Breast Cancer Consensus Subtypes: A system for subtyping breast cancer tumors based on gene expression.

Breast cancer is heterogeneous in prognoses and drug responses. To organize breast cancers by gene expression independent of statistical methodology, we identified the Breast Cancer Consensus Subtypes (BCCS) as the consensus groupings of six different subtyping methods. Our classification software identified seven BCCS subtypes in a study cohort of publicly available data (n = 5950) including METABRIC, TCGA-BRCA, and data assayed by Affymetrix arrays. All samples were fresh-frozen from primary tumors. The estrogen receptor-positive (ER+) BCCS subtypes were: PCS1 (18%) good prognosis, stromal infiltration; PCS2 (15%) poor prognosis, highly proliferative; PCS3 (13%) poor prognosis, highly proliferative, activated IFN-gamma signaling, cytotoxic lymphocyte infiltration, high tumor mutation burden; PCS4 (18%) good prognosis, hormone response genes highly expressed. The ER- BCCS subtypes were: NCS1 (11%) basal; NCS2 (10%) elevated androgen response; NCS3 (5%) cytotoxic lymphocyte infiltration; unclassified tumors (9%). HER2+ tumors were heterogeneous with respect to BCCS.

ColoType: a forty gene signature for consensus molecular subtyping of colorectal cancer tumors using whole-genome assay or targeted RNA-sequencing.

Colorectal cancer (CRC) tumors can be partitioned into four biologically distinct consensus molecular subtypes (CMS1-4) using gene expression. Evidence is accumulating that tumors in different subtypes are likely to respond differently to treatments. However, to date, there is no clinical diagnostic test for CMS subtyping. In this study, we used novel methodology in a multi-cohort training domain (n = 1,214) to develop the ColoType scores and classifier to predict CMS1-4 based on expression of 40 genes. In three validation cohorts (n = 1,744, in total) representing three distinct gene-expression measurement technologies, ColoType predicted gold-standard CMS subtypes with accuracies 0.90, 0.91, 0.88, respectively. To accommodate for potential intratumoral heterogeneity and tumors of mixed subtypes, ColoType was designed to report continuous scores measuring the prevalence of each of CMS1-4 in a tumor, in addition to specifying the most prevalent subtype. For analysis of clinical specimens, ColoType was also implemented with targeted RNA-sequencing (Illumina AmpliSeq). In a series of formalin-fixed, paraffin-embedded CRC samples (n = 49), ColoType by targeted RNA-sequencing agreed with subtypes predicted by two independent methods with accuracies 0.92, 0.82, respectively. With further validation, ColoType by targeted RNA-sequencing, may enable clinical application of CMS subtyping with widely-available and cost-effective technology.

Independent Validation of EarlyR Gene Signature in BIG 1-98: A Randomized, Double-Blind, Phase III Trial Comparing Letrozole and Tamoxifen as Adjuvant Endocrine Therapy for Postmenopausal Women With Hormone Receptor-Positive, Early Breast Cancer.

BACKGROUND: EarlyR gene signature in estrogen receptor-positive (ER+) breast cancer is computed from the expression values of ESPL1, SPAG5, MKI67, PLK1, and PGR. EarlyR has been validated in multiple cohorts profiled using microarrays. This study sought to verify the prognostic features of EarlyR in a case-cohort sample from BIG 1-98, a randomized clinical trial of ER+ postmenopausal breast cancer patients treated with adjuvant endocrine therapy (letrozole or tamoxifen). METHODS: Expression of EarlyR gene signature was estimated by Illumina cDNA-mediated Annealing, Selection, and Ligation assay of RNA from formalin-fixed, paraffin-embedded primary breast cancer tissues in a case-cohort subset of ER+ women (N = 1174; 216 cases of recurrence within 8 years) from BIG 1-98. EarlyR score and prespecified risk strata (≤25 = low, 26-75 = intermediate, >75 = high) were "blindly" computed. Analysis endpoints included distant recurrence-free interval and breast cancer-free interval at 8 years after randomization. Hazard ratios (HRs) and test statistics were estimated with weighted analysis methods. RESULTS: The distribution of the EarlyR risk groups was 67% low, 19% intermediate, and 14% high risk in this ER+ cohort. EarlyR was prognostic for distant recurrence-free interval; EarlyR high-risk patients had statistically increased risk of distant recurrence within 8 years (HR = 1.73, 95% confidence interval = 1.14 to 2.64) compared with EarlyR low-risk patients. EarlyR was also prognostic of breast cancer-free interval (HR = 1.74, 95% confidence interval = 1.21 to 2.62). CONCLUSIONS: This study confirmed the prognostic significance of EarlyR using RNA from formalin-fixed, paraffin-embedded tissues from a case-cohort sample of BIG 1-98. EarlyR identifies a set of high-risk patients with relatively poor prognosis who may be considered for additional treatment. Further studies will focus on analyzing the predictive value of EarlyR signature.

EarlyR: A Robust Gene Expression Signature for Predicting Outcomes of Estrogen Receptor-Positive Breast Cancer.

INTRODUCTION: Early stage estrogen receptor (ER)-positive breast cancer may be treated with chemotherapy in addition to hormone therapy. Currently available molecular signatures assess the risk of recurrence and the benefit of chemotherapy; however, these tests may have large intermediate risk groups, limiting their usefulness. METHODS: The EarlyR prognostic score was developed using integrative analysis of microarray data sets and formalin-fixed, paraffin-embedded-based quantitative real-time PCR assay and validated in Affymetrix data sets and METABRIC cohort using Cox proportional hazards models and Kaplan-Meier survival analysis. Concordance index was used to measure the probability of prognostic score agreement with outcome. RESULTS: The EarlyR score and categorical risk strata (EarlyR-Low, EarlyR-Int, EarlyR-High) derived from expression of ESPL1, MKI67, SPAG5, PLK1 and PGR was prognostic of 8-year distant recurrence-free interval in Affymetrix (categorical P = 3.5 × 10-14; continuous P = 8.8 × 10-15) and METABRIC (categorical P < 2.2 × 10-16; continuous P < 10-16) data sets of ER+ breast cancer. Similar results were observed for the breast cancer-free interval end point. At most 13% of patients were intermediate risk and at least 66% patients were low risk in both ER+ cohorts. The EarlyR score was significantly prognostic (distant recurrence-free interval; P < .001) in both lymph node-negative and lymph node-positive patients and was independent from clinical factors. EarlyR and surrogates of current molecular signatures were comparable in prognostic significance by concordance index. CONCLUSION: The 5-gene EarlyR score is a robust prognostic assay that identified significantly fewer patients as intermediate risk and more as low risk than currently available assays. Further validation of the assay in clinical trial-derived cohorts is ongoing.

EarlyR signature predicts response to neoadjuvant chemotherapy in breast cancer.

BACKGROUND: EarlyR gene signature uses ESPL1, SPAG5, MKI67, PLK1 and PGR to classify ER+ breast cancer (ER+ BC) into EarlyR-Low, EarlyR-Int, and EarlyR-High risk strata and is prognostic in patients treated with adjuvant chemotherapy. The ability of EarlyR to predict pathological complete response (pCR) and long-term survival following neoadjuvant chemotherapy (NACT) is evaluated herein. MATERIALS: The ability of EarlyR gene signature to predict pCR was assessed in publicly available Affymetrix microarray datasets (Cohort A; n = 659; 74 pCR events) derived from NACT-treated ER+ BC patients. Distant relapse-free survival (DRFS) results were analyzed in patients treated with NACT and adjuvant hormone therapy (AHT) (n = 281) and compared with patients treated with AHT alone (n = 455) (Cohort B; n = 736; 142 events). RESULTS: In cohort A, EarlyR was a significant predictor of pCR (p = 5.8 × 10-11) (EarlyR-Low, n = 400, pCR = 40, 5%; EarlyR-Int, n = 69, pCR = 7, 15% and EarlyR-High, n = 190, pCR = 47, 24%). In EarlyR-Low of Cohort B, the 5-year DRFS was not significantly (p = 0.55) different between NACT + AHT [0.81 (95%CI 0.73-0.90)] and AHT-only [0.85 (95%CI 0.81-0.90)]. In contrast, in EarlyR-High, the 5-year DRFS was higher (p = 0.019) in NACT + AHT [0.81 (95%CI 0.70-0.93)] as compared to AHT-only [0.60 (95%CI 0.51-0.71)]. CONCLUSIONS: High EarlyR is strongly associated with pCR in patients treated with neoadjuvant chemotherapy. EarlyR also predicts poor DRFS outcomes for patients in EarlyR-High not receiving NACT, and improved survival in NACT-treated EarlyR-High patients. EarlyR is not only a prognostic assay but also a predictive assay that identifies patients, who are also likely to respond to chemotherapy.

Investigating the deep supercooling ability of an Alaskan beetle, Cucujus clavipes puniceus, via high throughput proteomics.

Cucujus clavipes puniceus is a freeze avoiding beetle capable of surviving the long, extremely cold winters of the Interior of Alaska. Previous studies showed that some individuals typically supercool to mean values of approximately -40 °C, with some individuals supercooling to as low as -58 °C, but these non-deep supercooling (NDSC) individuals eventually freeze if temperatures drop below this. However, other larvae, especially if exposed to very cold temperatures, supercool even further. These deep supercooling (DSC) individuals do not freeze even if cooled to -100 °C. In addition, the body water of the DSC larvae vitrifies (turns to a glass) at glass transition temperatures of -58 to -70 °C. This study examines the proteomes of DSC and NDSC larvae to assess proteins that may contribute to or inhibit the DSC trait. Using high throughput proteomics, we identified 138 proteins and 513 Gene Ontology categories in the DSC group and 104 proteins and 573 GO categories in the NDSC group. GO categories enriched in DSC include alcohol metabolic process, cellular component morphogenesis, monosaccharide metabolic process, regulation of biological quality, extracellular region, structural molecule activity, and antioxidant activity. Proteins unique to DSC include alpha casein precursor, alpha-actinin, vimentin, tropomyosin, beta-lactoglobulin, immunoglobulins, tubulin, cuticle proteins and endothelins.

Elucidating the biochemical overwintering adaptations of Larval Cucujus clavipes puniceus, a nonmodel organism, via high throughput proteomics.

Cucujus clavipes puniceus (C.c.p.) is a nonmodel, freeze-avoiding beetle that overwinters as extremely cold-tolerant larvae in the interior boreal forests of Alaska to temperatures as low as -100 °C. Using a tandem MS-based approach, we compared the proteomes of winter- and summer-collected C.c.p. to identify proteins that may play functional roles in successful overwintering. Using Gene Ontology (GO) analysis and manual interpretation, we identified 104 proteins in winter and 128 proteins in summer samples. We found evidence to indicate a cytoskeletal rearrangement between seasons, with Winter NDSC possessing unique actin and myosin isoforms while summer larvae up-regulated α actinin, tubulin, and tropomyosin. We also detected a fortification of the cuticle in winter via unique cuticle proteins, specifically larval/pupal rigid cuticle protein 66 precursor and larval cuticle protein A2B. Also, of particular interest in the winter larvae was an up-regulation of proteins related to silencing of genes (bromodomain adjacent to zinc finger domain 2A and antisilencing protein 1), proteins involved with metabolism of amines (2-isopropylmalate synthase and dihydrofolate reductase), and immune system process (lysozyme C precursor), among others. This represents the first high throughput MS/MS analysis of a nonmodel, cold-tolerant organism without a concurrent microarray analysis.

Sulindac treatment alters collagen and matrilysin expression in adenomas of ApcMin/+ mice.

Non-steroidal anti-inflammatory drugs (NSAIDs) have shown potential as chemopreventive agents against cancer formation, especially colorectal cancers. However, the mechanisms by which these drugs act are not fully understood. In this study, Apc(Min/+) mice, a genetic model of human familial adenomatous polyposis, were treated with sulindac, and these mice demonstrated tumor reduction of >80%, consistent with previous reports. Gene microarray analyses of RNA from adenoma-derived dysplastic epithelial cells revealed that collagen genes, viz. Col1a2, Col5a2, Col6a2 and Col6a3, were upregulated, and matrilysin matrix metalloproteases-7 (Mmp7) was downregulated, in sulindac-treated mice. Reverse transcription-polymerase chain reaction validated gene expression of the Col6a2 subunit of collagen VI and of Mmp7. Confocal microscopy and immunofluorescence showed that within the tumors of non-treated mice, collagen VI was present in low amounts, but was enhanced within the tumors of sulindac-treated mice. Collagens I and V demonstrated similar patterns, but were not as prominent as collagen VI. Mmp7 was found in 'hot spot' areas within the tumors of Apc(Min/+) mice treated with the vehicle, but was greatly diminished in those mice treated with sulindac. Studies with Apc(Min/+)/Mmp7(-/-) double-deficient mice demonstrated the reciprocal relationships of Mmp7 expression and the levels of these three collagens in vivo. The results of this study demonstrated that sulindac was effective in increasing the expression of different collagens and decreasing the expression of Mmp7, effects that may contribute to altered tumor burden in cancer patients undergoing NSAIDs treatments.