Intra- and Interlaboratory Reproducibility of the Sensitivity to Endocrine Therapy Assay for Stage II/III Breast Cancer.

BACKGROUND: The sensitivity to endocrine therapy assay (SET2,3) predicts treatment outcomes in Stage II-III breast cancer. SET2,3 measures transcription related to estrogen and progesterone receptors (SETER/PR index) and the molecular subtype (RNA4: ESR1, PGR, ERBB2, AURKA) from formalin-fixed paraffin-embedded (FFPE) tissue sections. METHODS: We designed a nested study across 3 pathology laboratories, each testing 60 breast cancers twice in controlled batches. Laboratories macrodissected and directly homogenized the unstained FFPE tumor sections, then performed the QuantiGene Plex bead-based hybridization assay. SET2,3 was calculated centrally using predefined statistical R-scripts and applying pre-defined cutpoints. Concordance correlation coefficient (CCC) was calculated from continuous measurements and Kappa statistic from categorical results. A mixed-effects model estimated contributions to bias (fixed effects) and variance (random effects) from the replicated design. RESULTS: Intralaboratory (CCC 0.96-0.99) and interlaboratory (CCC 0.98-0.99) SET2,3 results were concordant, with rates of agreement for high/low categorization within (Kappa 0.83-0.93) and between laboratories (Kappa 0.87-0.88). The relative contributions to overall variance of SET2,3 measurements were 96.90% from biological differences between cancers, 0.67% from interlaboratory variability, and 2.44% from residual causes including intralaboratory replicates. Similar results were obtained with SETER/PR, the baseline prognostic index calculated using pathological or clinical tumor and nodal staging information, and the 4 individual genes (ESR1, PGR, ERBB2, and AURKA). CONCLUSION: Intra- and interpathology laboratory measurements of SET2,3 and its components were highly reproducible when tested from FFPE tumor sections.

Targeted RNAseq assay incorporating unique molecular identifiers for improved quantification of gene expression signatures and transcribed mutation fraction in fixed tumor samples.

BACKGROUND: Our objective was to assess whether modifications to a customized targeted RNA sequencing (RNAseq) assay to include unique molecular identifiers (UMIs) that collapse read counts to their source mRNA counts would improve quantification of transcripts from formalin-fixed paraffin-embedded (FFPE) tumor tissue samples. The assay (SET4) includes signatures that measure hormone receptor and PI3-kinase related transcriptional activity (SETER/PR and PI3Kges), and measures expression of selected activating point mutations and key breast cancer genes. METHODS: Modifications included steps to introduce eight nucleotides-long UMIs during reverse transcription (RT) in bulk solution, followed by polymerase chain reaction (PCR) of labeled cDNA in droplets, with optimization of the polymerase enzyme and reaction conditions. We used Lin's concordance correlation coefficient (CCC) to measure concordance, including precision (Rho) and accuracy (Bias), and nonparametric tests (Wilcoxon, Levene's) to compare the modified (NEW) SET4 assay to the original (OLD) SET4 assay and to whole transcriptome RNAseq using RNA from matched fresh frozen (FF) and FFPE samples from 12 primary breast cancers. RESULTS: The modified (NEW) SET4 assay measured single transcripts (p< 0.001) and SETER/PR (p=0.002) more reproducibly in technical replicates from FFPE samples. The modified SET4 assay was more precise for measuring single transcripts (Rho 0.966 vs 0.888, p< 0.01) but not multigene expression signatures SETER/PR (Rho 0.985 vs 0.968) or PI3Kges (Rho 0.985 vs 0.946) in FFPE, compared to FF samples. It was also more precise than wtRNAseq of FFPE for measuring transcripts (Rho 0.986 vs 0.934, p< 0.001) and SETER/PR (Rho 0.993 vs 0.915, p=0.004), but not PI3Kges (Rho 0.988 vs 0.945, p=0.051). Accuracy (Bias) was comparable between protocols. Two samples carried a PIK3CA mutation, and measurements of transcribed mutant allele fraction was similar in FF and FFPE samples and appeared more precise with the modified SET4 assay. Amplification efficiency (reads per UMI) was consistent in FF and FFPE samples, and close to the theoretically expected value, when the library size exceeded 400,000 aligned reads. CONCLUSIONS: Modifications to the targeted RNAseq protocol for SET4 assay significantly increased the precision of UMI-based and reads-based measurements of individual transcripts, multi-gene signatures, and mutant transcript fraction, particularly with FFPE samples.

Increasing accrual of minority patients in breast cancer clinical trials.

BACKGROUND: Black and Hispanic patients participate in clinical trials at lower rates than white patients nationally; lack of diversity in clinical trials prevents appropriate safety and efficacy testing of new treatments in these populations. METHODS: The Oncology Welcomes New Haven into Trials (OWN IT) initiative at the Yale Cancer Center used a multi-tiered approach to improve breast cancer minority clinical trial accrual through community focus groups, ongoing community outreach, institutional executive council representation, grand rounds presentation, and didactic lectures with healthcare providers. Eligibility criteria of breast cancer trials at Smilow Cancer Center were reviewed using clinicaltrials.gov. Also, an anonymous, 5-min survey was conducted at regular visits with Smilow Breast Center patients to gauge awareness of and access to clinical trials. Survey data were compared to the Yale Cancer Center Clinical Trials Office, Connecticut Tumor Registry, and U.S. Census records. Two-tailed Fisher's tests were used for all analyses. RESULTS: There was a significant increase in the number of minority patients who participated in clinical trials at Smilow Cancer Center from 2016 (95/750) to 2018 (155/944) (p = 0.0325). Two hundred patients participated in the survey; response rate 92%. There was no significant difference in the rate at which patients were invited to participate in clinical trials or the rate at which they declined to participate based on race or ethnicity. Black and Hispanic patients were significantly less likely to be aware of clinical trials than white patients (p < .001). The review of eligibility criteria showed that over half of the studies reviewed had restrictions regarding increased liver function tests, and many restricted the participation of patients with other chronic conditions. CONCLUSIONS: Low participation in clinical trials among black and Hispanic patients is likely multifaceted. This study indicated that there are likely structural factors at work which can be modified with institutional effort. The role of patient education regarding clinical trials and accrual should be studied further as should eligibility criteria as a potential barrier to participation.

Long-Term Survival of De Novo Stage IV Human Epidermal Growth Receptor 2 (HER2) Positive Breast Cancers Treated with HER2-Targeted Therapy.

BACKGROUND: An increasing proportion of human epidermal growth receptor 2 (HER2) positive (HER2+) metastatic breast cancer (MBC) is diagnosed as de novo stage IV disease. We hypothesize that a subset of these patients who achieve no evidence of disease (NED) status after multimodality HER2-targeted treatments may have prolonged progression-free survival (PFS) and overall survival (OS). MATERIALS AND METHODS: Patients with de novo stage IV, HER2+ MBC (n = 483) diagnosed between 1998 and 2015 were identified at two institutions (Yale and MD Anderson Cancer Centers). Clinical variables, treatment details, and survival outcomes were compared between those who achieved NED and those who did not. RESULTS: All patients received trastuzumab, and 20% also received pertuzumab as first-line therapy. The median OS was 5.5 years (95% confidence interval [Cl]: 4.8-6.2). Sixty-three patients (13.0%) achieved NED; their PFS and OS rates were 100% and 98% (95% CI: 94.6%-100%), respectively, at 5 years and remained the same at 10 years. For patients with no NED (n = 420), the PFS and OS rates were 12% (95% CI: 4.5%-30.4%) and 45% (95% CI: 38.4%-52.0%) at 5 years and 0% and 4% (95% CI, 1.3%-13.2%) at 10 years, respectively. NED patients more frequently had solitary metastasis (79% vs. 51%, p = .005) and surgery to resect cancer (59% vs. 22%, p ≤ .001). In multivariate analysis, NED status (hazard ratio [HR]: 0.014, p = .0002) and estrogen receptor positive status (HR: 0.72; p = .04) were associated with prolonged OS. CONCLUSION: Among patients with de novo stage IV, HER2+ MBC, those who achieve NED status have a very high PFS and OS. Further randomized studies are required to fully understand the impact of systemic or locoregional therapy on achieving these excellent long-term outcomes. IMPLICATIONS FOR PRACTICE: In this retrospective review at two institutions, it was demonstrated that 13% of patients with de novo stage IV, human epidermal growth receptor 2 positive metastatic breast cancer achieved no evidence of disease (NED) status with trastuzumab-based therapy plus/minus local therapies, and these patients had a very high progression-free survival (100%) and overall survival (98%) at both the 5- and 10-year time points. Achieving NED status may be an important therapeutic goal. However, further randomized studies are required to fully understand the impact of systemic or locoregional therapy on achieving these excellent long-term outcomes.

Immune microenvironment of triple-negative breast cancer in African-American and Caucasian women.

PURPOSE: African-American (AA) patients with triple-negative breast cancer (TNBC) are less likely to achieve pathologic complete response from neoadjuvant chemotherapy and have poorer prognosis than Caucasian patients with TNBC, suggesting potential biological differences by race. Immune infiltration is the most consistent predictive marker for chemotherapy response and improved prognosis in TNBC. In this study, we test the hypothesis that the immune microenvironment differs between AA and Caucasian patients. METHODS: RNA-seq expression data were obtained from The Cancer Genome Atlas (TCGA) database for 162 AA and 697 Caucasian breast cancers. Estrogen receptor (ER)-positive, human epidermal growth factor receptor-2 (HER2)-positive, and TNBC subtypes were included in the analyses. Tumor infiltrating lymphocyte (TIL) counts, immunomodulatory scores, and molecular subtypes were obtained from prior publications for a subset of the TNBC cases. Differences in immune cell distributions and immune functions, measured through gene expression and TIL counts, as well as neoantigen, somatic mutation, amplification, and deletion loads, were compared by race and tumor subtype. RESULTS: Immune metagene analysis demonstrated marginal immune attenuation in AA TNBC relative to Caucasian TNBC that did not reach statistical significance. The distributions of immune cell populations, lymphocyte infiltration, molecular subtypes, and genomic aberrations between AA and Caucasian subtypes were also not significantly different. The MHC1 metagene demonstrated increased expression in AA ER-positive cancers relative to Caucasian ER-positive cancers. CONCLUSIONS: This study suggests that the immunological differences between AA and Caucasian breast cancers represented by TCGA data are subtle, if they exist at all. We observed no consistent racial differences in immune gene expression or TIL counts in TNBC by race. However, this study cannot rule out small differences in immune cell subtype distribution and activity status that may not be apparent in bulk RNA analysis.

Examining the cost-effectiveness of baseline left ventricular function assessment among breast cancer patients undergoing anthracycline-based therapy.

BACKGROUND: There is a lack of consensus to guide which breast cancer patients require left ventricular function assessment (LVEF) prior to anthracycline therapy; the cost-effectiveness of screening this patient population has not been previously evaluated. METHODS: We performed a retrospective analysis of the Yale Nuclear Cardiology Database, including 702 patients with baseline equilibrium radionuclide angiography (ERNA) scan prior to anthracycline and/or trastuzumab therapy. We sought to examine associations between abnormal baseline LVEF and potential cardiac risk factors. Additionally, we designed a Markov model to determine the incremental cost-effectiveness ratio (ICER) of ERNA screening for women aged 55 with stage I-III breast cancer from a payer perspective over a lifetime horizon. RESULTS: An abnormal LVEF was observed in 2% (n = 14) of patients. There were no significant associations on multivariate analysis performed on self-reported risk factors. Our analysis showed LVEF screening is cost-effective with ICER of $45,473 per QALY gained. For a willingness-to-pay threshold of $100,000/ QALY, LVEF screening had an 81.9% probability of being cost-effective. Under the same threshold, screening was cost-effective for non-anthracycline cardiotoxicity risk of RR ≤ 0.58, as compared to anthracycline regimens. CONCLUSIONS: Age, preexisting cardiac risk factors and coronary artery disease did not predict a baseline abnormal LVEF. While the prevalence of an abnormal baseline LVEF is low in patients with breast cancer, our results suggest that cardiac screening prior to anthracycline is cost-effective.

The impact of communication style on patient satisfaction.

BACKGROUND: Communication between patients and health providers influences patient satisfaction, but it is unknown whether similarity in communication styles results in higher patient satisfaction. METHODS: This study was conducted in the Smilow Cancer Hospital Breast Center. During routine follow-up visits, patients completed a Communication Styles Assessment (CSA), health survey (SF-12), Princess Margaret Hospital Satisfaction with Doctor Questionnaire, and brief demographic form. Physicians and Advanced Practice Providers were also asked to complete the CSA. Patients and providers were blinded to each other's responses. A communication styles concordance score was calculated as the Pearson correlation between 80 binary CSA items for each provider/patient pair. Factors affecting patient satisfaction scores were assessed in mixed-effects models. RESULTS: In total, 330 patients were invited to participate; of these 289 enrolled and 245 returned surveys. One hundred seventy-four completed all survey components, and 18 providers completed the CSA. Among the factors considered, physical health score (effect size = 0.0058, 95% CI 0.00051 to 0.0011, p = 0.032) and employment status (0.12, 95% CI - 0.0094 to 0.25, p = 0.069) had the greatest impact on patient satisfaction. However, patients who were not employed and less physically healthy had significantly elevated satisfaction scores when their communication style was more similar to their provider's (1.52, 95% CI 0.66 to 2.38, p = 0.0016). CONCLUSIONS: Patients who were physically healthy and employed were generally more satisfied with their care. The similarity in communication styles of patients and providers had a greater impact on patient satisfaction for patients who were less physically healthy and not employed.

The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues.

BACKGROUND: Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. METHODS: Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. RESULTS: Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63-0.66) and between technical replicates (median expression difference 0.13-0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31-0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91-0.96). CONCLUSIONS: The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

An integrative bioinformatics approach reveals coding and non-coding gene variants associated with gene expression profiles and outcome in breast cancer molecular subtypes.

BACKGROUND: Sequence variations in coding and non-coding regions of the genome can affect gene expression and signalling pathways, which in turn may influence disease outcome. METHODS: In this study, we integrated somatic mutations, gene expression and clinical data from 930 breast cancer patients included in the TCGA database. Genes associated with single mutations in molecular breast cancer subtypes were identified by the Mann-Whitney U-test and their prognostic value was evaluated by Kaplan-Meier and Cox regression analyses. Results were confirmed using gene expression profiles from the Metabric data set (n = 1988) and whole-genome sequencing data from the TCGA cohort (n = 117). RESULTS: The overall mutation rate in coding and non-coding regions were significantly higher in ER-negative/HER2-negative tumours (P = 2.8E-03 and P = 2.4E-07, respectively). Recurrent sequence variations were identified in non-coding regulatory regions of several cancer-associated genes, including NBPF1, PIK3CA and TP53. After multivariate regression analysis, gene signatures associated with three coding mutations (CDH1, MAP3K1 and TP53) and two non-coding variants (CRTC3 and STAG2) in cancer-related genes predicted prognosis in ER-positive/HER2-negative tumours. CONCLUSIONS: These findings demonstrate that sequence alterations influence gene expression and oncogenic pathways, possibly affecting the outcome of breast cancer patients. Our data provide potential opportunities to identify non-coding variations with functional and clinical relevance in breast cancer.

First international TNBC conference meeting report.

Recently, Georgia State University's Centennial Hall was the premier location for the 2017 International Conference on Triple Negative Breast Cancer (TNBC): Illuminating Actionable Biology, which was held from Sept. 18 to 20, 2017, in Atlanta, USA. The conference featured a stellar line-up of domestic and international speakers and diverse participants including TNBC survivors, luminaries in breast cancer research, medical students and fellows, clinicians, translational researchers, epidemiologists, biostatisticians, bioinformaticians, and representatives from the industry. This report distills the burning questions that spiked the event and summarizes key themes, findings, unique opportunities and future directions that emerged from this confluence of thought leaders.

Single-arm, neoadjuvant, phase II trial of pertuzumab and trastuzumab administered concomitantly with weekly paclitaxel followed by 5-fluoruracil, epirubicin, and cyclophosphamide (FEC) for stage I-III HER2-positive breast cancer.

PURPOSE: The purpose of this two-cohort Phase II trial was to estimate the pathologic complete response (pCR: ypT0/is ypN0) rate when trastuzumab plus pertuzumab are administered concurrently during both the taxane and anthracycline phases of paclitaxel and 5-fluorouracil/epirubicin/cyclophosphamide (FEC) neoadjuvant chemotherapy. METHODS: The pCR rates were assessed separately in hormone receptor (HR) positive and negative cases following Simon's two-stage design, aiming to detect a 20% absolute improvement in pCR rates from 50 to 70 and 70 to 90% in the HR-positive and HR-`negative cohorts, respectively. RESULTS: The HR-negative cohort completed full accrual of 26 patients; pCR rate was 80% (95% CI 60-91%). The HR+ cohort was closed early after 24 patients due to lower than expected pCR rate of 26% (95% CI 13-46%) at interim analysis. Overall, 44% of patients (n = 22/50) experienced grade 3/4 adverse events. The most common were neutropenia (n = 10) and diarrhea (n = 7). There was no symptomatic heart failure, but 28% (n = 14) had ≥ 10% asymptomatic decrease in LVEF; in one patient, LVEF decreased to < 50%. Cardiac functions returned to baseline by the next assessment in 57% (8/14) of cases. CONCLUSIONS: Eighty percent of HR-negative, HER2-positive breast cancers achieve pCR with paclitaxel/FEC neoadjuvant chemotherapy administered concomitantly with pertuzumab and trastuzumab. These results are similar to pCR rates seen in trials using HER2-targeted therapy during the taxane phase only of sequential taxane-anthracycline regimens and suggest that we have reached a therapeutic plateau with HER2-targeted therapies combined with chemotherapy in the neoadjuvant setting.

Co-targeting the HER and IGF/insulin receptor axis in breast cancer, with triple targeting with endocrine therapy for hormone-sensitive disease.

PURPOSE: Interactions between HER2, estrogen receptor (ER), and insulin-like growth factor I receptor (IGF1R) are implicated in resistance to monotherapies targeting these receptors. We have previously shown in pre-clinical studies synergistic anti-tumor effects for co-targeting each pairwise combination of HER2, IGF1R, and ER. Strikingly, synergy for HER2/IGF1R targeting occurred not only in a HER2+ model, but also in a HER2-normal model. The purpose of the current study was therefore to determine the generalizability of synergistic anti-tumor effects of co-targeting HER2/IGF1R, the anti-tumor activity of triple-targeting HER2/IGF1R/ER in hormone-dependent cell lines, and the effect of using the multi-targeting drugs neratinib (pan-HER) and BMS-754807 (dual IGF1R/insulin receptor). METHODS: Proliferation and apoptosis assays were performed in a large panel of cell lines representing varying receptor expression levels. Mechanistic effects were studied using phospho-protein immunoblotting. Analyses of drug interaction effects were performed using linear mixed-effects regression models. RESULTS: Enhanced anti-proliferative effects of HER/IGF-insulin co-targeting were seen in most, though not all, cell lines, including HER2-normal lines. For ER+ lines, triple targeting with inclusion of anti-estrogen generally resulted in the greatest anti-tumor effects. Double or triple targeting generally resulted in marked increases in apoptosis in the sensitive lines. Mechanistic studies demonstrated that the synergy between drugs was correlated with maximal inhibition of Akt and ERK pathway signaling. CONCLUSIONS: Dual HER/IGF-insulin targeting, and triple targeting with inclusion of anti-estrogen drugs, shows striking anti-tumor activity across breast cancer types, and drugs with broader receptor specificity may be more effective than single receptor selective drugs, particularly for ER- cells.

Association of LN Evaluation with Survival in Women Aged 70 Years or Older With Clinically Node-Negative Hormone Receptor Positive Breast Cancer.

BACKGROUND: Some suggest that lymph node (LN) evaluation not be performed routinely in women aged ≥70 years with clinically (c) LN-negative (-), hormone receptor (HR)-positive (+) breast cancer. We sought to determine the association of omission of LN evaluation on survival. METHODS: Patients who met the above criteria and were diagnosed from 2004 to 2012 were identified in the NCDB and SEER databases. Overall survival (OS) and breast cancer-specific survival (BCSS) were determined. RESULTS: Using the NCDB, we identified 157,584 cLN- HR+ patients aged ≥70 years in whom survival and LN evaluation data were available. A total of 126,638 patients (80.2%) had regional LN surgery. With a median follow-up of 41.6 months, there was a significant difference in OS between those who had LN evaluation and those who did not (median OS: 100.5 vs. 70.9 months, respectively, p < 0.001). After adjusting for patient age, race, insurance, income, comorbidities, tumor characteristics and treatment, patients who had undergone LN evaluation still had a lower hazard rate for death than those who had not (hazard ratio = 0.633; 95% confidence interval [CI] 0.613-0.654, p < 0.001). We then did a parallel analysis using SEER data that showed LN evaluation was associated with a lower hazard rate for both BCSS (hazard ratio = 0.452; 95% CI 0.427-0.479, p < 0.001) and non-BCSS (hazard ratio = 0.465; 95% CI 0.447-0.482, p < 0.001). CONCLUSIONS: Roughly 20% of patients older than aged 70 years with cLN-, HR+ breast cancer did not have LN evaluation. Those who did had better OS controlling for sociodemographic, pathologic, and treatment variables; however, this may be due to patient selection.

Testing violations of the exponential assumption in cancer clinical trials with survival endpoints.

Personalized cancer therapy requires clinical trials with smaller sample sizes compared to trials involving unselected populations that have not been divided into biomarker subgroups. The use of exponential survival modeling for survival endpoints has the potential of gaining 35% efficiency or saving 28% required sample size (Miller, 1983), making personalized therapy trials more feasible. However, the use of exponential survival has not been fully accepted in cancer research practice due to uncertainty about whether or not the exponential assumption holds. We propose a test for identifying violations of the exponential assumption using a reduced piecewise exponential approach. Compared with an alternative goodness-of-fit test, which suffers from inflation of type I error rate under various censoring mechanisms, the proposed test maintains the correct type I error rate. We conduct power analysis using simulated data based on different types of cancer survival distribution in the SEER registry database, and demonstrate the implementation of this approach in existing cancer clinical trials.

Quantitative assessment of the spatial heterogeneity of tumor-infiltrating lymphocytes in breast cancer.

BACKGROUND: Tumor-infiltrating lymphocyte (TIL) count in breast cancer carries prognostic information and represents a potential predictive marker for emerging immunotherapies. However, the distribution of the lymphocyte subpopulations is not well defined. The goals of this study were to examine intratumor heterogeneity in TIL subpopulation counts in different fields of view (FOV) within each section, in different sections from the same biopsy, and between biopsies from different regions of the same cancer using quantitative immunofluorescence (QIF). METHODS: We used multiplexed QIF to quantify cytokeratin-positive epithelial cells, and CD3-positive, CD8-positive and CD20-positive lymphocytes in tissue sections from multiple biopsies obtained from different areas of 31 surgically resected primary breast carcinomas (93 samples total). Log2-transformed QIF scores or concordance and variance component analyses with linear mixed-effects models were used. Cohen's kappa index [k] of high versus low scores, defined as above and below the median, was used to measure sample similarity between areas. RESULTS: We found a strong positive correlation between CD3 and CD8 levels across all patients (Pearson correlation coefficient [CC] = 0.827). CD3 and CD8 showed a weaker but significant association with CD20 (CC = 0.446 and 0.363, respectively). For each marker, the variation between different FOVs in the same section was higher than the variation between sections or between biopsies of the same cancer. The intraclass correlation coefficients (ICC) were 0.411 for CD3, 0.324 for CD8, and 0.252 for CD20. In component analysis, 66-69 % of the variance was attributable to differences between FOVs in the same section and 30-33 % was due to differences between biopsies from different areas of the same cancer. Section to section differences were negligible. Concordance for low versus high marker status assignment in single biopsies compared to all three biopsies combined yielded k = 0.705 for CD3, k = 0.655 for CD8, and k = 0.603 for CD20. CONCLUSIONS: T and B lymphocytes show more heterogeneity across the dimensions of a single section than between different sections or regions of a given breast tumor. This observation suggests that the average lymphocyte score from a single biopsy of a tumor is reasonably representative of the whole cancer.

Predictors of Chemosensitivity in Triple Negative Breast Cancer: An Integrated Genomic Analysis.

BACKGROUND: Triple negative breast cancer (TNBC) is a highly heterogeneous and aggressive disease, and although no effective targeted therapies are available to date, about one-third of patients with TNBC achieve pathologic complete response (pCR) from standard-of-care anthracycline/taxane (ACT) chemotherapy. The heterogeneity of these tumors, however, has hindered the discovery of effective biomarkers to identify such patients. METHODS AND FINDINGS: We performed whole exome sequencing on 29 TNBC cases from the MD Anderson Cancer Center (MDACC) selected because they had either pCR (n = 18) or extensive residual disease (n = 11) after neoadjuvant chemotherapy, with cases from The Cancer Genome Atlas (TCGA; n = 144) and METABRIC (n = 278) cohorts serving as validation cohorts. Our analysis revealed that mutations in the AR- and FOXA1-regulated networks, in which BRCA1 plays a key role, are associated with significantly higher sensitivity to ACT chemotherapy in the MDACC cohort (pCR rate of 94.1% compared to 16.6% in tumors without mutations in AR/FOXA1 pathway, adjusted p = 0.02) and significantly better survival outcome in the TCGA TNBC cohort (log-rank test, p = 0.05). Combined analysis of DNA sequencing, DNA methylation, and RNA sequencing identified tumors of a distinct BRCA-deficient (BRCA-D) TNBC subtype characterized by low levels of wild-type BRCA1/2 expression. Patients with functionally BRCA-D tumors had significantly better survival with standard-of-care chemotherapy than patients whose tumors were not BRCA-D (log-rank test, p = 0.021), and they had significantly higher mutation burden (p < 0.001) and presented clonal neoantigens that were associated with increased immune cell activity. A transcriptional signature of BRCA-D TNBC tumors was independently validated to be significantly associated with improved survival in the METABRIC dataset (log-rank test, p = 0.009). As a retrospective study, limitations include the small size and potential selection bias in the discovery cohort. CONCLUSIONS: The comprehensive molecular analysis presented in this study directly links BRCA deficiency with increased clonal mutation burden and significantly enhanced chemosensitivity in TNBC and suggests that functional RNA-based BRCA deficiency needs to be further examined in TNBC.

Assessing cost-utility of predictive biomarkers in oncology: a streamlined approach.

Evaluation of cost-utility is critical in assessing the medical utility of predictive or prognostic biomarkers. Current methods involve complex state-transition models, requiring comprehensive data inputs. We propose a simplified decision-analytic tool to explore the relative effect of factors contributing to the cost-utility of a biomarker. We derived a cost-utility metric, the "test incremental cost-effectiveness ratio" (TICER) for biomarker-guided treatment compared to no biomarker use. This method uses data inputs readily accessible through clinical literature. We compared our results with traditional cost-effectiveness analysis of predictive biomarkers for established (HER2-guided trastuzumab, ALK-guided crizotinib, OncotypeDX-guided adjuvant chemotherapy) and emerging (ROS1-guided crizotinib) targeted treatments. We conducted sensitivity analysis to determine which factors had the greatest impact on TICER estimates. Base case TICER for HER2 was $149,600/quality-adjusted life year (QALY), for ALK was $22,200/QALY, and for OncotypeDX was $11,600/QALY, consistent with literature-reported estimates ($180,000/QALY, $202,800/QALY, $8900/QALY, respectively). Base case TICER for ROS1-guided crizotinib was $205,900/QALY. Generally, when treatment cost is considerably greater than biomarker testing costs, TICER is driven by clinical outcomes and health-related quality of life, while biomarker prevalence and treatment cost have a lesser effect. Our simplified decision-analytic approach produces values consistent with existing cost-effectiveness analyses. Our results suggest that biomarker value is mostly driven by the clinical efficacy of the targeted agent. A user-friendly web tool for complete TICER analysis has been made available for open use at http://medicine.yale.edu/lab/pusztai/ticer/ .

Seven Countries Study cohort in Crete, Greece: gluteal adipose-tissue fatty-acid profiles of survivors, at 2010.

OBJECTIVE: To analyse the gluteal adipose-tissue fatty-acid profiles from Cretan cohort survivors of the Seven Countries Study (SCS) at 2010 and to compare them with those of survivors assessed in 2000, as well as with literature data on male Cretans at 1965. DESIGN: We analysed data concerning the gluteal adipose-tissue fatty acids (analysed by GC) from three studies. SETTING: The island of Crete (rural areas and the city of Heraklion). SUBJECTS: Twenty-two of the 2010 SCS survivors aged 90 years and over; seventy-eight men aged 80 years of the 2000 SCS survivors; and 280 men assessed in 1965. RESULTS: In comparison to 1965 and 2000, the SCS survivors in 2010 had a higher amount of 18:1n-9 (P<0·05) in their gluteal adipose tissue and a lower amount of PUFA (P<0·05). On the other hand, a constant decrease in adipose-tissue 14:1n-5 and 16:1n-7 was recorded between 1965 and 2010 (P<0·001), and between 2000 and 2010 (P<0·05), while 18:2n-6 appeared to decrease between the 1965 and 2010 assessments (P<0·001). CONCLUSIONS: Comparison with a 1965 representative Cretan sample and 2000 SCS survivors indicated an increased concentration of oleic acid (known for its protective role against mortality) and a decreased concentration of PUFA (known for their susceptibility to oxidation) in our surviving sample at 2010. These changes may reflect internal physiological processes due to diet change within these years and/or ageing.

Multigene prognostic tests in breast cancer: past, present, future.

There is growing consensus that multigene prognostic tests provide useful complementary information to tumor size and grade in estrogen receptor (ER)-positive breast cancers. The tests primarily rely on quantification of ER and proliferation-related genes and combine these into multivariate prediction models. Since ER-negative cancers tend to have higher proliferation rates, the prognostic value of current multigene tests in these cancers is limited. First-generation prognostic signatures (Oncotype DX, MammaPrint, Genomic Grade Index) are substantially more accurate to predict recurrence within the first 5 years than in later years. This has become a limitation with the availability of effective extended adjuvant endocrine therapies. Newer tests (Prosigna, EndoPredict, Breast Cancer Index) appear to possess better prognostic value for late recurrences while also remaining predictive of early relapse. Some clinical prediction problems are more difficult to solve than others: there are no clinically useful prognostic signatures for ER-negative cancers, and drug-specific treatment response predictors also remain elusive. Emerging areas of research involve the development of immune gene signatures that carry modest but significant prognostic value independent of proliferation and ER status and represent candidate predictive markers for immune-targeted therapies. Overall metrics of tumor heterogeneity and genome integrity (for example, homologue recombination deficiency score) are emerging as potential new predictive markers for platinum agents. The recent expansion of high-throughput technology platforms including low-cost sequencing of circulating and tumor-derived DNA and RNA and rapid reliable quantification of microRNA offers new opportunities to build extended prediction models across multiplatform data.

Phase 1b study of the mammalian target of rapamycin inhibitor sirolimus in combination with nanoparticle albumin-bound paclitaxel in patients with advanced solid tumors.

BACKGROUND: The optimal weekly oral dose of sirolimus and intravenous nanoparticle albumin-bound paclitaxel (nab-paclitaxel) were evaluated. METHODS: A phase 1b study was performed to evaluate escalating doses of oral sirolimus (5-60 mg) on days 2, 9, and 16 with intravenous nab-paclitaxel (100 mg/m(2) ) on days 1, 8, and 15 in a 28-day cycle. A run-in treatment of nab-paclitaxel (day -14) and sirolimus (day -7) was administered for pharmacokinetic and pharmacodynamic assessments. Clinical trial endpoints included dose-limiting toxicities (DLTs), maximum tolerated doses, and response rates. Pharmacodynamics included immunohistochemistry for phosphatase and tensin homolog, mammalian target of rapamycin (mTOR), AKT, phosphorylated AKT, S6K1, and phosphorylated S6K1; exploratory gene expression analysis; and [(18) F]fludeoxyglucose (FDG) positron emission tomography. RESULTS: Twenty-three patients with advanced solid tumors were treated. Fifteen patients had prior taxane therapy. Twenty-two patients were evaluable for responses. One patient had a complete response, and 5 patients had a partial response (3 confirmed). DLTs were seen in 1 patient each at 10 (grade 3 dyspnea/hypoxia) and 40 mg (grade 4 leukopenia/neutropenia) and in 2 patients at 60 mg (grade 3 fatigue and grade 4 pericardial effusion). Patients with higher expression of posttreatment AKT and a greater decline in FDG activity were more likely to have a treatment response or stable disease. CONCLUSIONS: Sirolimus showed an acceptable safety profile at a weekly dose of 40 mg with weekly intravenous nab-paclitaxel at 100 mg/m(2) on days 1, 8, and 15 every 28 days. The posttreatment AKT score and changes in FDG activity may have roles as early predictors of responses to mTOR inhibitors.