M-FISH evaluation of chromosome aberrations to examine for historical exposure to ionising radiation due to participation at British nuclear test sites.

Veterans of the British nuclear testing programme represent a population of ex-military personnel who had the potential to be exposed to ionising radiation through their participation at nuclear testing sites in the 1950s and 1960s. In the intervening years, members of this population have raised concerns about the status of their health and that of their descendants, as a consequence. Radiation dose estimates based on film badge measurements of external dose recorded at the time of the tests suggest any exposure to be limited for the majority of personnel, however, only ∼20% of personnel were monitored and no measurement for internalised exposure are on record. Here, to in-part address families concerns, we assay for chromosomal evidence of historical radiation exposure in a group of aged nuclear test (NT) veterans, using multiplexin situhybridisation (M-FISH), for comparison with a matched group of veterans who were not present at NT sites. In total, we analysed 9379 and 7698 metaphase cells using M-FISH (24-colour karyotyping) from 48 NT and 38 control veteran samples, representing veteran servicemen from the army, Royal Airforce and Royal Navy. We observed stable and unstable simple- and complex-type chromosome aberrations in both NT and control veterans' samples, however find no significant difference in yield of any chromosome aberration type between the two cohorts. We do observe higher average frequencies of complex chromosome aberrations in a very small subset of veterans previously identified as having a higher potential for radiation exposure, which may be indicative of internalised contamination to long-lived radionuclides from radiation fallout. By utilising recently published whole genome sequence analysis data of a sub-set of the same family groups, we examined for but found no relationship between paternal chromosome aberration burden, germline mutation frequency and self-reported concerns of adverse health in family members, suggesting that the previously reported health issues by participants in this study are unlikely to be associated with historical radiation exposure. We did observe a small number of families, representing both control and NT cohorts, showing a relationship between paternal chromosome aberrations and germline mutation sub-types which should be explored in future studies. In conclusion, we find no cytogenetic evidence of historical radiation exposure in the cohort of nuclear veterans sampled here, offering reassurance that attendance at NTs sites by the veterans sampled here, was not associated with significant levels of exposure to radiation.

Etiology of hormone receptor positive breast cancer differs by levels of histologic grade and proliferation.

Limited epidemiological evidence suggests that the etiology of hormone receptor positive (HR+) breast cancer may differ by levels of histologic grade and proliferation. We pooled risk factor and pathology data on 5,905 HR+ breast cancer cases and 26,281 controls from 11 epidemiological studies. Proliferation was determined by centralized automated measures of KI67 in tissue microarrays. Odds ratios (OR), 95% confidence intervals (CI) and p-values for case-case and case-control comparisons for risk factors in relation to levels of grade and quartiles (Q1-Q4) of KI67 were estimated using polytomous logistic regression models. Case-case comparisons showed associations between nulliparity and high KI67 [OR (95% CI) for Q4 vs. Q1 = 1.54 (1.22, 1.95)]; obesity and high grade [grade 3 vs. 1 = 1.68 (1.31, 2.16)] and current use of combined hormone therapy (HT) and low grade [grade 3 vs. 1 = 0.27 (0.16, 0.44)] tumors. In case-control comparisons, nulliparity was associated with elevated risk of tumors with high but not low levels of proliferation [1.43 (1.14, 1.81) for KI67 Q4 vs. 0.83 (0.60, 1.14) for KI67 Q1]; obesity among women ≥50 years with high but not low grade tumors [1.55 (1.17, 2.06) for grade 3 vs. 0.88 (0.66, 1.16) for grade 1] and HT with low but not high grade tumors [3.07 (2.22, 4.23) for grade 1 vs. 0.85 (0.55, 1.30) for grade 3]. Menarcheal age and family history were similarly associated with HR+ tumors of different grade or KI67 levels. These findings provide insights into the etiologic heterogeneity of HR+ tumors.

FFPE breast tumour blocks provide reliable sources of both germline and malignant DNA for investigation of genetic determinants of individual tumour responses to treatment.

BACKGROUND: Bio-banked formalin-fixed paraffin-embedded (FFPE) tissues provide an excellent opportunity for translational genomic research. Historically matched blood has not always been collected as a source of germline DNA. This project aimed to establish if normal FFPE breast tissue could be used as an alternative to blood. METHODS: Exome sequencing was carried out on matched tumour tissue, normal breast tissue and blood on five patients in the START trial. Retrieved samples had been archived at different centres for at least 13 years. Following tissue macro-dissection and DNA extraction, targeted exome capture was performed using SureSelect Human All Exome v5 reagents (Agilent). Illumina paired-end libraries were prepared from the captured target regions and sequenced on a HiSeq2500 (Illumina) acquiring 2 × 75 bp reads. Somatic variants were called using the MuTect software analysis tool and copy number abnormalities (CNA) were identified using CNVkit. Targeted sequencing and droplet digital PCR were used to validate somatic variants and CNA, respectively. RESULTS: Overlap of somatic variants and CNA called on tumour versus blood and tumour versus normal breast tissue was good. Agreement in somatic variant calling ranged from 76.9 to 93.6%. Variants with an allele frequency lower than 10% were more difficult to validate irrespective of the type of germline DNA used. Pearson's correlation coefficients for paired comparisons of CNA using blood or normal tissue as reference ranged from 0.70 to 0.94. CONCLUSIONS: There is good correlation between the somatic mutations and CNA called using archived blood or normal breast tissue as germline reference material.

SOX11 promotes invasive growth and ductal carcinoma in situ progression.

Here, we show that SOX11, an embryonic mammary marker that is normally silent in postnatal breast cells, is expressed in many oestrogen receptor-negative preinvasive ductal carcinoma in situ (DCIS) lesions. Mature mammary epithelial cells engineered to express SOX11 showed alterations in progenitor cell populations, including an expanded basal-like population with increased aldehyde dehydrogenase (ALDH) activity, and increased mammosphere-forming capacity. DCIS.com cells engineered to express SOX11 showed increased ALDH activity, which is a feature of cancer stem cells. The CD44+/CD24-/ALDH+ cell population was increased in DCIS.com cells that expressed SOX11. Upregulating SOX11 expression in DCIS.com cells led to increased invasive growth both in vitro and when they were injected intraductally in a mouse model of DCIS that recapitulates human disease. Invasive lesions formed sooner and tumour growth was augmented in vivo, suggesting that SOX11 contributes to the progression of DCIS to invasive breast cancer. We identified potential downstream effectors of SOX11 during both microinvasive and invasive tumour growth stages, including several with established links to regulation of progenitor cell function and prenatal developmental growth. Our findings suggest that SOX11 is a potential biomarker for DCIS lesions containing cells harbouring distinct biological features that are likely to progress to invasive breast cancer. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Vessel co-option is common in human lung metastases and mediates resistance to anti-angiogenic therapy in preclinical lung metastasis models.

Anti-angiogenic therapies have shown limited efficacy in the clinical management of metastatic disease, including lung metastases. Moreover, the mechanisms via which tumours resist anti-angiogenic therapies are poorly understood. Importantly, rather than utilizing angiogenesis, some metastases may instead incorporate pre-existing vessels from surrounding tissue (vessel co-option). As anti-angiogenic therapies were designed to target only new blood vessel growth, vessel co-option has been proposed as a mechanism that could drive resistance to anti-angiogenic therapy. However, vessel co-option has not been extensively studied in lung metastases, and its potential to mediate resistance to anti-angiogenic therapy in lung metastases is not established. Here, we examined the mechanism of tumour vascularization in 164 human lung metastasis specimens (composed of breast, colorectal and renal cancer lung metastasis cases). We identified four distinct histopathological growth patterns (HGPs) of lung metastasis (alveolar, interstitial, perivascular cuffing, and pushing), each of which vascularized via a different mechanism. In the alveolar HGP, cancer cells invaded the alveolar air spaces, facilitating the co-option of alveolar capillaries. In the interstitial HGP, cancer cells invaded the alveolar walls to co-opt alveolar capillaries. In the perivascular cuffing HGP, cancer cells grew by co-opting larger vessels of the lung. Only in the pushing HGP did the tumours vascularize by angiogenesis. Importantly, vessel co-option occurred with high frequency, being present in >80% of the cases examined. Moreover, we provide evidence that vessel co-option mediates resistance to the anti-angiogenic drug sunitinib in preclinical lung metastasis models. Assuming that our interpretation of the data is correct, we conclude that vessel co-option in lung metastases occurs through at least three distinct mechanisms, that vessel co-option occurs frequently in lung metastases, and that vessel co-option could mediate resistance to anti-angiogenic therapy in lung metastases. Novel therapies designed to target both angiogenesis and vessel co-option are therefore warranted. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Three-dimensional modelling identifies novel genetic dependencies associated with breast cancer progression in the isogenic MCF10 model.

The initiation and progression of breast cancer from the transformation of the normal epithelium to ductal carcinoma in situ (DCIS) and invasive disease is a complex process involving the acquisition of genetic alterations and changes in gene expression, alongside microenvironmental and recognized histological alterations. Here, we sought to comprehensively characterise the genomic and transcriptomic features of the MCF10 isogenic model of breast cancer progression, and to functionally validate potential driver alterations in three-dimensional (3D) spheroids that may provide insights into breast cancer progression, and identify targetable alterations in conditions more similar to those encountered in vivo. We performed whole genome, exome and RNA sequencing of the MCF10 progression series to catalogue the copy number and mutational and transcriptomic landscapes associated with progression. We identified a number of predicted driver mutations (including PIK3CA and TP53) that were acquired during transformation of non-malignant MCF10A cells to their malignant counterparts that are also present in analysed primary breast cancers from The Cancer Genome Atlas (TCGA). Acquisition of genomic alterations identified MYC amplification and previously undescribed RAB3GAP1-HRAS and UBA2-PDCD2L expressed in-frame fusion genes in malignant cells. Comparison of pathway aberrations associated with progression showed that, when cells are grown as 3D spheroids, they show perturbations of cancer-relevant pathways. Functional interrogation of the dependency on predicted driver events identified alterations in HRAS, PIK3CA and TP53 that selectively decreased cell growth and were associated with progression from preinvasive to invasive disease only when cells were grown as spheroids. Our results have identified changes in the genomic repertoire in cell lines representative of the stages of breast cancer progression, and demonstrate that genetic dependencies can be uncovered when cells are grown in conditions more like those in vivo. The MCF10 progression series therefore represents a good model with which to dissect potential biomarkers and to evaluate therapeutic targets involved in the progression of breast cancer. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Prognostic value of automated KI67 scoring in breast cancer: a centralised evaluation of 8088 patients from 10 study groups.

BACKGROUND: The value of KI67 in breast cancer prognostication has been questioned due to concerns on the analytical validity of visual KI67 assessment and methodological limitations of published studies. Here, we investigate the prognostic value of automated KI67 scoring in a large, multicentre study, and compare this with pathologists' visual scores available in a subset of patients. METHODS: We utilised 143 tissue microarrays containing 15,313 tumour tissue cores from 8088 breast cancer patients in 10 collaborating studies. A total of 1401 deaths occurred during a median follow-up of 7.5 years. Centralised KI67 assessment was performed using an automated scoring protocol. The relationship of KI67 levels with 10-year breast cancer specific survival (BCSS) was investigated using Kaplan-Meier survival curves and Cox proportional hazard regression models adjusted for known prognostic factors. RESULTS: Patients in the highest quartile of KI67 (>12 % positive KI67 cells) had a worse 10-year BCSS than patients in the lower three quartiles. This association was statistically significant for ER-positive patients (hazard ratio (HR) (95 % CI) at baseline = 1.96 (1.31-2.93); P = 0.001) but not for ER-negative patients (1.23 (0.86-1.77); P = 0.248) (P-heterogeneity = 0.064). In spite of differences in characteristics of the study populations, the estimates of HR were consistent across all studies (P-heterogeneity = 0.941 for ER-positive and P-heterogeneity = 0.866 for ER-negative). Among ER-positive cancers, KI67 was associated with worse prognosis in both node-negative (2.47 (1.16-5.27)) and node-positive (1.74 (1.05-2.86)) tumours (P-heterogeneity = 0.671). Further classification according to ER, PR and HER2 showed statistically significant associations with prognosis among hormone receptor-positive patients regardless of HER2 status (P-heterogeneity = 0.270) and among triple-negative patients (1.70 (1.02-2.84)). Model fit parameters were similar for visual and automated measures of KI67 in a subset of 2440 patients with information from both sources. CONCLUSIONS: Findings from this large-scale multicentre analysis with centrally generated automated KI67 scores show strong evidence in support of a prognostic value for automated KI67 scoring in breast cancer. Given the advantages of automated scoring in terms of its potential for standardisation, reproducibility and throughput, automated methods appear to be promising alternatives to visual scoring for KI67 assessment.

Immunophenotypic and genomic characterization of papillary carcinomas of the breast.

Papillary carcinomas are a special histological type of breast cancer and have a relatively good outcome. We characterized the genomic and phenotypic characteristics of papillary carcinomas to determine whether they would constitute an entity distinct from grade- and oestrogen receptor (ER)-matched invasive ductal carcinomas of no special type (IDC-NSTs). The phenotype of 63 papillary carcinomas of the breast and grade- and ER-matched IDC-NSTs was determined by immunohistochemistry. DNA of sufficient quality was extracted from 49 microdissected papillary carcinomas and 49 microdissected grade- and ER-matched IDC-NSTs. These samples were subjected to high-resolution microarray-based comparative genomic hybridization (aCGH) and Sequenom MassARRAY sequencing analysis of 19 known oncogenes. Papillary carcinomas were predominantly of low histological grade, expressed immunohistochemical markers consistent with a luminal phenotype, and a lower rate of lymph node metastasis and p53 expression than grade- and ER-matched IDC-NSTs. Papillary carcinomas displayed less genomic aberrations than grade- and ER-matched IDC-NSTs; however, the patterns of gene copy number aberrations found in papillary carcinomas were similar to those of ER- and grade-matched IDC-NSTs, including 16q losses. Furthermore, PIK3CA mutations were found in 43% and 29% of papillary carcinomas and grade- and ER-matched IDC-NSTs, respectively. The genomic profiles of encapsulated, solid and invasive papillary carcinomas, the three morphological subtypes, were remarkably similar. Our results demonstrate that papillary carcinomas are a homogeneous special histological type of breast cancer. The similarities in the genomic profiles of papillary carcinomas and grade- and ER-matched IDC-NSTs suggest that papillary carcinomas may be best positioned as part of the spectrum of ER-positive breast cancers, rather than as a distinct entity. Furthermore, the good prognosis of papillary carcinomas may stem from the low rates of lymph node metastasis and p53 expression, low number of gene copy number aberrations and high prevalence of PIK3CA mutations.

Multi-candidate immunohistochemical markers to assess radiation response and prognosis in prostate cancer: results from the CHHiP trial of radiotherapy fractionation.

BACKGROUND: Protein markers of cellular proliferation, hypoxia, apoptosis, cell cycle checkpoints, growth factor signalling and inflammation in localised prostate tumours have previously shown prognostic ability. A translational substudy within the CHHiP trial of radiotherapy fractionation evaluated whether these could improve prediction of prognosis and assist treatment stratification following either conventional or hypofractionated radiotherapy. METHODS: Using case:control methodology, patients with biochemical or clinical failure after radiotherapy (BCR) were matched to patients without recurrence according to established prognostic factors (Gleason score, presenting PSA, tumour-stage) and fractionation schedule. Immunohistochemical (IHC) staining of diagnostic biopsy sections was performed and scored for HIF1α, Bcl-2, Ki67, Geminin, p16, p53, p-chk1 and PTEN. Univariable and multivariable conditional logistic regression models, adjusted for matching strata and age, estimated the prognostic value of each IHC biomarker, including interaction terms to determine BCR prediction according to fractionation. FINDINGS: IHC results were available for up to 336 tumours. PTEN, Geminin, mean Ki67 and max Ki67 were prognostic after adjusting for multiple comparisons and were fitted in a multivariable model (n = 212, 106 matched pairs). Here, PTEN and Geminin showed significant prediction of prognosis. No marker predicted BCR according to fractionation. INTERPRETATION: Geminin or Ki67, and PTEN, predicted response to radiotherapy independently of established prognostic factors. These results provide essential independent external validation of previous findings and confirm a role for these markers in treatment stratification. FUNDING: Cancer Research UK (BIDD) grant (A12518), Cancer Research UK (C8262/A7253), Department of Health, Prostate Cancer UK, Movember Foundation, NIHR Biomedical Research Centre at Royal Marsden/ICR.

Operable non-small cell lung cancer: correlation of volumetric helical dynamic contrast-enhanced CT parameters with immunohistochemical markers of tumor hypoxia.

PURPOSE: To assess the relationship between helical dynamic contrast material-enhanced (DCE) computed tomographic (CT) parameters and immunohistochemical markers of hypoxia in patients with operable non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: After institutional review board approval was obtained, 20 prospective patients who were suspected of having NSCLC underwent whole-tumor DCE CT with kinetic modeling (Patlak analysis) 24 hours before scheduled surgery. Flow-extraction product (in milliliters per 100 milliliters per minute) and blood volume (in milliliters per 100 milliliters) were derived. After surgery, matched whole-tumor sections were stained for exogenous and endogenous markers of hypoxia (pimonidazole infused intravenously 24 hours before surgery, immediately after DCE CT; glucose transporter protein). Correlation between DCE CT parameters and immunohistochemical markers was assessed by using the Spearman rank correlation. DCE CT parameters and immunohistochemical markers were also compared according to pathologic subtype, grade, stage, and nodal status by using the Mann-Whitney test. P values less than .05 indicated a statistically significant difference. RESULT: Fourteen patients with confirmed primary NSCLC underwent resection. There were negative correlations between blood volume and pimonidazole staining (r = -0.48, P = .004), and between flow-extraction product and glucose transporter protein expression (r = -0.50, P = .002). Flow-extraction product was significantly higher in adenocarcinomas than in squamous cell tumors (17.73 vs 11.46; P = .043). Glucose transporter protein expression was significantly lower for adenocarcinomas than for squamous tumors (14.07 vs 33.03; P < .001) and in node negative than in node positive tumors (15.63 vs 23.85; P = .005). CONCLUSION: Blood volume and flow-extraction product derived at DCE CT correlated negatively with pimonidazole and glucose transporter protein expression, indicating the potential of these CT parameters as imaging biomarkers of hypoxia.

3D Functional Genomics Screens Identify CREBBP as a Targetable Driver in Aggressive Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBC) are resistant to standard-of-care chemotherapy and lack known targetable driver gene alterations. Identification of novel drivers could aid the discovery of new treatment strategies for this hard-to-treat patient population, yet studies using high-throughput and accurate models to define the functions of driver genes in TNBC to date have been limited. Here, we employed unbiased functional genomics screening of the 200 most frequently mutated genes in breast cancer, using spheroid cultures to model in vivo-like conditions, and identified the histone acetyltransferase CREBBP as a novel tumor suppressor in TNBC. CREBBP protein expression in patient tumor samples was absent in 8% of TNBCs and at a high frequency in other tumors, including squamous lung cancer, where CREBBP-inactivating mutations are common. In TNBC, CREBBP alterations were associated with higher genomic heterogeneity and poorer patient survival and resulted in upregulation and dependency on a FOXM1 proliferative program. Targeting FOXM1-driven proliferation indirectly with clinical CDK4/6 inhibitors (CDK4/6i) selectively impaired growth in spheroids, cell line xenografts, and patient-derived models from multiple tumor types with CREBBP mutations or loss of protein expression. In conclusion, we have identified CREBBP as a novel driver in aggressive TNBC and identified an associated genetic vulnerability in tumor cells with alterations in CREBBP and provide a preclinical rationale for assessing CREBBP alterations as a biomarker of CDK4/6i response in a new patient population. SIGNIFICANCE: This study demonstrates that CREBBP genomic alterations drive aggressive TNBC, lung cancer, and lymphomas and may be selectively treated with clinical CDK4/6 inhibitors.

Homologous recombination DNA repair deficiency and PARP inhibition activity in primary triple negative breast cancer.

Triple negative breast cancer (TNBC) encompasses molecularly different subgroups, with a subgroup harboring evidence of defective homologous recombination (HR) DNA repair. Here, within a phase 2 window clinical trial, RIO trial (EudraCT 2014-003319-12), we investigate the activity of PARP inhibitors in 43 patients with untreated TNBC. The primary end point, decreased Ki67, occured in 12% of TNBC. In secondary end point analyses, HR deficiency was identified in 69% of TNBC with the mutational-signature-based HRDetect assay. Cancers with HRDetect mutational signatures of HR deficiency had a functional defect in HR, assessed by impaired RAD51 foci formation on end of treatment biopsy. Following rucaparib treatment there was no association of Ki67 change with HR deficiency. In contrast, early circulating tumor DNA dynamics identified activity of rucaparib, with end of treatment ctDNA levels suppressed by rucaparib in mutation-signature HR-deficient cancers. In ad hoc analysis, rucaparib induced expression of interferon response genes in HR-deficient cancers. The majority of TNBCs have a defect in DNA repair, identifiable by mutational signature analysis, that may be targetable with PARP inhibitors.

Colorectal tumor vascularity: quantitative assessment with multidetector CT--do tumor perfusion measurements reflect angiogenesis?

PURPOSE: To establish the relationships between quantitative perfusion computed tomography (CT) parameters-specifically, primary tumor blood flow, blood volume, transit time, and permeability surface-area product-and immunohistologic markers of angiogenesis in colorectal cancer. MATERIALS AND METHODS: After institutional review board approval and informed patient consent were obtained for this prospective study, 23 patients (11 men, 12 women; mean age, 68.4 years; age range, 34.8-87.1 years) with colorectal adenocarcinoma underwent a 65-second perfusion CT examination, and tumor blood flow, blood volume, mean transit time, and permeability surface-area product were determined. After surgery, resected specimens were sectioned and stained immunohistochemically to identify CD34 for quantification of microvessel density (MVD), to identify smooth muscle actin for assessment of pericyte coverage index, to identify vascular endothelial growth factor (VEGF), and to identify glucose transporter protein (GLUT-1). Perfusion CT measurements were correlated with MVD, pericyte coverage index, VEGF expression, and GLUT-1 expression by using Pearson or Spearman rank correlation analysis, with significance assigned at the 5% level. RESULTS: Mean blood flow, blood volume, transit time, and permeability surface-area product values were 72.1 mL/min/100 g of tissue +/- 28.4 (standard deviation), 6.2 mL/100 g of tissue +/- 1.4, 9.3 seconds +/- 3.9, and 13.9 mL/min/100 g of tissue +/- 3.2, respectively. Blood volume (r = 0.59, P = .002) and permeability surface-area product (r = 0.46, P = .03) correlated positively with MVD, but blood flow (r = 0.27, P = .22) and transit time (r = -0.18, P = .44) did not. There were no significant associations between any perfusion CT parameter and pericyte coverage index (r .05), VEGF score (rho or= .15), or GLUT-1 score (rho < 0.21, P >or= .33). CONCLUSION: Tumor permeability surface-area product and blood volume correlate positively with MVD and may reflect the microvascularity of colorectal tumors.

Ki67 Is an Independent Predictor of Recurrence in the Largest Randomized Trial of 3 Radiation Fractionation Schedules in Localized Prostate Cancer.

PURPOSE: To assess whether the cellular proliferation marker Ki67 provides prognostic information and predicts response to radiation therapy fractionation in patients with localized prostate tumors participating in a randomized trial of 3 radiation therapy fractionation schedules (74 Gy/37 fractions vs 60 Gy/20 fractions vs 57 Gy/19 fractions). METHODS AND MATERIALS: A matched case-control study design was used; patients with biochemical/clinical failure >2 years after radiation therapy (BCR) were matched 1:1 to patients without recurrence using established prognostic factors (Gleason score, prostate-specific antigen, tumor stage) and fractionation schedule. Immunohistochemistry was used to stain diagnostic biopsy specimens for Ki67, which were scored using the unweighted global method. Conditional logistic regression models estimated the prognostic value of mean and maximum Ki67 scores on BCR risk. Biomarker-fractionation interaction terms determined whether Ki67 was predictive of BCR by fractionation. RESULTS: Using 173 matched pairs, the median for mean and maximum Ki67 scores were 6.6% (interquartile range, 3.9%-9.8%) and 11.0% (interquartile range, 7.0%-15.0%) respectively. Both scores were significant predictors of BCR in models adjusted for established prognostic factors. Conditioning on matching variables and age, the odds of BCR were estimated to increase by 9% per 1% increase in mean Ki67 score (odds ratio 1.09; 95% confidence interval 1.04-1.15, P = .001). Interaction terms between Ki67 and fractionation schedules were not statistically significant. CONCLUSIONS: Diagnostic Ki67 did not predict BCR according to fractionation schedule in CHHiP; however, it was a strong independent prognostic factor for BCR.

Optimised ARID1A immunohistochemistry is an accurate predictor of ARID1A mutational status in gynaecological cancers.

ARID1A is a tumour suppressor gene that is frequently mutated in clear cell and endometrioid carcinomas of the ovary and endometrium and is an important clinical biomarker for novel treatment approaches for patients with ARID1A defects. However, the accuracy of ARID1A immunohistochemistry (IHC) as a surrogate for mutation status has not fully been established for patient stratification in clinical trials. Here we tested whether ARID1A IHC could reliably predict ARID1A mutations identified by next-generation sequencing. Three commercially available antibodies - EPR13501 (Abcam), D2A8U (Cell Signaling), and HPA005456 (Sigma) - were optimised for IHC using cell line models and human tissue, and screened across a cohort of 45 gynaecological tumours. IHC was scored independently by three pathologists using an immunoreactive score. ARID1A mutation status was assessed using two independent sequencing platforms and the concordance between ARID1A mutation and protein expression was evaluated using Receiver Operating Characteristic statistics. Overall, 21 ARID1A mutations were identified in 14/43 assessable tumours (33%), the majority of which were predicted to be deleterious. Mutations were identified in 6/17 (35%) ovarian clear cell carcinomas, 5/8 (63%) ovarian endometrioid carcinomas, 2/5 (40%) endometrial carcinomas, and 1/7 (14%) carcinosarcomas. ROC analysis identified greater than 95% concordance between mutation status and IHC using a modified immunoreactive score for all three antibodies allowing a definitive cut-point for ARID1A mutant status to be calculated. Comprehensive assessment of concordance of ARID1A IHC and mutation status identified EPR13501 as an optimal antibody, with 100% concordance between ARID1A mutation status and protein expression, across different gynaecological histological subtypes. It delivered the best inter-rater agreement between all pathologists, as well as a clear cost-benefit advantage. This could allow patients to be accurately stratified based on their ARID1A IHC status into early phase clinical trials.

Evaluation of CDK12 Protein Expression as a Potential Novel Biomarker for DNA Damage Response-Targeted Therapies in Breast Cancer.

Disruption of Cyclin-Dependent Kinase 12 (CDK12) is known to lead to defects in DNA repair and sensitivity to platinum salts and PARP1/2 inhibitors. However, CDK12 has also been proposed as an oncogene in breast cancer. We therefore aimed to assess the frequency and distribution of CDK12 protein expression by IHC in independent cohorts of breast cancer and correlate this with outcome and genomic status. We found that 21% of primary unselected breast cancers were CDK12 high, and 10.5% were absent, by IHC. CDK12 positivity correlated with HER2 positivity but was not an independent predictor of breast cancer-specific survival taking HER2 status into account; however, absent CDK12 protein expression significantly correlated with a triple-negative phenotype. Interestingly, CDK12 protein absence was associated with reduced expression of a number of DDR proteins including ATR, Ku70/Ku80, PARP1, DNA-PK, and γH2AX, suggesting a novel mechanism of CDK12-associated DDR dysregulation in breast cancer. Our data suggest that diagnostic IHC quantification of CDK12 in breast cancer is feasible, with CDK12 absence possibly signifying defective DDR function. This may have important therapeutic implications, particularly for triple-negative breast cancers. Mol Cancer Ther; 17(1); 306-15. ©2017 AACR.

Association of p16 expression with prognosis varies across ovarian carcinoma histotypes: an Ovarian Tumor Tissue Analysis consortium study.

We aimed to validate the prognostic association of p16 expression in ovarian high-grade serous carcinomas (HGSC) and to explore it in other ovarian carcinoma histotypes. p16 protein expression was assessed by clinical-grade immunohistochemistry in 6525 ovarian carcinomas including 4334 HGSC using tissue microarrays from 24 studies participating in the Ovarian Tumor Tissue Analysis consortium. p16 expression patterns were interpreted as abnormal (either overexpression referred to as block expression or absence) or normal (heterogeneous). CDKN2A (which encodes p16) mRNA expression was also analyzed in a subset (n = 2280) mostly representing HGSC (n = 2010). Association of p16 expression with overall survival (OS) was determined within histotypes as was CDKN2A expression for HGSC only. p16 block expression was most frequent in HGSC (56%) but neither protein nor mRNA expression was associated with OS. However, relative to heterogeneous expression, block expression was associated with shorter OS in endometriosis-associated carcinomas, clear cell [hazard ratio (HR): 2.02, 95% confidence (CI) 1.47-2.77, p < 0.001] and endometrioid (HR: 1.88, 95% CI 1.30-2.75, p = 0.004), while absence was associated with shorter OS in low-grade serous carcinomas (HR: 2.95, 95% CI 1.61-5.38, p = 0.001). Absence was most frequent in mucinous carcinoma (50%), and was not associated with OS in this histotype. The prognostic value of p16 expression is histotype-specific and pattern dependent. We provide definitive evidence against an association of p16 expression with survival in ovarian HGSC as previously suggested. Block expression of p16 in clear cell and endometrioid carcinoma should be further validated as a prognostic marker, and absence in low-grade serous carcinoma justifies CDK4 inhibition.

Hypoxia in prostate cancer: correlation of BOLD-MRI with pimonidazole immunohistochemistry-initial observations.

PURPOSE: To investigate the ability of blood oxygen level-dependent (BOLD) MRI to depict clinically significant prostate tumor hypoxia. METHODS AND MATERIALS: Thirty-three patients with prostate carcinoma undergoing radical prostatectomy were studied preoperatively, using gradient echo sequences without and with contrast medium enhancement, to map relative tissue oxygenation according to relaxivity rates and relative blood volume (rBV). Pimonidazole was administered preoperatively, and whole-mount sections of selected tumor-bearing slices were stained for pimonidazole fixation and tumor and nontumor localization. Histologic and imaging parameters were independently mapped onto patient prostate outlines. Using 5-mm grids, 861 nontumor grid locations were compared with 237 tumor grids (with >50% tumor per location) using contingency table analysis with respect to the ability of imaging to predict pimonidazole staining. RESULTS: Twenty patients completed the imaging and histologic protocols. Pimonidazole staining was found in 33% of nontumor and in 70% of tumor grids. The sensitivity of the MR relaxivity parameter R(2)* in depicting tumor hypoxia was high (88%), improving with the addition of low rBV information (95%) without changing specificity (36% and 29%, respectively). High R(2)* increased the positive predictive value for hypoxia by 6% (70% to 76%); conversely, low R(2)* decreased the likelihood of hypoxia being present by 26% (70% to 44%) and by 41% (71% to 30%) when combined with rBV information. CONCLUSION: R(2)* maps from BOLD-MRI have high sensitivity but low specificity for defining intraprostatic tumor hypoxia. This together with the negative predictive value of 70% when combined with blood volume information makes BOLD-MRI a potential noninvasive technique for mapping prostatic tumor hypoxia.

Preclinical evaluation of the PARP inhibitor BMN-673 for the treatment of ovarian clear cell cancer.

PURPOSE: To determine if models of ovarian clear cell carcinomas (OCCCs) harbouring defects in homologous recombination (HR) DNA repair of double strand breaks (DSBs) are sensitive to cisplatin and/or PARP inhibition. EXPERIMENTAL DESIGN: The HR status of 12 OCCC cell lines was determined using RAD51/γH2AX foci formation assays. Sensitivity to cisplatin and the PARP inhibitor BMN-673 was correlated with HR status. BRCA1, BRCA2, MRE11 and PTEN loss of expression was investigated as a potential determinant of BMN-673 sensitivity. A tissue microarray containing 50 consecutive primary OCCC was assessed for PTEN expression using immunohistochemistry. RESULTS: A subset of OCCC cells displayed reduced RAD51 foci formation in the presence of DNA DSBs, suggestive of HR defects. HR-defective OCCC cells, with the exception of KOC-7c, had higher sensitivity to cisplatin/ BMN-673 than HR-competent OCCC cell lines (Log10 SF50 -9.4 (SD +/- 0.29) vs -8.1 (SD +/- 0.35), mean difference 1.3, p < 0.01). Of the cell lines studied, two, TOV-21G and KOC-7c, showed loss of PTEN expression. In primary OCCCs, loss of PTEN expression was observed in 10% (5/49) of cases. CONCLUSIONS: A subset of OCCC cells are sensitive to PARP inhibition in vitro, which can be predicted by HR defects as defined by γH2AX/RAD51 foci formation. These results provide a rationale for the testing of HR deficiency and PARP inhibitors as a targeted therapy in a subset of OCCCs.

Epidermal growth factor receptor expression in pretreatment biopsies from head and neck squamous cell carcinoma as a predictive factor for a benefit from accelerated radiation therapy in a randomized controlled trial.

PURPOSE: Accelerated repopulation is a main reason for locoregional failure after fractionated radiotherapy for head and neck squamous cell carcinoma (HNSCC). Epidermal growth factor receptor (EGFR) is a key controller of cellular proliferation in HNSCC, which stimulated the current study to look for a direct link between EGFR status and a possible clinical advantage of accelerated radiotherapy. PATIENTS AND METHODS: Immunohistochemical staining for EGFR was performed in 304 patients with available pretreatment tumor biopsy material among 918 patients randomized to receive continuous hyperfractionated accelerated radiotherapy versus conventionally fractionated radiotherapy. The EGFR index was estimated as the proportion of tumor cells with EGFR membrane staining. RESULTS: Significant benefit in locoregional tumor control from continuous hyperfractionated accelerated radiotherapy was seen in patients with HNSCC with high EGFR expression (2P = .010) but not in those with low EGFR expression (2P = .85). EGFR status had no significant effect on survival or rate of distant metastases. The EGFR index was significantly associated with histologic grade and microvessel density. There was moderate support for an association between EGFR status and subsite within the head and neck region but no significant association with Ki-67 index, Ki-67 pattern, p53 index, p53 intensity, bcl-2 expression, or cyclin D1 index. CONCLUSION: This study indicates a key role for the EGFR receptor in determining the proliferative cellular response to fractionated radiotherapy in HNSCC. It also shows that we can select the dose-fractionation regime that has the greatest chance of benefiting the patient. These results also encourage further development of EGFR targeting combined with fractionated radiotherapy in HNSCC.