Germline ERBB3 mutation in familial non-small-cell lung carcinoma: expanding ErbB's role in oncogenesis.

Lung cancer is the commonest cause of cancer deaths worldwide. Although strongly associated with smoking, predisposition to lung cancer is also heritable, with multiple common risk variants identified. Rarely, dominantly inherited non-small-cell lung cancer (NSCLC) has been reported due to somatic mutations in EGFR/ErbB1 and ERBB2. Germline exome sequencing was performed in a multi-generation family with autosomal dominant NSCLC, including an affected child. Tumour samples were also sequenced. Full-length wild-type (wtErbB3) and mutant ERBB3 (mutErbB3) constructs were transfected into HeLa cells. Protein expression, stability, and subcellular localization were assessed, and cellular proliferation, pAkt/Akt and pERK levels determined. A novel germline variant in ERBB3 (c.1946 T > G: p.Iso649Arg), coding for receptor tyrosine-protein kinase erbB-3 (ErbB3), was identified, with appropriate segregation. There was no loss-of-heterozygosity in tumour samples. Both wtErbB3 and mutErbB3 were stably expressed. MutErbB3-transfected cells demonstrated an increased ratio of the 80 kDa form (which enhances proliferation) compared with the full-length (180 kDa) form. MutErbB3 and wtErbB3 had similar punctate cytoplasmic localization pre- and post-epidermal growth factor stimulation; however, epidermal growth factor receptor (EGFR) levels decreased faster post-stimulation in mutErbB3-transfected cells, suggesting more rapid processing of the mutErbB3/EGFR heterodimer. Cellular proliferation was increased in mutErbB3-transfected cells compared with wtErbB3 transfection. MutErbB3-transfected cells also showed decreased pAkt/tAkt ratios and increased pERK/tERK 30 min post-stimulation compared with wtErbB3 transfection, demonstrating altered signalling pathway activation. Cumulatively, these results support this mutation as tumorogenic. This is the first reported family with a germline ERBB3 mutation causing heritable NSCLC, furthering understanding of the ErbB family pathway in oncogenesis.

An ErbB2/c-Src axis links bioenergetics with PRC2 translation to drive epigenetic reprogramming and mammary tumorigenesis.

Dysregulation of histone modifications promotes carcinogenesis by altering transcription. Breast cancers frequently overexpress the histone methyltransferase EZH2, the catalytic subunit of Polycomb Repressor Complex 2 (PRC2). However, the role of EZH2 in this setting is unclear due to the context-dependent functions of PRC2 and the heterogeneity of breast cancer. Moreover, the mechanisms underlying PRC2 overexpression in cancer are obscure. Here, using multiple models of breast cancer driven by the oncogene ErbB2, we show that the tyrosine kinase c-Src links energy sufficiency with PRC2 overexpression via control of mRNA translation. By stimulating mitochondrial ATP production, c-Src suppresses energy stress, permitting sustained activation of the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), which increases the translation of mRNAs encoding the PRC2 subunits Ezh2 and Suz12. We show that Ezh2 overexpression and activity are pivotal in ErbB2-mediated mammary tumourigenesis. These results reveal the hitherto unknown c-Src/mTORC1/PRC2 axis, which is essential for ErbB2-driven carcinogenesis.

Examining the contribution of smoking and HPV towards the etiology of oral cavity squamous cell carcinoma using high-throughput sequencing: A prospective observational study.

Oral cavity Squamous Cell Carcinoma (OCSCC) is a common form of head and neck cancer throughout the developed and developing world. However, the etiology of OCSCC is still unclear. Here, we explored the extent to which tobacco use, Human Papillomavirus (HPV) infection and genetic and transcriptomic changes contributed to the oncogenesis of OCSCC. In a prospective observational study, we analysed fresh tissue biopsies from 45 OCSCC collected from 51 subjects presenting with OCSCC to the Brisbane Head and Neck Clinics between 2013 and 2015. Exploration of the genetic and transcriptomic landscape of the biopsies were performed using RNA sequencing (RNA-seq) and whole exome sequencing. HPV associated tumours were determined using p16 staining of histological sections and RNA sequencing. Patient demographics including tumor location within the oral cavity, and history of tobacco and alcohol use were correlated with genomic and transcriptomics analyses. About 4.5% of OCSCC were HPV associated. The most frequent mutations in the OCSCC samples were in the TP53 and CDKN2A genes, but no association of specific mutations with HPV or tobacco use was observed. Using weighted gene co-expression network analysis to explore the RNA-seq data, tumors from participants with a history of tobacco use showed a significant trend towards increased mammalian target of Rapamycin (mTOR) signaling and decreased mitochondrial respiration. In conclusion, HPV was shown to be an uncommon association with OCSCC and changes in TP53 transcriptional regulation, mTOR signaling and mitochondrial function were associated with a history of tobacco use. Larger data sets will be required to enable detection of differences which may help with development of personalized therapeutics in the future.

Punch 'scoring': a technique that facilitates melanoma diagnosis of clinically suspicious pigmented lesions.

AIMS: Early recognition and accurate diagnosis underpins melanoma survival. Identifying early melanomas arising in association with pre-existing lesions is often challenging. Clinically suspicious foci, however small, must be identified and examined histologically. This study assessed the accuracy of punch biopsy 'scoring' of suspicious foci in excised atypical pigmented skin lesions to identify early melanomas. METHODS AND RESULTS: Forty-one excised pigmented skin lesions with a clinically/dermoscopically focal area of concern for melanoma, with the suspicious focus marked prior to excision with a punch biopsy 'score' (a partial incision into the skin surface), were analysed. Melanoma was diagnosed in nine of 41 cases (22%). In eight of nine cases (89%) the melanoma was associated with a naevus, and in seven of nine (88%) cases the melanoma was identified preferentially by the scored focus. In six of nine cases (67%), the melanoma was entirely encompassed by the scored focus. In one case of melanoma in situ, the diagnostic material was identified only on further levelling through the scored focus. In 28 of 32 of non-melanoma cases (88%), the scored focus identified either diagnostic features of a particular lesion or pathological features that correlated with the clinical impression of change/atypia including altered architecture or distribution of pigmentation, features of irritation or regression. CONCLUSIONS: The 'punch scoring technique' allows direct clinicopathological correlation and facilitates early melanoma diagnosis by focusing attention on clinically suspicious areas. Furthermore, it does not require special expertise in ex-vivo clinical techniques for implementation. Nevertheless, in some cases examination of the lesion beyond the scored focus is also necessary to make a diagnosis of melanoma.

Pulmonary schistosomiasis mimicking IgG4-related lung disease.

IgG4-related disease (IgG4-RD) is a systemic fibroinflammatory disease characterized by tumefactive lesions in various organ systems, including the lungs. Clinical and radiological manifestations of IgG4-RD are relatively non-specific, and we report a case highlighting the importance of histopathological confirmation in cases of suspected IgG4-related lung disease. A 44-year-old male with significantly elevated serum IgG4 levels, patchy consolidation on thoracic CT imaging, and cough was referred with suspected IgG4-related lung disease. However, surgical lung biopsy revealed an unexpected diagnosis of pulmonary schistosomiasis, and following treatment with praziquantel, cough resolved and IgG4 levels significantly declined. This case highlights the potentially diverse array of conditions that may mimic IgG4-related lung disease and the importance of comprehensive evaluation including histopathological confirmation where possible.

Programmed death ligand 1 expression in triple-negative breast cancer is associated with tumour-infiltrating lymphocytes and improved outcome.

AIMS: Triple-negative breast cancer (TNBC) patients generally have a poor outcome; there is a pressing need to identify more effective therapeutic strategies. Clinical trials targeting programmed death 1/programmed death ligand 1 (PD1/PDL1) in melanoma and non-small-cell lung cancer have reported high response rates, and tumoral PDL1 expression has been suggested as a potential biomarker to enrich for patient response to these treatments. There are only very limited data to date reporting the expression of PDL1 in TNBC. METHODS AND RESULTS: PDL1 immunohistochemistry was performed on 161 primary TNBCs and assessed in the tumour as well as immune cells in the stromal compartment. PDL1 expression was very common in TNBC, expressed in the tumour cell membrane (64%), cytoplasm (80%) and stromal (93%) cellular compartments. Cytoplasmic tumoral expression of PDL1 was associated with a lower risk of breast cancer-specific death [hazard ratio (HR) 0.45, P = 0.035] while stromal PDL1 expression was associated with a lower rate of deaths from all causes (HR 0.305, P = 0.0042). Membranous expression of PDL1 was not associated with outcome. While both PDL1 expression and tumour-infiltrating lymphocytes were associated with a better outcome, only lymphovascular invasion and high tumour-infiltrating lymphocytes were independently prognostic for breast cancer-specific death. CONCLUSION: While PDL1 expression is frequent in TNBC, it was not independently prognostic. There were differences in outcome depending on the cellular compartment of PDL1 expression. These data provide further impetus for investigating the utility of immune checkpoint therapies in TNBC, given the clinical significance of tumour-infiltrating lymphocytes (TILs) and PDL1 expression in this cohort.

MicroRNA-Related DNA Repair/Cell-Cycle Genes Independently Associated With Relapse After Radiation Therapy for Early Breast Cancer.

PURPOSE: Local recurrence and distant failure after adjuvant radiation therapy for breast cancer remain significant clinical problems, incompletely predicted by conventional clinicopathologic markers. We had previously identified microRNA-139-5p and microRNA-1274a as key regulators of breast cancer radiation response in vitro. The purpose of this study was to investigate standard clinicopathologic markers of local recurrence in a contemporary series and to establish whether putative target genes of microRNAs involved in DNA repair and cell cycle control could better predict radiation therapy response in vivo. METHODS AND MATERIALS: With institutional ethics board approval, local recurrence was measured in a contemporary, prospectively collected series of 458 patients treated with radiation therapy after breast-conserving surgery. Additionally, independent publicly available mRNA/microRNA microarray expression datasets totaling >1000 early-stage breast cancer patients, treated with adjuvant radiation therapy, with >10 years of follow-up, were analyzed. The expression of putative microRNA target biomarkers--TOP2A, POLQ, RAD54L, SKP2, PLK2, and RAG1--were correlated with standard clinicopathologic variables using 2-sided nonparametric tests, and to local/distant relapse and survival using Kaplan-Meier and Cox regression analysis. RESULTS: We found a low rate of isolated local recurrence (1.95%) in our modern series, and that few clinicopathologic variables (such as lymphovascular invasion) were significantly predictive. In multiple independent datasets (n>1000), however, high expression of RAD54L, TOP2A, POLQ, and SKP2 significantly correlated with local recurrence, survival, or both in univariate and multivariate analyses (P<.001). Low RAG1 expression significantly correlated with local recurrence (multivariate, P=.008). Additionally, RAD54L, SKP2, and PLK2 may be predictive, being prognostic in radiation therapy-treated patients but not in untreated matched control individuals (n=107; P<.05). CONCLUSIONS: Biomarkers of DNA repair and cell cycle control can identify patients at high risk of treatment failure in those receiving radiation therapy for early breast cancer in independent cohorts. These should be further investigated prospectively, especially TOP2A and SKP2, for which targeted therapies are available.

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

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.

The retinoid signalling molecule, TRIM16, is repressed during squamous cell carcinoma skin carcinogenesis in vivo and reduces skin cancer cell migration in vitro.

Retinoid therapy is used for chemo-prevention in immuno-suppressed patients at high risk of developing skin cancer. The retinoid signalling molecule, tripartite motif protein 16 (TRIM16), is a regulator of keratinocyte differentiation and a tumour suppressor in retinoid-sensitive neuroblastoma. We sought to determine the role of TRIM16 in skin squamous cell carcinoma (SCC) pathogenesis. We have shown that TRIM16 expression was markedly reduced during the histological progression from normal skin to actinic keratosis and SCC. SCC cell lines exhibited lower cytoplasmic and nuclear TRIM16 expression compared with primary human keratinocyte (PHK) cells due to reduced TRIM16 protein stability. Overexpressed TRIM16 translocated to the nucleus, inducing growth arrest and cell differentiation. In SCC cells, TRIM16 bound to and down regulated nuclear E2F1, this is required for cell replication. Retinoid treatment increased nuclear TRIM16 expression in retinoid-sensitive PHK cells, but not in retinoid-resistant SCC cells. Overexpression of TRIM16 reduced SCC cell migration, which required the C-terminal RET finger protein (RFP)-like domain of TRIM16. The mesenchymal intermediate filament protein, vimentin, was directly bound and down-regulated by TRIM16 and was required for TRIM16-reduced cell migration. Taken together, our data suggest that loss of TRIM16 expression plays an important role in the development of cutaneous SCC and is a determinant of retinoid sensitivity.

Hedgehog overexpression is associated with stromal interactions and predicts for poor outcome in breast cancer.

Hedgehog (Hh) signaling plays an important role in several malignancies but its clinical significance in breast cancer is unclear. In a cohort of 279 patients with invasive ductal carcinoma of the breast, expression of Hh ligand was significantly associated with increased risk of metastasis, breast cancer-specific death, and a basal-like phenotype. A paracrine signature, encompassing high epithelial Hh ligand and high stromal Gli1, was an independent predictor for overall survival in multivariate analysis. In 2 independent histological progression series (n = 301), Hh expression increased with atypia. Hh ligand overexpression in a mouse model of basal breast cancer increased growth, induced a poorly differentiated phenotype, accelerated metastasis, and reduced survival. A stromal requirement for these effects was supported by the lack of similar Hh-mediated changes in vitro, and by stromal-specific expression of Hh target genes in vivo. Furthermore, inhibition of Hh ligand with a monoclonal antibody (5E1) inhibited tumor growth and metastasis. These data suggest that epithelial-stromal Hh signaling, driven by ligand expression in carcinoma cells, promotes breast cancer growth and metastasis. Blockade of Hh signaling to peritumoral stromal cells may represent a novel therapeutic approach in some basal-like breast cancers.

Retroperitoneal inflammatory liposarcoma in a patient with non-hodgkin lymphoma: a report highlighting diagnostic pitfalls.

Well differentiated liposarcoma (WDLS) is the commonest subtype of liposarcoma. Recognised subtypes of WDLSs are lipoma-like, sclerosing, spindle cell and inflammatory. The inflammatory variant of WDLS also known as "lymphocyte-rich liposarcoma" is rare. We present a case of inflammatory WDLS occurring in the retroperitoneum, in a patient with a past history of non-Hodgkin lymphoma. We outline the histological features, discuss the differential diagnoses and highlight the diagnostic pitfalls in interpretation of this lesion on fine needle biopsy.

The prognostic value of tumor mitotic rate and other clinicopathologic factors in patients with locoregional recurrences of melanoma.

BACKGROUND: Tumor mitotic rate (MRP) is an independent prognostic factor in clinically localized primary cutaneous melanoma, but the prognostic importance of mitotic rate in melanoma recurrences (MRR) is not known. In this study, we sought to determine the prognostic value of MRR and other clinicopathologic factors in recurrent melanoma. METHODS: Patients with primary cutaneous melanoma diagnosed between 1979 and 2006, who subsequently developed recurrence(s), were studied. Histologic sections of primary and first locoregional recurrences of melanoma were examined, and MRP and MRR were measured. Relationships between MRR, known prognostic parameters in melanoma, time to first recurrence (TTR), and postrecurrence survival were analyzed. RESULTS: A total of 279 patients (172 men, 107 women) had AJCC stage I (n = 97) or stage II (n = 182) melanoma. Median MRP and MRR were 4/mm(2) (0-34) and 4/mm(2) (0-51), respectively. There was weak association between MRP and MRR (R (2) = 0.02, p = 0.02). Independent predictors of poorer postrecurrence survival were shorter TTR (hazard ratio, 0.74; 95% confidence interval, 0.61-0.90, p = 0.003) and recurrence type (10-year postrecurrence survival for local, lymph node, and in-transit recurrences: 70%, 21.5%, and 11.1%, respectively; p = 0.04). MRR >0 was associated with poorer 10-year postrecurrence survival (39.1%) than if MRR = 0 (51.2%), but the difference did not reach statistical significance (p = 0.15). However, the difference in survival between patients with MRR >0 and those with MRR = 0 increased with time. CONCLUSIONS: TTR is an independent predictor of postrecurrence survival. Because survival in patients with MRR >0 decreases with time (relative to those with MRR = 0), MRR should be routinely measured so that future studies can determine whether MRR has any independent predictive value.

Synchronous and metachronous malignancies in patients with melanoma: a clinicopathologic study highlighting the role of fine-needle biopsy cytology and potential diagnostic pitfalls.

Fine-needle biopsy (FNB) is commonly used in the investigation of patients with a history of melanoma who present with possible metastatic disease. Non-melanoma malignancies (NMM) are common in the general population and not infrequent in patients with melanoma. Such tumors may be difficult to distinguish from metastatic melanoma on FNB. We sought to determine the types of NMMs that occur in melanoma patients, to document the frequency with which they were diagnosed by FNB, and to highlight potential pitfalls in cytologic diagnosis. NMMs occurring in 1416 consecutive melanoma patients who underwent FNB of 2204 clinically suspicious lesions between 1992 and 2002 at a single center were reviewed and analyzed. The sites of FNB included lymph nodes (36.9%), skin and subcutis (25.1%), visceral locations (17.9%), and other sites (20.0%). Of the 1416 melanoma patients investigated by FNB, 116 (8.2%) had a metachronous or synchronous NMM; the NMM was diagnosed by the FNB in 17 (14.7%) patients. The most common NMMs were epithelial tumors (69.4%, most commonly carcinomas of large bowel, breast and prostate) and hematologic malignancies (21.8%). As NMMs are not infrequent in patients with melanoma, they should always be considered in the differential diagnosis of clinically suspicious masses in patients with a history of melanoma, as well as in patients at high risk of melanoma. Careful assessment of the FNB cytologic features and directed use of ancillary studies should enable accurate diagnosis in most cases and facilitate appropriate patient management.