of the salivary glands

Objective Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy, with a proportion harboring MAML2 rearrangement. This study evaluates the diagnostic and prognostic utility of MAML2 rearrangement in MEC. Study Design Salivary gland malignancies at a single institution (1989-2014) were reviewed to identify MECs. Histopathologic evaluation, immunohistochemistry, and fluorescent in situ hybridization (FISH) were performed. Results Forty-one cases of MEC were identified, with mean age of 47 years and mean tumor size of 21 mm. Seven locoregional recurrences and five MEC-related deaths were seen over a 22-year follow-up period. Thirty-eight cases were suitable for FISH, and 31 (82%) cases were positive for MAML2 rearrangement, including the oncocytic and clear cell variants of MEC. FISH was negative in the morphologic mimics of MEC. MAML2 rearrangement was significantly associated with longer survival. Conclusions MAML2 rearrangement is common and specific for MEC, which makes it a useful diagnostic tool. MAML2 rearrangement also predicts a favorable prognosis