Sandra A. O’Toole

PD-L1 expression is a favorable prognostic factor in early stage non-small cell carcinoma

OBJECTIVES Immune checkpoint blockade using inhibitors of programmed death-1 have shown promise in early phase clinical trials in NSCLC and programmed death-ligand 1 (PD-L1) tumoral expression could potentially be a useful predictive marker. Data reporting the prevalence of PD-L1 expression in NSCLC and clinicopathologic associations is very limited. We sought to determine the frequency of PD-L1 expression in NSCLC and investigate associations with clinicopathologic features and patient outcome. MATERIALS AND METHODS PD-L1 expression was analyzed using immunohistochemistry (Merck; clone 22C3) in 678 stages I-III NSCLC and 52 paired nodal metastases using tissue microarrays. Tumors with ≥50% cells showing positive membrane staining were considered to have high expression of PD-L1. RESULTS PD-L1 expression of any intensity was identified in 32.8% of cases. High PD-L1 expression was found in 7.4% of NSCLC. Squamous cell carcinomas (8.1%) and large cell carcinomas (12.1%) showed high PD-L1 expression more commonly than adenocarcinomas (5.1%) but this was not statistically significant (p=0.072). High PD-L1 expression was associated with younger patient age and high tumor grade (p<0.05). There was no association with gender, tumor size, stage, nodal status, EGFR or KRAS mutation status. In multivariate analysis, patients with high PD-L1 expression had significantly longer overall survival (p<0.05). CONCLUSIONS PD-L1 is expressed at high levels in a significant proportion of NSCLC and appears to be a favorable prognostic factor in early stage disease. As there are potential sampling limitations using tissue microarrays to assess heterogeneously expressed biomarkers, and as the results may differ in advanced stage disease, further studies are recommended.

BRAF inhibitor activity in V600R metastatic melanoma

Activating mutations in the BRAF gene occur in approximately 50% of melanomas. More than 70% of BRAF mutations are V600E and 10-30% are V600K. Potent and selective BRAF inhibitors have demonstrated significant clinical benefits in patients with V600E and V600K BRAF-mutated melanoma. V600R mutations constitute approximately 3-7% of all BRAF mutations and the activity of BRAF inhibitors in patients with this mutation is unknown. We have treated 45 patients with V600 mutated melanoma including patients with V600R mutation between July 2011 and October 2012 with the selective BRAF inhibitor dabrafenib (n=43) or vemurafenib (n=2) via a compassionate access programme. The overall response rate was 50% for the whole population with a progression-free survival of 5.5 months. Five objective responses were seen in six assessable patients with V600R BRAF mutation (n=9). Our experience suggests that patients with V600R BRAF mutations can be treated successfully with oral BRAF inhibitors, and molecular diagnostic assays should include detection of this type of mutation.

Transcription factor ATF3 links host adaptive response to breast cancer metastasis

Host response to cancer signals has emerged as a key factor in cancer development; however, the underlying molecular mechanism is not well understood. In this report, we demonstrate that activating transcription factor 3 (ATF3), a hub of the cellular adaptive response network, plays an important role in host cells to enhance breast cancer metastasis. Immunohistochemical analysis of patient tumor samples revealed that expression of ATF3 in stromal mononuclear cells, but not cancer epithelial cells, is correlated with worse clinical outcomes and is an independent predictor for breast cancer death. This finding was corroborated by data from mouse models showing less efficient breast cancer metastasis in Atf3-deficient mice than in WT mice. Further, mice with myeloid cell-selective KO of Atf3 showed fewer lung metastases, indicating that host ATF3 facilitates metastasis, at least in part, by its function in macrophage/myeloid cells. Gene profiling analyses of macrophages from mouse tumors identified an ATF3-regulated gene signature that could distinguish human tumor stroma from distant stroma and could predict clinical outcomes, lending credence to our mouse models. In conclusion, we identified ATF3 as a regulator in myeloid cells that enhances breast cancer metastasis and has predictive value for clinical outcomes.

Fibroblast growth factor receptor 1 (FGFR1) copy number is an independent prognostic factor in non-small cell lung cancer

Fibroblast growth factor receptor 1 (FGFR1) is an oncogene that can potentially be targeted by tyrosine kinase inhibitors. We aimed to investigate the prevalence and prognostic significance of alterations in FGFR1 copy number in non-small cell lung cancer (NSCLC). FGFR1 status was evaluated by chromogenic silver in situ hybridisation (ISH) in tissue microarray sections from a retrospective cohort of 304 surgically resected NSCLCs and results were correlated with the clinicopathological features and overall survival. High FGFR1 gene copy number (amplification or high-level polysomy) was significantly more frequent in squamous cell carcinomas (SCC) (24.8%) and large cell carcinomas (LCC) (25%) compared to adenocarcinomas (11.3%) (p = 0.01 and p = 0.03 respectively). Among NSCLC there was no significant correlation between FGFR1-positive status and other clinicopathological features including age, gender, smoking history, tumour size, lymph node status, stage, grade, vascular, lymphatic or perineural invasion. FGFR1-positive patients showed a tendency to longer overall survival in univariate analysis (p = 0.14). Multivariate survival analysis using Cox regression model confirmed FGFR1-positive patients had a significant reduction in the risk of death compared to FGFR1-negative patients (HR 0.6; p = 0.02). High FGFR1 gene copy number is a common finding in SCC and LCC and is an independent favourable prognostic factor.

High Notch1 protein expression is an early event in breast cancer development and is associated with the HER‐2 molecular subtype

Zardawi S J, Zardawi I, McNeil C M, Millar E K A, McLeod D, Morey A L, Crea P, Murphy N C, Pinese M, Lopez‐Knowles E, Oakes S R, Ormandy C J, Qiu M R, Hamilton A, Spillane A, Soon Lee C, Sutherland R L, Musgrove E A & O’Toole S A
(2010) Histopathology56, 286–296

ERG induces taxane resistance in castration-resistant prostate cancer

Taxanes are the only chemotherapies used to treat patients with metastatic castration-resistant prostate cancer (CRPC). Despite the initial efficacy of taxanes in treating CRPC, all patients ultimately fail due to the development of drug resistance. In this study, we show that ERG overexpression in in vitro and in vivo models of CRPC is associated with decreased sensitivity to taxanes. ERG affects several parameters of microtubule dynamics and inhibits effective drug-target engagement of docetaxel or cabazitaxel with tubulin. Finally, analysis of a cohort of 34 men with metastatic CRPC treated with docetaxel chemotherapy reveals that ERG-overexpressing prostate cancers have twice the chance of docetaxel resistance than ERG-negative cancers. Our data suggest that ERG plays a role beyond regulating gene expression and functions outside the nucleus to cooperate with tubulin towards taxane insensitivity. Determining ERG rearrangement status may aid in patient selection for docetaxel or cabazitaxel therapy and/or influence co-targeting approaches.

What’s new in non-small cell lung cancer for pathologists the importance of accurate subtyping, EGFR mutations and ALK rearrangements

In the past, the only critical point of distinction in the pathological diagnosis of lung cancer was between small cell and non-small cell lung cancer (NSCLC). The emergence of new targeted therapies and clinical trials demonstrating differing efficacy and toxicity of treatments according to specific histological subtypes of NSCLC, has resulted in an increasing need for improvements in pathological diagnosis. Accurate distinction between adenocarcinoma and squamous cell carcinoma is now critical as histological subtyping has the potential to influence clinical decision making and impact on patient outcome. While morphological criteria remain the most important feature to distinguish NSCLC subtypes, use of mucin and immunohistochemical stains (TTF-1, p63 and CK5/6) can be of assistance in difficult small biopsy cases. With the emergence of selective kinase inhibitors targeting epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK), there is a corresponding need to identify the subset of NSCLCs harbouring specific genetic mutations associated with sensitivity to these agents, almost all of which are found in adenocarcinomas. In this review, the importance of accurately subtyping NSCLC is discussed, along with a suggested approach for distinguishing histological subtypes in small biopsy specimens. The significance of EGFR and ALK mutations in NSCLC and the impact of these genotypes on pathology and clinical practice are also reviewed.

Classification, morphology and molecular pathology of premalignant lesions of the pancreas

Summary Over the past few years there have been substantial advances in our knowledge of premalignant lesions of the pancreas. Given the dismal prognosis of untreated pancreatic cancer, and the small proportion of patients who are operative candidates, an understanding of these premalignant lesions is essential for the development of strategies for early diagnosis and prevention. The 2010 WHO classification has added new entities, including intraductal tubular papillary neoplasms (ITPNs), and clarified the nomenclature and grading of previously recognised precursor lesions of pancreatic adenocarcinoma, such as intraductal papillary mucinous neoplasms (IPMNs), mucinous cystic neoplasms (MCNs) and pancreatic intraepithelial neoplasia (PanIN). In particular, there has been an upsurge of interest in the natural history of IPMN, driven partly by improvements in imaging modalities and the consequent apparent increase in their incidence, and partly by recognition that subtypes based on location or histological appearance define groups with significantly different behaviours. In mid 2012 revised international guidelines for the classification and management of IPMNs and MCNs were published, although in several respects these guidelines represent a consensus view rather than being evidence-based. In recent years major advances in molecular technologies, including whole-exome sequencing, have significantly enhanced our knowledge of pancreatic premalignancy and have identified potentially highly specific diagnostic biomarkers such as mutations in GNAS and RNF43 that could be used to pre-operatively assess pancreatic cysts.

A Detailed Clinicopathologic Study of ALK-translocated Papillary Thyroid Carcinoma

Pathogenic ALK translocations have been reported in papillary thyroid carcinoma (PTC). We developed and validated a screening algorithm based on immunohistochemistry (IHC), followed by fluorescence in situ hybridization (FISH) in IHC-positive cases to identify ALK-rearranged PTC. IHC and FISH were performed in a cohort of 259 thyroid carcinomas enriched for aggressive variants. IHC was positive in 8 cases, 6 confirmed translocated by FISH (specificity 75%). All 251 IHC-negative cases were FISH negative (sensitivity 100%). Having validated this approach, we performed screening IHC, followed by FISH in IHC-positive cases in an expanded cohort. ALK translocations were identified in 11 of 498 (2.2%) of all consecutive unselected PTCs and 3 of 23 (13%) patients with diffuse sclerosing variant PTCs. No ALK translocations were identified in 36 PTCs with distant metastases, 28 poorly differentiated (insular) carcinomas, and 20 anaplastic carcinomas. All 14 patients with ALK translocations were female (P=0.0425), and translocations occurred at a younger age (mean 38 vs. 48 y, P=0.0289 in unselected patients). ALK translocation was an early clonal event present in all neoplastic cells and mutually exclusive with BRAFV600E mutation. ALK translocation was not associated with aggressive clinicopathologic features (size, stage, metastasis, vascular invasion, extrathyroidal extension, multifocality, risk for recurrence, radioiodine resistance). We conclude that 2.2% of PTCs are ALK-translocated and can be identified by screening IHC followed by FISH. ALK translocations may be more common in young females and diffuse sclerosing variant PTC but do not connote more aggressive disease.

MicroRNA profiling of the pubertal mouse mammary gland identifies miR-184 as a candidate breast tumour suppressor gene

IntroductionThe study of mammalian development has offered many insights into the molecular aetiology of cancer. We previously used analysis of mammary morphogenesis to discover a critical role for GATA-3 in mammary developmental and carcinogenesis. In recent years an important role for microRNAs (miRNAs) in a myriad of cellular processes in development and in oncogenesis has emerged.MethodsmicroRNA profiling was conducted on stromal and epithelial cellular subsets microdissected from the pubertal mouse mammary gland. miR-184 was reactivated by transient or stable overexpression in breast cancer cell lines and examined using a series of in vitro (proliferation, tumour-sphere and protein synthesis) assays. Orthotopic xenografts of breast cancer cells were used to assess the effect of miR-184 on tumourigenesis as well as distant metastasis. Interactions between miR-184 and its putative targets were assessed by quantitative PCR, microarray, bioinformatics and 3′ untranslated region Luciferase reporter assay. The methylation status of primary patient samples was determined by MBD-Cap sequencing. Lastly, the clinical prognostic significance of miR-184 putative targets was assessed using publicly available datasets.ResultsA large number of microRNA were restricted in their expression to specific tissue subsets. MicroRNA-184 (miR-184) was exclusively expressed in epithelial cells and markedly upregulated during differentiation of the proliferative, invasive cells of the pubertal terminal end bud (TEB) into ductal epithelial cells in vivo. miR-184 expression was silenced in mouse tumour models compared to non-transformed epithelium and in a majority of breast cancer cell line models. Ectopic reactivation of miR-184 inhibited the proliferation and self-renewal of triple negative breast cancer (TNBC) cell lines in vitro and delayed primary tumour formation and reduced metastatic burden in vivo. Gene expression studies uncovered multi-factorial regulation of genes in the AKT/mTORC1 pathway by miR-184. In clinical breast cancer tissues, expression of miR-184 is lost in primary TNBCs while the miR-184 promoter is methylated in a subset of lymph node metastases from TNBC patients.ConclusionsThese studies elucidate a new layer of regulation in the PI3K/AKT/mTOR pathway with relevance to mammary development and tumour progression and identify miR-184 as a putative breast tumour suppressor.