Trina Lum

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 BRAFV600E protein expression in patients with multiple tumors. Sixty-four patients with 171 tumors at various stages of disease progression had tumor BRAFV600E protein expression immunohistochemically (IHC) assessed using the BRAFV600E 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 BRAFV600E 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 BRAFV600E 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 BRAFV600E 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.

Oligoastrocytomas: throwing the baby out with the bathwater?

biology, clinical trial design, and future treatment deci-sions. OA should not be ignored in the proposed new clas-sification guidelines [2].Sahm and colleagues studied 43 cases diagnosed as OA based on histology. But in 30 of these cases, immunostain-ing for IDH1 (R132H) mutation was restricted to oligo-dendroglial areas only. The astrocytic component of these tumours was re-interpreted to be reactive in nature [4], and they were reclassified as oligodendroglioma. We propose that this finding indicates that ‘true’ OAs (i.e. tumours com-posed of two distinct

Dynamics of Chemokine, Cytokine, and Growth Factor Serum Levels in BRAF-Mutant Melanoma Patients during BRAF Inhibitor Treatment

The purpose of this study is to profile the changes in the serum levels of a range of chemokines, cytokines, and growth and angiogenic factors in MAPK inhibitor–treated metastatic melanoma patients and to correlate these changes with clinical outcome and changes in melanoma tissue biopsies taken from the same patients. Forty-two chemokine, cytokine, angiogenic, and growth factors were measured in the sera of 20 BRAF inhibitor–treated and four combination BRAF and MEK inhibitor–treated metastatic melanoma patients using a multiplex chemokine assay. The changes were correlated with Ki-67 and CD8+ tumor-infiltrating lymphocytes in the tumor biopsies taken at the same time points, as well as clinical outcome, including response rate, progression-free survival, and overall survival. Serum levels of IFN-γ, CCL4, and TNF-α were significantly increased, whereas CXCL8 significantly decreased from pretreatment (PRE) to early during treatment (EDT) serum samples. The decrease in serum CXCL8 levels from PRE to EDT significantly correlated with decreases in markers of melanoma proliferation (Ki-67) and increases in cytotoxic tumor-infiltrating T cells in corresponding tumor biopsies. In addition, a greater fold reduction in CXCL8 serum levels from PRE to EDT serum samples was associated with decreased overall survival. These results suggest that BRAF inhibition causes decreased CXCL8 secretion from melanoma cells and induce an immune response against the tumor associated with increased IFN-γ, CCL4, and TNF-α. Further studies are needed to determine if CXCL8 is predictive of response and to confirm the functions of these chemokine and cytokine in BRAF-mutant melanoma under BRAF inhibition.

Salivary duct carcinoma: Clinicopathologic features, morphologic spectrum, and somatic mutations.

Accurate diagnosis of salivary duct carcinoma requires a high index of suspicion and clinicopathologic correlation. Hallmark genetic changes that may provide novel therapeutic options are being explored.

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.

EGFR mutant-specific immunohistochemistry has high specificity and sensitivity for detecting targeted activating EGFR mutations in lung adenocarcinoma

Aim We assessed the diagnostic accuracy of epidermal growth factor receptor (EGFR) mutant-specific antibodies for detecting two common activating EGFR mutations. Methods Immunohistochemical expression of mutation-specific antibodies against EGFR exon 19 deletion E746-A750 ((c.2235_2249del15 or c.2236_2250del15, p. Glu746_Ala750del) and exon 21 L858R point mutation (c.2573T>G, p.Leu858Arg) were assessed in a cohort of 204 resected early stage node negative lung adenocarcinomas, and protein expression was compared with DNA analysis results from mass spectrometry analysis. Results Of seven cases with L858R point mutation, six were positive by immunohistochemistry (IHC). There were three false positive cases using L858R IHC (sensitivity 85.7%, specificity 98.5%, positive predictive value 66.7%, negative predictive value 99.5%). All seven E746-A750 exon 19 deletions identified by mutation analysis were positive by IHC. Four additional cases were positive for exon 19 IHC but negative by mutation analysis. The sensitivity of exon 19 IHC for E746-A750 was 100%, specificity 98.0%, positive predictive value 63.6% and negative predictive value 100%. Conclusions Mutant-specific EGFR IHC has good specificity and sensitivity for identifying targeted activating EGFR mutations. Although inferior to molecular genetic analysis of the EGFR gene, IHC is highly specific and sensitive for the targeted EGFR mutations. The antibodies are likely to be of clinical value in cases where limited tumour material is available, or in situations where molecular genetic analysis is not readily available.

Molecular assays in breast cancer pathology

Recent advances in understanding the molecular pathology of breast cancer offer significant potential to identify patients 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 as predictive or prognostic 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, with particular 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 readily performed 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 their potential 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.

Programmed cell death-ligand 1 expression in oral squamous cell carcinoma is associated with an inflammatory phenotype

Phase 2 clinical trials utilising novel anti-PD1/PD-L1 antibodies are being conducted in oral cavity squamous cell carcinoma (OSCC) patients. However, data regarding PD-L1 expression in OSCC is limited. The aim of this study was to characterise the PD-L1 immunohistochemical expression in OSCC and its association with clinicopathological factors. Clinicopathological review of 217 patients with OSCC was performed, including quantifying tumour-infiltrating lymphocytes. Immunohistochemistry with PD-L1, CD4 and CD8 was performed. Forty (18.3%) cases showed PD-L1 expression. Expression was significantly more frequent in females (p=0.013), tongue/buccal mucosal SCCs (p=0.05), and in tumours with a high lymphocytic infiltrate (p>0.001). Intratumoural heterogeneity of PD-L1 expression was observed in 30% of the cases. PD-L1 expression was not significantly associated with disease-free (p=0.82) or overall survival (p=0.93). PD-L1 expression occurred in a significant minority of OSCC and can be heterogeneous. Frequent PD-L1 expression in OSCCs in females and in tumours with high lymphocytic infiltrate may assist in the selection of patients who may respond to anti-PD1/PD-L1 therapies.

Tumor Suppressor microRNAs Contribute to the Regulation of PD-L1 Expression in Malignant Pleural Mesothelioma

Introduction: The upregulation of programmed death ligand 1 (PD‐L1) is found in many cancers and contributes to evasion of the hosts immune defense. In malignant pleural mesothelioma (MPM), PD‐L1 expression is associated with the nonepithelioid histological subtype and poor prognosis, but the pathways involved in control of PD‐L1 expression in MPM are poorly understood. To address one possible means of PD‐L1 regulation we investigated the relationship between dysregulated microRNA levels and PD‐L1 expression. Methods: PD‐L1 expression was analyzed by immunohistochemistry in tissue microarrays prepared from samples from patients undergoing an operation (pleurectomy with or without decortication). MicroRNA expression was analyzed by reverse‐transcriptase quantitative polymerase chain reaction. Regulation of PD‐L1 expression in cell lines was assessed after transfection with microRNA mimics and small interfering RNAs. Interaction between microRNAs and PD‐L1 was analyzed by using argonaute‐2 immunoprecipitation and a luciferase reporter assay. Results: In a series of 72 patients with MPM, 18 (25%) had positive PD‐L1 staining, and this was more common in patients with the nonepithelioid subtype (p = 0.01). PD‐L1 expression was associated with poor survival (median overall survival 4.0 versus 9.2 months with positive versus negative PD‐L1 expression [p < 0.001]), and in multivariate analyses, PD‐L1 expression remained a significant adverse prognostic indicator (hazard ratio = 2.2, 95% confidence interval: 1.2–4.1, p < 0.01). In the same patient series, PD‐L1 expression was also associated with downregulation of microRNAs previously shown to have tumor suppressor activity in MPM. The median microRNA expression levels of miR‐15b, miR‐16, miR‐193a‐3p, miR‐195, and miR‐200c were significantly lower in the PD‐L1–positive samples. Transfecting MPM cell lines with mimics corresponding to miR‐15a and miR‐16, both of which are predicted to target PD‐L1, led to downregulation of PD‐L1 mRNA and protein. In addition, miR‐193a‐3p, with an alternative G‐U–containing target site, also caused PD‐L1 downregulation. Conclusions: Together, these data suggest that tumor suppressor microRNAs contribute to the regulation of PD‐L1 expression in MPM.

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

Summary 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 BRAFV600E 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 BRAFV600E 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.