The 27-gene IO score is associated with efficacy of PD-1/L1 inhibitors independent of FGFR expression in a real-world metastatic urothelial carcinoma cohort.

Multiple targeted therapeutics have been approved by the FDA for mUC, including immune checkpoint inhibitors (ICIs) and more recently targeted agents for both FGFR and Nectin-4. FGFR3-aberrant and Nectin-4 expressing cells have been associated with an immunosuppressed phenotype. Given that less than half of all patients respond to these agents as monotherapies and less than 20% are eligible to receive salvage therapy, effective personalized treatment plans are critical. Typical biomarkers for ICIs such as PD-L1 and TMB have not been definitive in mUC, yet a biomarker-driven optimization of first-line therapy and subsequent sequencing have the potential to achieve higher and more durable response rates. The IO score is a 27-gene tumor immune microenvironment (TIME) classifier that has been associated with the clinical benefits of ICIs in multiple cancer types, including mUC. This study demonstrates that the IO score was associated with both progression-free survival (PFS) and overall survival (OS) in a real-world cohort of mUC patients treated with ICIs. Furthermore, the IO score was independent of and provided information incremental to TMB. Interestingly, the IO score predicted benefit in patients with high FGFR expression, despite conflicting data regarding response rates among the FGFR aberrant population. Taken together, these results demonstrate that the IO score assessment of the TIME is associated with a clinical benefit from ICI therapy and that this novel biomarker may inform therapeutic sequencing decisions in mUC, potentially improving outcomes for this notoriously difficult-to-treat disease.

5-Aza-2'-deoxycytidine advances the epithelial-mesenchymal transition of breast cancer cells by demethylating Sipa1 promoter-proximal elements.

Human breast cancer cells exhibit considerable diversity in the methylation status of genomic DNA CpGs that regulate metastatic transcriptome networks. In this study, we identified human Sipa1 promoter-proximal elements that contained a CpG island and demonstrated that the methylation status of the CpG island was inversely correlated with SIPA1 protein expression in cancer cells. 5-Aza-2'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, promoted the expression of Sipa1 in the MCF7 breast cancer cells with a low level of SIPA1 expression. On the contrary, in MDA-MB-231 breast cancer cells with high SIPA1 expression levels, hypermethylation of the CpG island negatively regulated the transcription of Sipa1 In addition, the epithelial-mesenchymal transition (EMT) was reversed after knocking down Sipa1 in MDA-MB-231 cells. However, the EMT was promoted in MCF7 cells with over-expression of SIPA1 or treated with 5-Aza-CdR. Taken together, hypomethylation of the CpG island in Sipa1 promoter-proximal elements could enhance SIPA1 expression in breast cancer cells, which could facilitate EMT of cancer cells, possibly increasing a risk of cancer cell metastasis in individuals treated with 5-Aza-CdR.

Translation of the 27-gene immuno-oncology test (IO score) to predict outcomes in immune checkpoint inhibitor treated metastatic urothelial cancer patients.

BACKGROUND: The IO Score is a 27-gene immuno-oncology (IO) classifier that has previously predicted benefit to immune checkpoint inhibitor (ICI) therapy in triple negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC). It generates both a continuous score and a binary result using a defined threshold that is conserved between breast and lung. Herein, we aimed to evaluate the IO Score's binary threshold in ICI-naïve TCGA bladder cancer patients (TCGA-BLCA) and assess its clinical utility in metastatic urothelial cancer (mUC) using the IMvigor210 clinical trial treated with the ICI, atezolizumab. METHODS: We identified a list of tumor immune microenvironment (TIME) related genes expressed across the TCGA breast, lung squamous and lung adenocarcinoma cohorts (TCGA-BRCA, TCGA-LUSQ, and TCGA-LUAD, 939 genes total) and then examined the expression of these 939 genes in TCGA-BLCA, to identify patients as having high inflammatory gene expression. Using this as a test of classification, we assessed the previously established threshold of IO Score. We then evaluated the IO Score with this threshold in the IMvigor210 cohort for its association with overall survival (OS). RESULTS: In TCGA-BLCA, IO Score positive patients had a strong concordance with high inflammatory gene expression (p < 0.0001). Given this concordance, we applied the IO Score to the ICI treated IMvigor210 patients. IO Score positive patients (40%) had a significant Cox proportional hazard ratio (HR) of 0.59 (95% CI 0.45-0.78 p < 0.001) for OS and improved median OS (15.6 versus 7.5 months) compared to IO Score negative patients. The IO Score remained significant in bivariate models combined with all other clinical factors and biomarkers, including PD-L1 protein expression and tumor mutational burden. CONCLUSION: The IMvigor210 results demonstrate the potential for the IO Score as a clinically useful biomarker in mUC. As this is the third tumor type assessed using the same algorithm and threshold, the IO Score may be a promising candidate as a tissue agnostic marker of ICI clinical benefit. The concordance between IO Score and inflammatory gene expression suggests that the classifier is capturing common features of the TIME across cancer types.

Generation of an algorithm based on minimal gene sets to clinically subtype triple negative breast cancer patients.

BACKGROUND: Recently, a gene expression algorithm, TNBCtype, was developed that can divide triple-negative breast cancer (TNBC) into molecularly-defined subtypes. The algorithm has potential to provide predictive value for TNBC subtype-specific response to various treatments. TNBCtype used in a retrospective analysis of neoadjuvant clinical trial data of TNBC patients demonstrated that TNBC subtype and pathological complete response to neoadjuvant chemotherapy were significantly associated. Herein we describe an expression algorithm reduced to 101 genes with the power to subtype TNBC tumors similar to the original 2188-gene expression algorithm and predict patient outcomes. METHODS: The new classification model was built using the same expression data sets used for the original TNBCtype algorithm. Gene set enrichment followed by shrunken centroid analysis were used for feature reduction, then elastic-net regularized linear modeling was used to identify genes for a centroid model classifying all subtypes, comprised of 101 genes. The predictive capability of both this new "lean" algorithm and the original 2188-gene model were applied to an independent clinical trial cohort of 139 TNBC patients treated initially with neoadjuvant doxorubicin/cyclophosphamide and then randomized to receive either paclitaxel or ixabepilone to determine association of pathologic complete response within the subtypes. RESULTS: The new 101-gene expression model reproduced the classification provided by the 2188-gene algorithm and was highly concordant in the same set of seven TNBC cohorts used to generate the TNBCtype algorithm (87%), as well as in the independent clinical trial cohort (88%), when cases with significant correlations to multiple subtypes were excluded. Clinical responses to both neoadjuvant treatment arms, found BL2 to be significantly associated with poor response (Odds Ratio (OR) =0.12, p=0.03 for the 2188-gene model; OR = 0.23, p < 0.03 for the 101-gene model). Additionally, while the BL1 subtype trended towards significance in the 2188-gene model (OR = 1.91, p = 0.14), the 101-gene model demonstrated significant association with improved response in patients with the BL1 subtype (OR = 3.59, p = 0.02). CONCLUSIONS: These results demonstrate that a model using small gene sets can recapitulate the TNBC subtypes identified by the original 2188-gene model and in the case of standard chemotherapy, the ability to predict therapeutic response.

Mitochondrial mutations associated with aminoglycoside ototoxicity and hearing loss susceptibility identified by meta-analysis.

BACKGROUND: Genetic variations, including mitochondrial mutations, are important contributors to hearing loss, especially in children, and newborn genetic screens for hearing loss mutations are becoming increasingly common. Mitochondrial mutations have been linked with ototoxic responses to common antibiotics, therefore understanding the association of these mutations with hearing loss is of special importance. To address the usefulness of screening for these mutations in a clinical setting, we formed a collaboration of clinicians and geneticists to analyse the association of mitochondrial mutations with non-syndromic hearing loss, including the effect of ethnicity, audiological test methods and aminoglycoside exposure. METHODS: This survey identified 122 variants in 43 studies that have been assessed for an association with hearing loss, and meta-analysis was performed on clinically relevant subsets. RNA folding and conservation analysis further explored possible relevance of these variants. RESULTS: Among all studies, eight variants were found to have significant associations with hearing loss. A partially overlapping set of six variants had significant association with hearing loss when aminoglycoside exposure was assessed. Five of these variants predictive of sensitivity to aminoglycoside spatially co-localise in an RNA folding model. There was little effect of the audiological test method used to assess hearing loss on the association with the variants. CONCLUSIONS: Our results found a small set of studied variants had reproducible association with hearing loss, which will help clarify mutations useful in genetic screens for hearing loss. Several of the aminoglycoside exposure-associated mutations may co-localise on folded 12S rRNA, suggesting a functional association between these loci and aminoglycoside-induced hearing loss.

Expression of a-Tocopherol-Associated protein (TAP) is associated with clinical outcome in breast cancer patients.

BACKGROUND: The role of vitamin E in breast cancer prevention and treatment has been widely investigated, and the different tocopherols that comprise this nutrient have been shown to have divergent associations with cancer outcome. Our previous studies have shown that α-Tocopherol-associated protein (TAP), a vitamin E binding protein, may function as a tumor suppressor-like factor in breast carcinogenesis. The current study addresses the association of TAP expression with breast cancer clinical outcomes. METHODS: Immunohistochemical stain for TAP was applied to a tissue microarray from a breast cancer cohort consisting of 271 patients with a median follow-up time of 5.2 years. The expression of TAP in tumor cells was compared with patient's clinical outcome at 5 years after diagnosis. The potential role of TAP in predicting outcome was also assessed in clinically relevant subsets of the cohort. In addition, we compared TAP expression and Oncotype DX scores in an independent breast cancer cohort consisting of 71 cases. RESULTS: We demonstrate that the expression of TAP was differentially expressed within the breast cancer cohort, and that ER+/PR ± tumors were more likely to exhibit TAP expression. TAP expression was associated with an overall lower recurrence rate and a better 5-year survival rate. This association was primarily in patients with ER+ tumors; exploratory analysis showed that this association was strongest in patients with node-positive tumors and was independent of stage and treatment with chemotherapy. TAP expression in ER/PR negative or triple negative tumors had no association with clinical outcome. In addition, we did not observe an association between TAP expression and Oncotype DX recurrence score. CONCLUSIONS: The significant positive association we found for α-Tocopherol-associated protein with outcome in breast cancer may help to better define and explain studies addressing α-tocopherol's association with cancer risk and outcome. Additionally, further studies to validate and extend these findings may allow TAP to serve as a breast-specific prognostic marker in breast cancer patients, especially in those patients with ER+ tumors.

Angiosarcoma: a study of 98 cases with immunohistochemical evaluation of TLE3, a recently described marker of potential taxane responsiveness.

Angiosarcomas may be primary in the skin, primary in soft tissue or viscera, or secondary to irradiation. All angiosarcomas have a poor prognosis. Taxanes may have efficacy in the treatment of angiosarcoma. Expression of TLE3 has been associated with improved outcome in taxane-treated breast cancers. We studied a series of angiosarcoma with TLE3 immunohistochemistry. Cases of angiosarcoma (98 total cases; 37 cutaneous, 48 soft tissue/visceral and 13 post-irradiation) were retrieved and follow up was obtained. Tumors were classified as 'vasoformative', 'spindled', 'epithelioid' and 'mixed'. TLE3 immunohistochemistry was performed. Statistical analyses were performed. Patients (50 males and 48 females) had a median age of 60.2 years. Tumors had a median size 7.5 cm and were vasoformative (N = 43, 44%), spindled (N = 21, 21%), epithelioid (N = 16, 16%) and mixed (N = 18, 18%). Follow up was available for 89/98 patients (91%): 32 (36%) were dead due to disease, 36 (41%) were dead due to other causes and 21 (24%) remained alive. The median time to death was 2.1 years. TLE3 reactivity was observed in 0/37 (0%) cutaneous angiosarcomas, in 28/48 (58%) cases from soft tissue/viscera and in 4/13 (31%) post-irradiation angiosarcomas. (p = <0.0001). Improved 5-year survival was seen in vasoformative angiosarcomas (p = 0.03). TLE3 expression was not associated with taxane response. However, only a subset of patients was treated with taxane. Our study confirms the poor prognosis of angiosarcoma. Vasoformative angiosarcoma may have a more favorable prognosis. A lack of TLE3 expression in cutaneous angiosarcoma may reflect differing pathogenesis.

A novel immuno-oncology algorithm measuring tumor microenvironment to predict response to immunotherapies.

Immune checkpoint inhibitor (ICI) therapies can improve clinical outcomes for patients with solid tumors, but relatively few patients respond. Because ICI therapies support an adaptive immune response, patients with an active tumor microenvironment (TME) may be more likely to respond, and thus biomarkers capable of discerning an active from a quiescent TME may be useful in patient selection. We developed an algorithm optimized for genes expressed in the mesenchymal and immunomodulatory subtypes of a 101-gene triple negative breast cancer model (Ring, BMC Cancer, 2016, 16:143) as a means to capture the immunological state of the TME. We compared the outcome of the algorithm (IO score) with the 101-gene model and found 88% concordance, indicating the models are correlated but not identical, and may be measuring different TME features. We found 92.5% correlation between IO scores of matched tumor epithelial and adjacent stromal tissues, indicating the IO score is not specific to these tissues, but reflects the TME as a whole. We observed a significant difference in IO score (p = 0.0092) between samples with high tumor-infiltrating lymphocytes and samples with increased neutrophil load, demonstrating agreement between IO score and these two prognostic markers. Finally, among non-small cell lung cancer patients receiving immunotherapy, we observed a significant difference in IO score (p = 0.0035) between responders and non-responders, and a significant odds ratio (OR = 5.76, 95% CI 1.30-25.51, p = 0.021), indicating the IO score can predict patient response. The immuno-oncology algorithm may offer independent and incremental predictive value over current biomarkers in the clinic.

Intron 3 16 bp duplication polymorphism of TP53 contributes to cancer susceptibility: a meta-analysis.

A few genetic polymorphisms of TP53 are known to have a significant effect on cancer susceptibility. Intron 3 16 bp duplication polymorphism of TP53 has been reported to be associated with breast cancer, colorectal cancer, lung cancer and other cancers, but the reported results remain inconclusive. The present study, a meta-analysis including a total of 9801 cases and 10,391 controls from 26 studies, revealed that the 16 bp insertion (Ins) allele is significantly associated with an increased cancer risk in overall analysis [Ins/Ins + deletion (Del)/Ins versus Del/Del: odds ratio (OR) = 1.14, 95% confidence interval (CI) = 1.02-1.27, P = 0.02; Ins/Ins versus Del/Del: OR = 1.35, 95% CI = 1.11-1.63, P = 0.002; Del/Ins versus Del/Del: OR = 1.10, 95% CI = 0.98-1.23, P = 0.11.), particularly in breast cancer subgroup (Ins/Ins + Del/Ins versus Del/Del: OR = 1.16, 95% CI = 1.03-1.31, P = 0.02; Ins/Ins versus Del/Del: OR = 1.81, 95% CI = 1.30-2.52, P < 0.001; Del/Ins versus Del/Del: OR = 1.10, 95% CI = 0.97-1.25, P = 0.13). The relative risks to the colorectal and lung cancers increased but their association power was relatively weak, which may result from a limited number of studies of these two cancer types. These results suggest that intron 3 16 bp duplication polymorphism of TP53 is potentially an important and clinically relevant genetic marker contributing to cancer susceptibility.

Mammostrat as a tool to stratify breast cancer patients at risk of recurrence during endocrine therapy.

INTRODUCTION: Patients with early-stage breast cancer, treated with endocrine therapy, have approximately 90% 5-year disease-free survival. However, for patients at higher risk of relapse despite endocrine therapy, additional adjuvant therapy, such as chemotherapy, may be indicated. The challenge is to prospectively identify such patients. The Mammostrat® test uses five immunohistochemical markers to stratify patients on tamoxifen therapy into risk groups to inform treatment decisions. We tested the efficacy of this panel in a mixed population of cases treated in a single center with breast-conserving surgery and long-term follow-up. METHODS: Tissue microarrays from a consecutive series (1981 to 1998) of 1,812 women managed by wide local excision and postoperative radiotherapy were collected following appropriate ethical review. Of 1,390 cases stained, 197 received no adjuvant hormonal or chemotherapy, 1,044 received tamoxifen only, and 149 received a combination of hormonal therapy and chemotherapy. Median age at diagnosis was 57, 71% were postmenopausal, 23.9% were node-positive and median tumor size was 1.5 cm. Samples were stained using triplicate 0.6 mm2 tissue microarray cores, and positivity for p53, HTF9C, CEACAM5, NDRG1 and SLC7A5 was assessed. Each case was assigned a Mammostrat risk score, and distant recurrence-free survival (DRFS), relapse-free survival (RFS) and overall survival (OS) were analyzed by marker positivity and risk score. RESULTS: Increased Mammostrat scores were significantly associated with reduced DRFS, RFS and OS in estrogen receptor (ER)-positive breast cancer (P < 0.00001). In multivariate analyses the risk score was independent of conventional risk factors for DRFS, RFS and OS (P < 0.05). In node-negative, tamoxifen-treated patients, 10-year recurrence rates were 7.6 ± 1.5% in the low-risk group versus 20.0 ± 4.4% in the high-risk group. Further, exploratory analyses revealed associations with outcome in both ER-negative and untreated patients. CONCLUSIONS: This is the fifth independent study providing evidence that Mammostrat can act as an independent prognostic tool for ER-positive, tamoxifen-treated breast cancer. In addition, this study revealed for the first time a possible association with outcome regardless of node status and ER-negative tumors. When viewed in the context of previous results, these data provide further support for this antibody panel as an aid to patient management in early-stage breast cancer.

Three common TP53 polymorphisms in susceptibility to breast cancer, evidence from meta-analysis.

The association of polymorphisms of tumor suppressor gene TP53 with breast cancer has widely been reported; however, the results are inconsistent. Here, we selected three commonly studied TP53 polymorphisms: codon 72 Arg > Pro, IVS3 16 bp Del/Ins, and IVS6 + 62A > G to explore their association with breast cancer risk by meta-analysis of published case-control studies. The results showed that codon 72 was not associated with breast cancer risk among 37 combined case-control studies (23,567 cases and 25,995 controls). However, a significant association with decreased risk of breast cancer was found in the Mediterranean studies (PP + PR vs. RR: OR = 0.32, 95% CI = 0.24-0.44, P < 0.001; PP vs. RR: OR = 0.35, 95% CI = 0.21-0.60, P < 0.001). IVS3 16 bp Del/Ins was significantly associated with an increased risk of developing breast cancer in a pooled 8 study dataset (2,470 cases and 2,825 controls; Ins/Ins + Del/Ins vs. Del/Del: OR = 1.15, 95% CI = 1.01-1.30, P = 0.04; Ins/Ins vs. Del/Del: OR = 1.75, 95% CI = 1.20-2.37, P = 0.003). No significant association was observed between IVS6 + 62A > G and breast cancer risk in a total of 10 studies (8,537 cases and 9,586 controls). These results suggest that IVS3 16 bp Del/Ins is likely an important genetic marker contributing to susceptibility of breast cancer, and codon 72 has a potential role in association with breast cancer risk within certain populations or regions.

The expression of TRMT2A, a novel cell cycle regulated protein, identifies a subset of breast cancer patients with HER2 over-expression that are at an increased risk of recurrence.

BACKGROUND: Over-expression of HER2 in a subset of breast cancers (HER2+) is associated with high histological grade and aggressive clinical course. Despite these distinctive features, the differences in response of HER2+ patients to both adjuvant cytotoxic chemotherapy and targeted therapy (e.g. trastuzumab) suggests that unrecognized biologic and clinical diversity is confounding treatment strategies. Furthermore, the small but established risk of cardiac morbidity with trastuzumab therapy compels efforts towards the identification of biomarkers that might help stratify patients. METHODS: A single institution tissue array cohort assembled at the Clearview Cancer Institute of Huntsville (CCIH) was screened by immunohistochemistry staining using a large number of novel and commercially available antibodies to identify those with a univariate association with clinical outcome in HER2+ patients. Staining with antibody directed at TRMT2A was found to be strongly associated with outcome in HER2+ patients. This association with outcome was tested in two independent validation cohorts; an existing staining dataset derived from tissue assembled at the Cleveland Clinic Foundation (CCF), and in a new retrospective study performed by staining archived paraffin blocks available at the Roswell Park Cancer Institute (RPCI). RESULTS: TRMT2A staining showed a strong correlation with likelihood of recurrence at five years in 67 HER2+ patients from the CCIH discovery cohort (HR 7.0; 95% CI 2.4 to 20.1, p < 0.0004). This association with outcome was confirmed using 75 HER2+ patients from the CCF cohort (HR 3.6; 95% CI 1.3 to 10.2, p < 0.02) and 64 patients from the RPCI cohort (HR 3.4; 95% CI 1.3-8.9, p < 0.02). In bivariable analysis the association with outcome was independent of grade, tumor size, nodal status and the administration of conventional adjuvant chemotherapy in the CCIH and RPCI cohorts. CONCLUSIONS: Studies from three independent single institution cohorts support TRMT2A protein expression as a biomarker of increased risk of recurrence in HER2+ breast cancer patients. These results suggest that TRMT2A expression should be further studied in the clinical trial setting to explore its predictive power for response to adjuvant cytotoxic chemotherapy in combination with HER2 targeted therapy.

TLE3 as a candidate biomarker of response to taxane therapy.

INTRODUCTION: The addition of taxanes (Ts) to chemotherapeutic regimens has not demonstrated a consistent benefit in early-stage breast cancer. To date, no clinically relevant biomarkers that predict T response have been identified. METHODS: A dataset of immunohistochemistry stains in 411 patients was mined to identify potential markers of response. TLE3 emerged as a candidate marker for T response. To test the association with T sensitivity, an independent 'triple-negative' (TN) validation cohort was stained with anti-TLE3 antibody. RESULTS: TLE3 staining was associated with improved 5-year disease-free interval (DFI) in the overall cohort (n = 441, P < 0.004), in patients treated with cyclophosphamide (C), methotrexate, and 5-fluorouracil (n = 72, P < 0.02), and in those treated with regimens containing doxorubicin (A) and a T (n = 65, P < 0.04). However, no association was shown with outcome in untreated patients (n = 203, P = 0.49) or those treated with a regimen containing A only (n = 66, P = 0.97). In the TN cohort, TLE3 staining was significantly associated with improved 5-year DFI in all patients (n = 81, P < 0.015), in patients treated with AC + T (n = 45, P < 0.02), but not in patients treated with AC (n = 17, P = 0.81). TLE3 was independent of tumor size, nodal status, and grade by bivariable analysis in both cohorts. CONCLUSION: TLE3 staining is associated with improved DFI in T-treated patients in two independent cohorts. Since the validation study was performed in a TN cohort, TLE3 is not serving as a surrogate for estrogen receptor or HER2 expression. TLE3 should be studied in large clinical trial cohorts to establish its role in T chemotherapy selection.

A novel five-antibody immunohistochemical test for subclassification of lung carcinoma.

Malignant epithelial lung carcinoma can be subclassified by histology into several tumor types, including adenocarcinoma and squamous cell carcinoma. The need for a uniform method of classifying lung carcinomas is growing as clinical trials reveal treatment and side effect differences associated with histological subtypes. Diagnosis is primarily performed by morphological assessment. However, the increased use of needle biopsy has diminished the amount of tissue available for interpretation. These changes in how lung carcinomas are diagnosed and treated suggest that the development of improved molecular-based classification tools could improve patient management. We used a 551-patient surgical specimen lung carcinoma retrospective cohort from a regional hospital to assess the association of a large number of proteins with histological type by immunohistochemistry. Five of these antibodies, targeting the proteins TRIM29, CEACAM5, SLC7A5, MUC1, and CK5/6, were combined into one test using a weighted algorithm trained to discriminate adenocarcinoma from squamous cell carcinoma. Antibody-based classification on 600 muM tissue array cores with the five-antibody test was compared to standard histological evaluation on surgical specimens in three independent lung carcinoma cohorts (combined population of 1111 patients). In addition, the five-antibody test was tested against the two-marker panel thyroid transcription factor-1 (TTF-1) and TP63. Both the five-antibody test and TTF-1/TP63 panel had similarly low misclassification rates on the validation cohorts compared to morphological-based diagnosis (4.1 vs 3.5%). However the percentage of patients remaining unclassifiable by TTF-1/TP63 (22%, 95% CI: 20-25%) was twice that of the five-antibody test (11%, 95% CI: 8-13%). The results of this study suggest the five-antibody test may have an immediate function in the clinic for helping pathologists distinguish lung carcinoma histological types. The results also suggest that if validated in prospectively defined clinical trials this classifier might identify candidates for targeted therapy that are overlooked with current diagnostic approaches.

Chemosensitivity and stratification by a five monoclonal antibody immunohistochemistry test in the NSABP B14 and B20 trials.

PURPOSE: To test the association between risk stratification and outcome in a prospectively designed, blinded retrospective study using tissue arrays of available paraffin blocks from the estrogen receptor-expressing, node-negative samples from the National Surgical Adjuvant Breast and Bowel Project B14 and B20 tamoxifen and chemotherapy trials. EXPERIMENTAL DESIGN: Tissue arrays were stained by immunohistochemistry targeting p53, NDRG1, SLC7A5, CEACAM5, and HTF9C. Risk stratification was done using predefined scoring rules, algorithm for combining scores, and cutoff points for low-risk, moderate-risk, and high-risk patient strata. RESULTS: In a univariate Cox model, this test was significantly associated with recurrence-free interval [HR, 1.3 (95% confidence interval, 1.1-1.6); P = 0.006]. In a multivariate model it contributed information independent of age, tumor size, and menopausal status (P = 0.007). The Kaplan-Meier estimates of the proportion of recurrence-free after 10 years were 73%, 86%, and 85% for the high-risk, moderate-risk, and low-risk groups (P = 0.001). The Kaplan-Meier estimates of the breast-cancer-specific-death rate were 23%, 10%, and 9% (P < 0.0001). Exploratory analysis in patients >/=60 years old showed Kaplan-Meier estimates of the proportion of recurrence-free of 78%, 89%, and 92%. Both high-risk and low-risk groups showed significant improvement on treatment with cytotoxic chemotherapy. CONCLUSIONS: Immunohistochemistry using five monoclonal antibodies assigns breast cancer patients to a risk index that was significantly associated with clinical outcome among the estrogen receptor-expressing, node-negative tamoxifen-treated patients. It seems that the test may be able to identify patients who have greater absolute benefit from adjuvant chemotherapy compared with unstratified patient populations. Exploratory analysis suggests that this test will be most useful in clinical decision making for postmenopausal patients.

Transcript annotation in FANTOM3: mouse gene catalog based on physical cDNAs.

The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species.

Complex relationship between TAS2 receptor variations, bitterness perception, and alcohol consumption observed in a population of wine consumers.

Our ability to taste bitterness affects our food choices and alcohol consumption. Alleles in the taste 2 receptor member TAS2R38 have been linked to the ability to perceive bitterness in bitter-tasting compounds and in many foods, and people with these bitterness sensitivity alleles have been shown to be less likely to consume alcohol, presumably because of alcohol's bitter taste. In a survey of 519 participants, almost all of whom regularly consumed alcohol, we observed that genetic variants in TAS2R38 were significantly associated with both increased alcohol consumption and the ability to perceive bitterness in several foods and a bitter chemical. In total, we assayed 39 variants in 25 genes that have been implicated in the genetics of taste perception, and no other variants predicted alcohol consumption. Perception of bitterness in broccoli and a preference for black coffee were also positively associated with alcohol consumption. As the consumption of alcohol is a social activity there may be incentive to appreciate its bitter aspects, and increased perception of bitterness could therefore be associated with consumption of some bitter beverages. As this study's respondents were predominantly frequent consumers of alcohol, these findings may be consistent with previous studies that have seen that increased experience in the consumption of wine is associated with an increased perception of PROP bitterness. Further work elucidating the complex relationship between the genetics of bitter perception and alcohol consumption will better describe these connections.

Gene expression patterns within cell lines are predictive of chemosensitivity.

BACKGROUND: The NCI has undertaken a twenty-year project to characterize compound sensitivity patterns in a selected set of sixty tumor derived cell lines. Previous studies have explored the relationship between compound sensitivity patterns to gene expression, protein expression, and DNA copy number for these same cell lines. A strong correlation between the pattern of expression of a biomarker and sensitivity to a compound could suggest a clinically interesting biological relationship between the two. RESULTS: We isolated RNA's and measured expression of 40000 genes using cDNA microarrays from the fifty-nine publicly available cell lines. Analysis of this data set in comparison with published gene expression data sets demonstrates a high degree of reproducibility in expression level measurements even using completely independent RNA preparations and array technologies. Using the fifty-nine cell lines for discovery and an additional seven cell lines for which extensive compound sensitivity data were available as a test set, we determined that gene-compound pairs with a correlation coefficient above 0.6 had a false discovery rate of approximately 5%. Large scale features of the gene expression and chemosensitivity data, such as tissue of origin and other physiological factors, did not seem to explain the majority of correlations between gene and compound patterns. CONCLUSION: A comparison of gene expression and compound sensitivity in panels of cell lines was demonstrated to have a relatively high validation and low false discovery rate supporting the use of this approach and datasets for identifying candidate biomarkers and targeted biologically active compounds.

Large scale newborn deafness genetic screening of 142,417 neonates in Wuhan, China.

Almost one third of the three million people in China suffering severe deafness are children, and 50% of these cases are believed to have genetic components to their etiology. Newborn hearing genetic screening can complement Universal Neonatal Hearing Screening for the diagnosis of congenital hearing loss as well as identifying children at risk for late-onset and progressive hearing impairment. The aim of this joint academic and Ministry of Health project was to prototype a cost effective newborn genetic screen in a community health setting on a city-wide level, and to ascertain the prevalence of variation at loci that have been associated with non-syndromic hearing loss. With the participation of 143 local hospitals in the city of Wuhan, China we screened 142,417 neonates born between May 2014 and Dec. 2015. The variants GJB2 c.235delC, SLC26A4 c.919-2A>G, and mitochondrial variants m.1555A>G and m.1494C>T were assayed using real time PCR. Newborns found to carry a variant were re-assayed by sequencing in duplicate. Within a subset of 707 newborns we assayed using real-time PCR and ARMS-PCR to compare cost, sensitivity and operating procedure. The most frequent hearing loss associated allele detected in this population was the 235delC variant in GJB2 gene. In total, 4289 (3.01%) newborns were found to carry at least one allele of either GJB2 c.235delC, SLC26A4 c.919-2A>G or two assayed MT-RNR1 variants. There was complete accordance between the real-time PCR and the ARMS PCR, though the real-time PCR had a much lower failure rate. Real-time PCR had a lower cost and operating time than ARMS PCR. Ongoing collaboration with the participating hospitals will determine the specificity and sensitivity of the association of the variants with hearing loss at birth and arising in early childhood, allowing an estimation of the benefits of newborn hearing genetic screening in a large-scale community setting.