Dynamics of breast-cancer relapse reveal late-recurring ER-positive genomic subgroups.

The rates and routes of lethal systemic spread in breast cancer are poorly understood owing to a lack of molecularly characterized patient cohorts with long-term, detailed follow-up data. Long-term follow-up is especially important for those with oestrogen-receptor (ER)-positive breast cancers, which can recur up to two decades after initial diagnosis1-6. It is therefore essential to identify patients who have a high risk of late relapse7-9. Here we present a statistical framework that models distinct disease stages (locoregional recurrence, distant recurrence, breast-cancer-related death and death from other causes) and competing risks of mortality from breast cancer, while yielding individual risk-of-recurrence predictions. We apply this model to 3,240 patients with breast cancer, including 1,980 for whom molecular data are available, and delineate spatiotemporal patterns of relapse across different categories of molecular information (namely immunohistochemical subtypes; PAM50 subtypes, which are based on gene-expression patterns10,11; and integrative or IntClust subtypes, which are based on patterns of genomic copy-number alterations and gene expression12,13). We identify four late-recurring integrative subtypes, comprising about one quarter (26%) of tumours that are both positive for ER and negative for human epidermal growth factor receptor 2, each with characteristic tumour-driving alterations in genomic copy number and a high risk of recurrence (mean 47-62%) up to 20 years after diagnosis. We also define a subgroup of triple-negative breast cancers in which cancer rarely recurs after five years, and a separate subgroup in which patients remain at risk. Use of the integrative subtypes improves the prediction of late, distant relapse beyond what is possible with clinical covariates (nodal status, tumour size, tumour grade and immunohistochemical subtype). These findings highlight opportunities for improved patient stratification and biomarker-driven clinical trials.

Development of a deep-learning model tailored for HER2 detection in breast cancer to aid pathologists in interpreting HER2-low cases.

AIMS: Over 50% of breast cancer cases are "Human epidermal growth factor receptor 2 (HER2) low breast cancer (BC)", characterized by HER2 immunohistochemistry (IHC) scores of 1+ or 2+ alongside no amplification on fluorescence in situ hybridization (FISH) testing. The development of new anti-HER2 antibody-drug conjugates (ADCs) for treating HER2-low breast cancers illustrates the importance of accurately assessing HER2 status, particularly HER2-low breast cancer. In this study we evaluated the performance of a deep-learning (DL) model for the assessment of HER2, including an assessment of the causes of discordances of HER2-Null between a pathologist and the DL model. We specifically focussed on aligning the DL model rules with the ASCO/CAP guidelines, including stained cells' staining intensity and completeness of membrane staining. METHODS AND RESULTS: We trained a DL model on a multicentric cohort of breast cancer cases with HER2-IHC scores (n = 299). The model was validated on two independent multicentric validation cohorts (n = 369 and n = 92), with all cases reviewed by three senior breast pathologists. All cases underwent a thorough review by three senior breast pathologists, with the ground truth determined by a majority consensus on the final HER2 score among the pathologists. In total, 760 breast cancer cases were utilized throughout the training and validation phases of the study. The model's concordance with the ground truth (ICC = 0.77 [0.68-0.83]; Fisher P = 1.32e-10) is higher than the average agreement among the three senior pathologists (ICC = 0.45 [0.17-0.65]; Fisher P = 2e-3). In the two validation cohorts, the DL model identifies 95% [93% - 98%] and 97% [91% - 100%] of HER2-low and HER2-positive tumours, respectively. Discordant results were characterized by morphological features such as extended fibrosis, a high number of tumour-infiltrating lymphocytes, and necrosis, whilst some artefacts such as nonspecific background cytoplasmic stain in the cytoplasm of tumour cells also cause discrepancy. CONCLUSION: Deep learning can support pathologists' interpretation of difficult HER2-low cases. Morphological variables and some specific artefacts can cause discrepant HER2-scores between the pathologist and the DL model.

Multiscale deep learning framework captures systemic immune features in lymph nodes predictive of triple negative breast cancer outcome in large-scale studies.

The suggestion that the systemic immune response in lymph nodes (LNs) conveys prognostic value for triple-negative breast cancer (TNBC) patients has not previously been investigated in large cohorts. We used a deep learning (DL) framework to quantify morphological features in haematoxylin and eosin-stained LNs on digitised whole slide images. From 345 breast cancer patients, 5,228 axillary LNs, cancer-free and involved, were assessed. Generalisable multiscale DL frameworks were developed to capture and quantify germinal centres (GCs) and sinuses. Cox regression proportional hazard models tested the association between smuLymphNet-captured GC and sinus quantifications and distant metastasis-free survival (DMFS). smuLymphNet achieved a Dice coefficient of 0.86 and 0.74 for capturing GCs and sinuses, respectively, and was comparable to an interpathologist Dice coefficient of 0.66 (GC) and 0.60 (sinus). smuLymphNet-captured sinuses were increased in LNs harbouring GCs (p < 0.001). smuLymphNet-captured GCs retained clinical relevance in LN-positive TNBC patients whose cancer-free LNs had on average ≥2 GCs, had longer DMFS (hazard ratio [HR] = 0.28, p = 0.02) and extended GCs' prognostic value to LN-negative TNBC patients (HR = 0.14, p = 0.002). Enlarged smuLymphNet-captured sinuses in involved LNs were associated with superior DMFS in LN-positive TNBC patients in a cohort from Guy's Hospital (multivariate HR = 0.39, p = 0.039) and with distant recurrence-free survival in 95 LN-positive TNBC patients of the Dutch-N4plus trial (HR = 0.44, p = 0.024). Heuristic scoring of subcapsular sinuses in LNs of LN-positive Tianjin TNBC patients (n = 85) cross-validated the association of enlarged sinuses with shorter DMFS (involved LNs: HR = 0.33, p = 0.029 and cancer-free LNs: HR = 0.21 p = 0.01). Morphological LN features reflective of cancer-associated responses are robustly quantifiable by smuLymphNet. Our findings further strengthen the value of assessment of LN properties beyond the detection of metastatic deposits for prognostication of TNBC patients. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Folate receptor alpha in ovarian cancer tissue and patient serum is associated with disease burden and treatment outcomes.

BACKGROUND: Survival rates for ovarian cancer remain poor, and monitoring and prediction of therapeutic response may benefit from additional markers. Ovarian cancers frequently overexpress Folate Receptor alpha (FRα) and the soluble receptor (sFRα) is measurable in blood. Here we investigated sFRα as a potential biomarker. METHODS: We evaluated sFRα longitudinally, before and during neo-adjuvant, adjuvant and palliative therapies, and tumour FRα expression status by immunohistrochemistry. The impact of free FRα on the efficacy of anti-FRα treatments was evaluated by an antibody-dependent cellular cytotoxicity assay. RESULTS: Membrane and/or cytoplasmic FRα staining were observed in 52.7% tumours from 316 ovarian cancer patients with diverse histotypes. Circulating sFRα levels were significantly higher in patients, compared to healthy volunteers, specifically in patients sampled prior to neoadjuvant and palliative treatments. sFRα was associated with FRα cell membrane expression in the tumour. sFRα levels decreased alongside concurrent tumour burden in patients receiving standard therapies. High concentrations of sFRα partly reduced anti-FRα antibody tumour cell killing, an effect overcome by increased antibody doses. CONCLUSIONS: sFRα may present a non-invasive marker for tumour FRα expression, with the potential for monitoring patient response to treatment. Larger, prospective studies should evaluate FRα for assessing disease burden and response to systemic treatments.

Invadopodia play a role in prostate cancer progression.

BACKGROUND: Invadopodia, actin-rich structures that release metallo-proteases at the interface with extra-cellular matrix, in a punctate manner are thought to be important drivers of tumour invasion. Invadopodia formation has been observed in-vitro and in-vivo in numerous metastatic cell lines derived from multiple tumour types. However, prostate cancer cell lines have not been routinely reported to generate invadopodia and the few instances have always required external stimulation. METHODS: In this study, the invasive potential of primary prostate adenocarcinoma cell lines, which have never been fully characterised before, was investigated both in-vitro invadopodia assays and in-vivo zebrafish dissemination assay. Subsequently, circulating tumour cells from prostate cancer patients were isolated and tested in the invadopodia assay. RESULTS: Retention of E-cadherin and N-cadherin expression indicated a transitional state of EMT progression, consistent with the idea of partial EMT that has been frequently observed in aggressive prostate cancer. All cell lines tested were capable of spontaneous invadopodia formation and possess a significant degradative ability in-vitro under basal conditions. These cell lines were invasive in-vivo and produced visible metastasis in the zebrafish dissemination assay. Importantly we have proceeded to demonstrate that circulating tumour cells isolated from prostate cancer patients exhibit invadopodia-like structures and degrade matrix with visible puncta. This work supports a role for invadopodia activity as one of the mechanisms of dissemination employed by prostate cancer cells. CONCLUSION: The combination of studies presented here provide clear evidence that invadopodia activity can play a role in prostate cancer progression.

Real-time ex vivo perfusion of human lymph nodes invaded by cancer (REPLICANT): a feasibility study.

Understanding how breast cancer (BC) grows in axillary lymph nodes (ALNs), and refining how therapies might halt that process, is clinically important. However, modelling the complex ALN microenvironment is difficult, and no human models exist at present. We harvested ALNs from ten BC patients, and perfused them at 37 °C ex vivo for up to 24 h. Controlled autologous testing showed that ALNs remain viable after 24 h of ex vivo perfusion: haematoxylin and eosin-stained histological appearance and proliferation (by Ki67 immunohistochemistry) did not change significantly over time for any perfused ALN compared with a control from time-point zero. Furthermore, targeted gene expression analysis (NanoString PanCancer IO360 panel) showed that only 21/750 genes were differentially expressed between control and perfused ALNs (|log2 FC| > 1 and q < 0.1): none were involved in apoptosis and metabolism, but rather all 21 genes were involved in immune function and angiogenesis. During perfusion, tissue acid-base balance remained stable. Interestingly, the flow rate increased (p < 0.001) in cancer-replaced (i.e. metastasis occupied more than 90% of the surface area on multiple levels) compared to cancer-free nodes (i.e. nodes with no metastasis on multiple sections). CXCL11 transcripts were significantly more abundant in cancer-replaced nodes, while CXCL12 transcripts were significantly more abundant in cancer-free nodes. These cytokines were also detected in the circulating perfusate. Monoclonal antibodies (nivolumab and trastuzumab) were administered into a further three ALNs to confirm perfusion efficacy. These drugs saturated the nodes; nivolumab even induced cancer cell death. Normothermic ALN perfusion is not only feasible but sustains the tumour microenvironment ex vivo for scientific investigation. This model could facilitate the identification of actionable immuno-oncology targets. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Cohort profile: King's Health Partners bladder cancer biobank.

BACKGROUND: Bladder cancer (BC) is the 9th most common cancer worldwide, but little progress has been made in improving patient outcomes over the last 25 years. The King's Health Partners (KHP) BC biobank was established to study unanswered, clinically relevant BC research questions. Donors are recruited from the Urology or Oncology departments of Guy's Hospital (UK) and can be approached for consent at any point during their treatment pathway. At present, patients with bladder cancer are approached to provide their consent to provide blood, urine and bladder tissue. They also give access to medical records and linkage of relevant clinical and pathological data across the course of their disease. Between June 2017 and June 2019, 531 out of 997 BC patients (53.3%) gave consent to donate samples and data to the Biobank. During this period, the Biobank collected fresh frozen tumour samples from 90/178 surgical procedures (of which 73 were biopsies) and had access to fixed, paraffin embedded samples from all patients who gave consent. Blood and urine samples have been collected from 38 patients, all of which were processed into component derivatives within 1 to 2 h of collection. This equates to 193 peripheral blood mononuclear cell vials; 238 plasma vials, 224 serum vials, 414 urine supernatant vials and 104 urine cell pellets. This biobank population is demographically and clinically representative of the KHP catchment area. CONCLUSION: The King's Health Partners BC Biobank has assembled a rich data and tissue repository which is clinically and demographically representative of the local South East London BC population, making it a valuable resource for future BC research.

Frequency of pathogenic germline variants in BRCA1, BRCA2, PALB2, CHEK2 and TP53 in ductal carcinoma in situ diagnosed in women under the age of 50 years.

INTRODUCTION: Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive ductal breast cancer, and approximately 20% of screen-detected tumours are pure DCIS. Most risk factors for breast cancer have similar associations with DCIS and IDC; however, there is limited data on the prevalence of the known high and moderate penetrance breast cancer predisposition genes in DCIS and which women with DCIS should be referred for genetic screening. The aim of this study was to assess the frequency of germline variants in BRCA2, BRCA1, CHEK2, PALB2 and TP53 in DCIS in women aged less than 50 years of age. METHODS: After DNA extraction from the peripheral blood, Access Array technology (Fluidigm) was used to amplify all exons of these five known breast cancer predisposition genes using a custom made targeted sequencing panel in 655 cases of pure DCIS presenting in women under the age of 50 years together with 1611 controls. RESULTS: Case-control analysis revealed an excess of pathogenic variants in BRCA2 (OR = 27.96, 95%CI 6.56-119.26, P = 2.0 × 10-10) and CHEK2 (OR = 8.04, 95%CI 2.93-22.05, P = 9.0 × 10-6), with weaker associations with PALB2 (P = 0.003), BRCA1 (P = 0.007) and TP53 (P = 0.02). For oestrogen receptor (ER)-positive DCIS the frequency of pathogenic variants was 9% under the age of 50 (14% with a family history of breast cancer) and 29% under the age of 40 (42% with a family history of breast cancer). For ER-negative DCIS, the frequency was 9% (16% with a family history of breast cancer) and 8% (11% with a family history of breast cancer) under the ages of 50 and 40, respectively. CONCLUSIONS: This study has shown that breast tumourigenesis in women with pathogenic variants in BRCA2, CHEK2, PALB2, BRCA1 and TP53 can involve a DCIS precursor stage and that the focus of genetic testing in DCIS should be on women under the age of 40 with ER-positive DCIS.

MED12, TERT promoter and RBM15 mutations in primary and recurrent phyllodes tumours.

BACKGROUND: MED12 and TERT promoter mutations have been shown to be the most common somatic mutations in phyllodes tumours (PTs). The aims of this study were to determine the frequency of these mutations in recurrent PTs, assess whether TERT promoter mutations could be helpful in distinguishing fibroadenomas (FAs) from PTs and identify novel mutations that may be driving malignant progression. METHODS: MED12 and the TERT promoter were Sanger sequenced in 75 primary PTs, 21 recurrences, 19 single FAs and 2 cases of multiple FAs with benign PTs. Whole-exome sequencing was performed on one borderline PT. RESULTS: Recurrent PTs and multiple FAs showed temporal discordance in MED12 but not TERT. Recurrent samples did acquire TERT mutations, with recurrent benign PTs more likely to have mutations in both genes. TERT mutations were not helpful in differentiating between benign PTs and FAs in cases of multiple FAs/PTs. Exome sequencing revealed a nonsense mutation in RBM15 and Sanger sequencing revealed another three RBM15 mutations in malignant/borderline PTs. CONCLUSIONS: This study has shown that MED12 mutations can be heterogeneous in both synchronous and recurrent PTs unlike TERT mutations. We have also shown that RBM15 mutations may be important in the pathogenesis of borderline/malignant PTs.

Gene expression modules in primary breast cancers as risk factors for organotropic patterns of first metastatic spread: a case control study.

BACKGROUND: Metastases from primary breast cancers can involve single or multiple organs at metastatic disease diagnosis. Molecular risk factors for particular patterns of metastastic spread in a clinical population are limited. METHODS: A case-control design including 1357 primary breast cancers was used to study three distinct clinical patterns of metastasis, which occur within the first six months of metastatic disease: bone and visceral metasynchronous spread, bone-only, and visceral-only metastasis. Whole-genome expression profiles were obtained using whole genome (WG)-DASL assays from formalin-fixed paraffin-embedded (FFPE) samples. A systematic protocol was developed for handling FFPE samples together with stringent data quality controls to identify robust expression profiling data. A panel of published and novel gene sets were tested for association with these specific patterns of metastatic spread and odds ratios (ORs) were calculated. RESULTS: Metasynchronous metastasis to bone and viscera was found in all intrinsic breast cancer subtypes, while immunohistochemically (IHC)-defined receptor status and specific IntClust subgroups were risk factors for visceral-only or bone-only first metastases. Among gene modules, those related to proliferation increased the risk of metasynchronous metastasis (OR (95% CI) = 2.3 (1.1-4.8)) and visceral-only first metastasis (OR (95% CI) = 2.5 (1.2-5.1)) but not bone-only metastasis (OR (95% CI) = 0.97 (0.56-1.7)). A 21-gene module (BV) was identified in estrogen-receptor-positive breast cancers with metasynchronous metastasis to bone and viscera (area under the curve = 0.77), and its expression increased the risk of bone and visceral metasynchronous spread in this population. BV was further orthogonally validated with NanoString nCounter in primary breast cancers, and was reproducible in their matched lymph nodes metastases and an external cohort. CONCLUSION: This case-control study of WG-DASL global expression profiles from FFPE tumour samples, after careful quality control and RNA selection, revealed that gene modules in the primary tumour have differing risks for clinical patterns of metasynchronous first metastases. Moreover, a novel gene module was identified as a putative risk factor for metasynchronous bone and visceral first metastatic spread, with potential implications for disease monitoring and treatment planning.

PIK3CA mutations are common in lobular carcinoma in situ, but are not a biomarker of progression.

BACKGROUND: Lobular carcinoma in situ (LCIS) is a non-invasive breast lesion that is typically found incidentally on biopsy and is often associated with invasive lobular carcinoma (ILC). LCIS is considered by some to be a risk factor for future breast cancer rather than a true precursor lesion. The aim of this study was to identify genetic changes that could be used as biomarkers of progression of LCIS to invasive disease using cases of pure LCIS and comparing their genetic profiles to LCIS which presented contemporaneously with associated ILC, on the hypothesis that the latter represents LCIS that has already progressed. METHODS: Somatic copy number aberrations (SCNAs) were assessed by SNP array in three subgroups: pure LCIS, LCIS associated with ILC and the paired ILC. In addition exome sequencing was performed on seven fresh frozen samples of LCIS associated with ILC, to identify recurrent somatic mutations. RESULTS: The copy number profiles of pure LCIS and LCIS associated with ILC were almost identical. However, four SCNAs were more frequent in ILC than LCIS associated with ILC, including gain/amplification of CCND1. CCND1 protein over-expression assessed by immunohistochemical analysis in a second set of samples from 32 patients with pure LCIS and long-term follow up, was associated with invasive recurrence (P = 0.02, Fisher's exact test). Exome sequencing revealed that PIK3CA mutations were as frequent as CDH1 mutations in LCIS, but were not a useful biomarker of LCIS progression as they were as frequent in pure LCIS as in LCIS associated with ILC. We also observed heterogeneity of PIK3CA mutations and evidence of sub-clonal populations in LCIS irrespective of whether they were associated with ILC. CONCLUSIONS: Our data shows that pure LCIS and LCIS co-existing with ILC have very similar SCNA profiles, supporting the hypothesis that LCIS is a true precursor lesion. We have provided evidence that over-expression of CCND1 may identify a subgroup of patients with pure LCIS who are more likely to develop invasive disease, in contrast to PIK3CA mutations, which occur too early in lobular tumorigenesis to be informative.

Metformin and longevity (METAL): a window of opportunity study investigating the biological effects of metformin in localised prostate cancer.

BACKGROUND: Metformin is a biguanide oral hypoglycaemic agent commonly used for the treatment of type 2 diabetes mellitus. In addition to its anti-diabetic effect, metformin has also been associated with a reduced risk of cancer incidence of a number of solid tumours, including prostate cancer (PCa). However, the underlying biological mechanisms for these observations have not been fully characterised in PCa. One hypothesis is that the indirect insulin lowering effect may have an anti-neoplastic action as elevated insulin and insulin like growth factor - 1 (IGF-1) levels play a role in PCa development and progression. In addition, metformin is a potent activator of activated protein kinase (AMPK) which in turn inhibits the mammalian target of rapamycin (mTOR) and other signal transduction mechanisms. These direct effects can lead to reduced cell proliferation. Given its wide availability and tolerable side effect profile, metformin represents an attractive potential therapeutic option for men with PCa. Hence, the need for a clinical trial investigating its biological mechanisms in PCa. METHODS: METAL is a randomised, placebo-controlled, double-blind, window of opportunity study investigating the biological mechanism of metformin in PCa. 100 patients with newly-diagnosed, localised PCa scheduled for radical prostatectomy will be randomised 1:1 to receive metformin (1 g b.d.) or placebo for four weeks (+/- 1 week) prior to prostatectomy. Tissue will be collected from both diagnostic biopsy and prostatectomy specimens. The primary endpoint is the difference in expression levels of markers of the Fatty acid synthase (FASN)/AMPK pathway pre and post treatment between the placebo and metformin arms. Secondary endpoints include the difference in expression levels of indicators of proliferation (ki67 and TUNEL) pre and post treatment between the placebo and metformin arms. METAL is currently open to recruitment at Guy's and St Thomas' Hospital and the Royal Marsden Hospital, London. DISCUSSION: This randomised placebo-controlled double blinded trial of metformin vs. placebo in men with localised PCa due to undergo radical prostatectomy, aims to elucidate the mechanism of action of metformin in PCa cells, which should then enable further larger stratification trials to take place. TRIAL REGISTRATION: EudraCT number 2014-005193-11 . Registered on September 09, 2015.

Genetic predisposition to in situ and invasive lobular carcinoma of the breast.

Invasive lobular breast cancer (ILC) accounts for 10-15% of all invasive breast carcinomas. It is generally ER positive (ER+) and often associated with lobular carcinoma in situ (LCIS). Genome-wide association studies have identified more than 70 common polymorphisms that predispose to breast cancer, but these studies included predominantly ductal (IDC) carcinomas. To identify novel common polymorphisms that predispose to ILC and LCIS, we pooled data from 6,023 cases (5,622 ILC, 401 pure LCIS) and 34,271 controls from 36 studies genotyped using the iCOGS chip. Six novel SNPs most strongly associated with ILC/LCIS in the pooled analysis were genotyped in a further 516 lobular cases (482 ILC, 36 LCIS) and 1,467 controls. These analyses identified a lobular-specific SNP at 7q34 (rs11977670, OR (95%CI) for ILC = 1.13 (1.09-1.18), P = 6.0 × 10(-10); P-het for ILC vs IDC ER+ tumors = 1.8 × 10(-4)). Of the 75 known breast cancer polymorphisms that were genotyped, 56 were associated with ILC and 15 with LCIS at P<0.05. Two SNPs showed significantly stronger associations for ILC than LCIS (rs2981579/10q26/FGFR2, P-het = 0.04 and rs889312/5q11/MAP3K1, P-het = 0.03); and two showed stronger associations for LCIS than ILC (rs6678914/1q32/LGR6, P-het = 0.001 and rs1752911/6q14, P-het = 0.04). In addition, seven of the 75 known loci showed significant differences between ER+ tumors with IDC and ILC histology, three of these showing stronger associations for ILC (rs11249433/1p11, rs2981579/10q26/FGFR2 and rs10995190/10q21/ZNF365) and four associated only with IDC (5p12/rs10941679; rs2588809/14q24/RAD51L1, rs6472903/8q21 and rs1550623/2q31/CDCA7). In conclusion, we have identified one novel lobular breast cancer specific predisposition polymorphism at 7q34, and shown for the first time that common breast cancer polymorphisms predispose to LCIS. We have shown that many of the ER+ breast cancer predisposition loci also predispose to ILC, although there is some heterogeneity between ER+ lobular and ER+ IDC tumors. These data provide evidence for overlapping, but distinct etiological pathways within ER+ breast cancer between morphological subtypes.

Progression of breast cancer following locoregional ipsilateral recurrence: importance of interval time.

BACKGROUND: Studies comparing prognosis of breast cancer (BC) patients with and without locoregional recurrence (LR) present conflicting results. We aimed to improve our understanding of the impact of LR on prognosis by examining a large cohort of patients treated at Guy's and St Thomas' NHS Foundation Trust. METHODS: Risk factors associated with BC-specific death were investigated using Cox proportional hazards regression in 5199 women diagnosed between 1975 and 2007. Breast cancer-specific death following LR was assessed with Poisson regression. RESULTS: Overall, 552 women (11%) developed LR, with a median follow-up time of 4.28 years. Known factors associated with BC-specific death (tumour stage, grade, and nodal status) were of significance in our data. Women with a shorter disease-free interval had a worse prognosis. For instance, the HR for BC-specific death among women undergoing mastectomy with an LR 0.5-1 year after diagnosis of their primary tumour was 6.67 (95% CI: 3.71-11.99), when compared with women who did not experience LR. CONCLUSIONS: It often remains difficult to distinguish between a genuine LR and a new primary. The HRs for risk of BC-specific death following a second lesion suggest that they may act as a marker of systemic disease, large tumour burden, or depleted host defence. The clinically highly relevant impairment in prognosis calls for further research into the underlying mechanisms. We showed that for at least 15 years of follow-up, the prognosis in women following the occurrence of an LR may benefit from careful diagnostic and therapeutic management.

HOX transcription factors are potential targets and markers in malignant mesothelioma.

BACKGROUND: The HOX genes are a family of homeodomain-containing transcription factors that determine cellular identity during development and which are dys-regulated in some cancers. In this study we examined the expression and oncogenic function of HOX genes in mesothelioma, a cancer arising from the pleura or peritoneum which is associated with exposure to asbestos. METHODS: We tested the sensitivity of the mesothelioma-derived lines MSTO-211H, NCI-H28, NCI-H2052, and NCI-H226 to HXR9, a peptide antagonist of HOX protein binding to its PBX co-factor. Apoptosis was measured using a FACS-based assay with Annexin, and HOX gene expression profiles were established using RT-QPCR on RNA extracted from cell lines and primary mesotheliomas. The in vivo efficacy of HXR9 was tested in a mouse MSTO-211H flank tumor xenograft model. RESULTS: We show that HOX genes are significantly dysregulated in malignant mesothelioma. Targeting HOX genes with HXR9 caused apoptotic cell death in all of the mesothelioma-derived cell lines, and prevented the growth of mesothelioma tumors in a mouse xenograft model. Furthermore, the sensitivity of these lines to HXR9 correlated with the relative expression of HOX genes that have either an oncogenic or tumor suppressive function in cancer. The analysis of HOX expression in primary mesothelioma tumors indicated that these cells could also be sensitive to the disruption of HOX activity by HXR9, and that the expression of HOXB4 is strongly associated with overall survival. CONCLUSION: HOX genes are a potential therapeutic target in mesothelioma, and HOXB4 expression correlates with overall survival.

Big Data: the challenge for small research groups in the era of cancer genomics.

In the past decade, cancer research has seen an increasing trend towards high-throughput techniques and translational approaches. The increasing availability of assays that utilise smaller quantities of source material and produce higher volumes of data output have resulted in the necessity for data storage solutions beyond those previously used. Multifactorial data, both large in sample size and heterogeneous in context, needs to be integrated in a standardised, cost-effective and secure manner. This requires technical solutions and administrative support not normally financially accounted for in small- to moderate-sized research groups. In this review, we highlight the Big Data challenges faced by translational research groups in the precision medicine era; an era in which the genomes of over 75,000 patients will be sequenced by the National Health Service over the next 3 years to advance healthcare. In particular, we have looked at three main themes of data management in relation to cancer research, namely (1) cancer ontology management, (2) IT infrastructures that have been developed to support data management and (3) the unique ethical challenges introduced by utilising Big Data in research.

Digital imaging in the immunohistochemical evaluation of the proliferation markers Ki67, MCM2 and Geminin, in early breast cancer, and their putative prognostic value.

BACKGROUND: Immunohistochemical assessment of proliferation may provide additional prognostic information in early breast cancer. However, due to a lack of methodological standards proliferation markers are still not routinely used for determining therapy. Even for Ki67, one of the most widely-studied markers, disagreements over the optimal cutoff exist. Improvements in digital microscopy may provide new avenues to standardise and make data more reproducible. METHODS: We studied the immunohistochemical expression of three markers of proliferation: Ki67, Mini-Chromosome Maintenance protein 2 and Geminin, by conventional light microscope and digital imaging on triplicate TMAs from 309 consecutive cases of primary breast cancers. Differences between the average and the maximum percentage reactivity in tumour cell nuclei from the three TMA cores were investigated to assess the validity of the approach. Time-dependent Receiver Operating Characteristic curves were utilized to obtain optimal expression level cut-offs, which were then correlated with clinico-pathological features and survival. RESULTS: High concordance between conventional and digital scores was observed for all 3 markers (Ki67: rs = 0.87, P < 0.001; MCM2: rs = 0.94, P < 0.001; and Geminin: rs = 0.86, P < 0.001; Spearman's rank). There was no significant difference according to the number of TMA cores included for either Ki67 or MCM2; analysis of two or three cores produced comparable results. Higher levels of all three proliferation markers were significantly associated with higher grade (P < 0.001) and ER-negativity (P < 0.001). Optimal prognostic cut-offs for percentage expression in the tumour were 8 %, 12 and 2.33 % for Ki67, MCM2 and Geminin respectively. All 3 proliferation marker cutoffs were predictive of 15-year breast cancer-specific survival in univariable Cox regression analyses. In multivariable analysis only lymph node status (HR = 3.9, 95 % CI = 1.79-8.5, P = 0.0006) and histological grade (HR = 1.84, 95 % CI = 1-3.38, P = 0.05) remained significantly prognostic. CONCLUSIONS: Here we show that. MCM2 is a more sensitive marker of proliferation than Ki67 and should be examined in future studies, especially in the lymph node-negative, hormone receptor-positive subgroup. Further, digital microscopy can be used effectively as a high-throughput method to evaluate immunohistochemical expression.

Phosphorylation of NTRK1 at Y674/Y675 induced by TP53-dependent repression of PTPN6 expression: a potential novel prognostic marker for breast cancer.

We have identified a ligand-independent mechanism whereby the tumor suppressor, TP53, induces nerve growth factor receptor, NTRK1, phosphorylation at Y674/Y675 (NTRK1-pY674/pY675), via the repression of the NTRK1-phosphatase, PTPN6. This results in suppression of breast cancer cell proliferation. In this investigation, we aimed to establish whether perturbation of the wild-type TP53-NTRK1-pY674/pY675-PTPN6 pathway has an impact on disease-free survival of breast cancer patients without neo-adjuvant treatment. A total of 308 tumor samples were stained for NTRK1, NTRK1-pY674/pY675, PTPN6, and TP53 expression. Association between expression levels and disease-free survival was determined by the univariate/multivariate and Kaplan-Meir methods of analysis. DNA from tumors was sequenced to identify mutant or wild-type TP53. Tumors expressing NTRK1-pY674/pY675 but with undetectable or low levels of PTPN6 and TP53 were associated with prolonged 5, 10, and 15 years' disease-free survival by 48%, 36%, and 37%, respectively, in the multivariate analysis (P<0.05). A similar result was observed in tumors expressing wild-type TP53, NTRK1-pY674/pY675, and low or undetectable levels of PTPN6. Given that estrogen receptor-positive breast cancers encode wild-type TP53, we analyzed this expression pattern in these tumors. Multivariate analysis showed that it was significantly and independently predictive of prolonged survival by 66%, 70%, and 84%, respectively, (P<0.05). The Kaplan-Meir method demonstrated that NTRK1-pY674/pY675 together with undetectable or low levels of PTPN6 correlated with 59% probability of disease-free survival (median survival 15 years), compared with 7% probability of disease-free survival (median survival 4.5 years) when absent. In luminal A tumors, the presence of this pattern was estimated to have a 61% probability of disease-free survival (median survival 15 years), compared with 6% probability of disease-free survival (median survival 3 years) when it was absent. These results strongly suggest that expression of NTRK1-pY674/pY675 together with wild-type TP53 and low levels of PTPN6 expression are predictors of improved disease-free survival and that they could be useful biomarkers to predict clinical outcome.

Imaging tumour heterogeneity of the consequences of a PKCα-substrate interaction in breast cancer patients.

Breast cancer heterogeneity demands that prognostic models must be biologically driven and recent clinical evidence indicates that future prognostic signatures need evaluation in the context of early compared with late metastatic risk prediction. In pre-clinical studies, we and others have shown that various protein-protein interactions, pertaining to the actin microfilament-associated proteins, ezrin and cofilin, mediate breast cancer cell migration, a prerequisite for cancer metastasis. Moreover, as a direct substrate for protein kinase Cα, ezrin has been shown to be a determinant of cancer metastasis for a variety of tumour types, besides breast cancer; and has been described as a pivotal regulator of metastasis by linking the plasma membrane to the actin cytoskeleton. In the present article, we demonstrate that our tissue imaging-derived parameters that pertain to or are a consequence of the PKC-ezrin interaction can be used for breast cancer prognostication, with inter-cohort reproducibility. The application of fluorescence lifetime imaging microscopy (FLIM) in formalin-fixed paraffin-embedded patient samples to probe protein proximity within the typically <10 nm range to address the oncological challenge of tumour heterogeneity, is discussed.