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Background/aim: This study sought to determine whether an autoimmune background could identify patients with HER2-positive early breast cancer (EBC) who derive differential benefit from primary adjuvant trastuzumab-based therapy., Patients and Methods: HERA is an international randomized trial of 5,102 women with HER2-positive EBC, who were enrolled to either receive adjuvant trastuzumab or not. In this exploratory analysis, the interaction between autoimmune history and the magnitude of trastuzumab benefit was evaluated., Results: A total of 5,099 patients were included in the current analysis. Among them, 325 patients (6.4%) had autoimmune disease history, 295 of whom had active disease. Patients were randomly assigned to trastuzumab or no-trastuzumab groups. Similar reductions in the risk of events in patients with and without autoimmune history were observed (interaction p=0.95 for disease-free survival, and p=0.62 for overall survival)., Conclusion: No evidence of a differential benefit from trastuzumab in patients with a medical history of autoimmune disease was found., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Cyclin-dependent kinase 4 (CDK4) and CDK6 together with D-type cyclins (D1, D2 and D3) to promote cell cycle entry and progression through G1 by inactivating retinoblastoma protein (RB) by inhibiting an INK4 family of CDK inhibitors (CDKN2A/B). Selective cyclin-dependent kinase inhibitors are game changers in the clinical management of hormone receptor-positive/HER2-negative advanced breast cancers. There are currently three CDK4/6 inhibitors that have been approved by the US Food and Drug administration: palbociclib, ribociclib, and abemaciclib. Although, the bulk of the data supporting the use of selective CDK4/6 inhibitors is currently in breast cancer patients with other tumor types are expected to benefit as well from this class of agents, which can counter proliferative signaling pathways and arrest cell cycle in early G1 phase. Areas of active interest include identifying predictive biomarkers for CDK4/6 inhibitors, deciding whether to continue CDK4/6 inhibitor after disease progression, creating novel treatment combinations and expanding the use of CDK4/6 inhibitors beyond hormone receptor-positive/HER2-negative advanced breast cancer. Right now, CCND1 amplification and CDKN2A/B loss have not sorted out biomarkers useful for the purpose. One of the most important clinical questions is how to use a CDK4/6 inhibitor with other targeted therapies. Here we provide a rationale that oncologists can use to sequence the CDK4/6 inhibitors along with the PI3K-AKT-mTOR pathway-specific inhibitor(s); future data will better guide this approach. In this review we have tried to (a) describe the specific cellular signals initiated following alterations in the cell cycle pathway genes and the PI3K pathway genes, (b) interrogate how these alterations/co-alterations influence the action of PI3K and cell cycle pathway-targeted drugs in different clinical trials and (c) understand the role of co-alterations towards the development of cell cycle inhibitors induced drug-resistance in ER+ breast cancers., Competing Interests: None.

Fusions involving the oncogenic gene RET have been observed in thyroid and lung cancers. Here we report RET gene alterations, including amplification, missense mutations, known fusions, novel fusions, and rearrangements in breast cancer. Their frequency, oncogenic potential, and actionability in breast cancer are described. Two out of eight RET fusions (NCOA4-RET and a novel RASGEF1A-RET fusion) and RET amplification were functionally characterized and shown to activate RET kinase and drive signaling through MAPK and PI3K pathways. These fusions and RET amplification can induce transformation of non-tumorigenic cells, support xenograft tumor formation, and render sensitivity to RET inhibition. An index case of metastatic breast cancer progressing on HER2-targeted therapy was found to have the NCOA4-RET fusion. Subsequent treatment with the RET inhibitor cabozantinib led to a rapid clinical and radiographic response. RET alterations, identified by genomic profiling, are promising therapeutic targets and are present in a subset of breast cancers.

Purpose: The Herceptin Adjuvant study is an international multicenter randomized trial that compared 1 or 2 years of trastuzumab given every 3 weeks with observation in women with human epidermal growth factor 2-positive (HER2+) breast cancer after chemotherapy. Identification of biomarkers predictive of a benefit from trastuzumab will minimize overtreatment and lower health care costs., Methods: To identify possible single-gene biomarkers, an exploratory analysis of 3,669 gene probes not expected to be expressed in normal breast tissue was conducted. Disease-free survival (DFS) was used as the end point in a Cox regression model, with the interaction term between C8A mRNA and treatment as a categorical variable split on the cohort mean., Results: A significant interaction between C8A mRNA and treatment was detected ( P < .001), indicating a predictive response to trastuzumab treatment. For the C8A-low subgroup (mRNA expression lower than the cohort mean), no significant treatment benefit was observed ( P = .73). In the C8A-high subgroup, patients receiving trastuzumab experienced a lower hazard of a DFS event by approximately 75% compared with those in the observation arm (hazard ratio [HR], 0.25; P < .001). A significant prognostic effect of C8A mRNA also was seen ( P < .001) in the observation arm, where the C8A-high group hazard of a DFS event was three times the respective hazard of the C8A-low group (HR, 3.27; P < .001). C8A mRNA is highly prognostic in the Hungarian Academy of Science HER2+ gastric cancer cohort (HR, 1.72; P < .001)., Conclusion: C8A as a single-gene biomarker prognostic of DFS and predictive of a benefit from trastuzumab has the potential to improve the standard of care in HER2+ breast cancer if validated by additional studies. Understanding the advantage of overexpression of C8A related to the innate immune response can give insight into the mechanisms that drive cancer., Competing Interests: The contents of this article are solely the responsibility of the authors. Clinical trial information: NCT00045032.The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center. Scooter WillisNo relationship to discloseVarvara PolydoropoulouNo relationship to discloseYuliang SunNo relationship to discloseBrandon YoungNo relationship to discloseZoi TsourtiNo relationship to discloseDimitris KarlisNo relationship to discloseBradley LongNo relationship to discloseXiaoqian LinNo relationship to discloseStephanie TheelNo relationship to discloseJennifer CarlsonNo relationship to discloseBalazs GyőrffyConsulting or Advisory Role: Richter GedeonCasey WilliamsResearch Funding: Takeda Pharmaceuticals (Inst), TESARO (Inst)Mark AbramovitzNo relationship to discloseUrania DafniHonoraria: AmgenMitch DowsettHonoraria: Novartis, Pfizer, Myriad Genetics Consulting or Advisory Role: Genoptix, Roche, Genentech, Janssen Pharmaceuticals, GTx, Radius Health Research Funding: Roche (Inst), AstraZeneca (Inst), Novartis (Inst), Pfizer (Inst), Radius Health (Inst) Other Relationship: Institute of Cancer ResearchBrian Leyland-JonesStock and Other Ownership Interests: Catalyst Pharmaceuticals, ProGenix, Puma Biotechnology, Sucampo Pharmaceuticals, ARIAD Pharmaceuticals, Zogenix Consulting or Advisory Role: GlaxoSmithKline, Amgen Speakers’ Bureau: Genentech, Exelixis Research Funding: Takeda Pharmaceuticals, TESARO Expert Testimony: Amgen, (© 2018 by American Society of Clinical Oncology.)

Purpose: To determine the potential for detection of incidental germline cancer predisposition mutations through cell-free DNA (cfDNA) analyses in patients who underwent solid tumor somatic mutation evaluation., Patients and Methods: Data were evaluated from 10,888 unselected patients with advanced (stage III/IV) cancer who underwent Guardant360 testing between November 2015 and December 2016. The main outcome was prevalence of putative germline mutations identified among 16 actionable hereditary cancer predisposition genes., Results: More than 50 cancer types were studied, including lung (41%), breast (19%), colorectal (8%), prostate (6%), pancreatic (3%), and ovarian (2%). Average patient age was 63.5 years (range, 18 to 95 years); 43% were male. One hundred and fifty-six individuals (1.4%) had suspected hereditary cancer mutations in 11 genes. Putative germline mutations were more frequent in individuals younger than 50 years versus those 50 years and older (3.0% v 1.2%, respectively; P < .001). Highest yields of putative germline findings were in patients with ovarian (8.13%), prostate (3.46%), pancreatic (3.34%), and breast (2.2%) cancer. Putative germline mutation identification was consistent among 12 individuals with multiple samples. Patients with circulating tumor DNA copy number variation and/or reversion mutations suggestive of functional loss of the wild-type allele in the tumor DNA also are described., Conclusion: Detection of putative germline mutations from cfDNA is feasible across multiple genes and cancer types without prior mutation knowledge. Many mutations were found in cancers without clear guidelines for hereditary cancer genetic counseling/testing. Given the clinical significance of identifying hereditary cancer predisposition for patients and their families as well as targetable germline alterations such as in BRCA1 or BRCA2, research on the best way to validate and return potential germline results from cfDNA analysis to clinicians and patients is needed.

We present a protocol for live cancer cell-imaging by triple-fluorescent staining to test 3 crucial mechanisms of apoptosis; the enzymatic activity of executioner caspase3, caspase-dependent phosphatidylserine presentation on the cell surface and mitochondrial function. We standardized a protocol to co-stain live tumor cells with the NucView488-Casp3 substrate, CF594 AnnexinV, and MitoViewBlue. We validated this protocol following apoptosis induction with paclitaxel or in combination with BKM120. Fluorescent imaging of cells using simultaneous live/dead cell markers (CalceinAM green/EthD-1red) was used as internal control. We used quantitative confluence (Essen), AnnexinV-PE staining (Accuri C6), expression of cl-caspase3, Cl-PARP and mitochondrial potential (TMRE-A) as validation criteria in A2780 and OVK18 cells following drug treatment which decreased proliferation, & increased apoptotic signaling with mitochondrial depolarization. Treatment blocked cytoplasmic MitoViewBlue staining while increased both nuclear NucView488-Casp3 substrate and red membranous CF594 AnnexinV staining. Merged images showed 100% mutual exclusivity between MitoViewBlue and caspase3 or AnnexinV stains in control and treated cells as determined by overlap and colocalization coefficients. Caspase3 and AnnexinV staining in treated cells were both separate and overlapped (yellow fluorescence) indicating the sequence of apoptotic-events. The protocol will help in deciphering mechanistic involvement of different stages/features of apoptosis in tumor cell following anti-cancer drugs in real-time.

Background: While standard RNA expression tests stratify patients into risk groups, RNA-Seq can guide personalized drug selection based on expressed mutations, fusion genes, and differential expression (DE) between tumor and normal tissue. However, patient-matched normal tissue may be unavailable. Additionally, biological variability in normal tissue and technological biases may confound results. Therefore, we present normal expression reference data for two sequencing methods that are suitable for breast biopsies., Results: We identified breast cancer related and drug related genes that are expressed uniformly across our normal samples. Large subsets of these genes are identical for formalin fixed paraffin embedded samples and fresh frozen samples. Adipocyte signatures were detected in frozen compared to formalin samples, prepared by surgeons and pathologists, respectively. Gene expression confounded by adipocytes was identified using fat tissue samples. Finally, immune repertoire statistics were obtained for healthy breast, tumor and fat tissues., Conclusions: Our reference data can be used with patient tumor samples that are asservated and sequenced with a matching aforementioned method. Coefficients of variation are given for normal gene expression. Thus, potential drug selection can be based on confidently overexpressed genes and immune repertoire statistics., Materials and Methods: Normal expression from formalin and frozen healthy breast tissue samples using Roche Kapa RiboErase (total RNA) (19 formalin, 9 frozen) and Illumina TruSeq RNA Access (targeted RNA-Seq, aka TruSeq RNA Exome) (11 formalin, 1 frozen), and fat tissue (6 frozen Access). Tumor DE using 10 formalin total RNA tumor samples and 1 frozen targeted RNA tumor sample., Competing Interests: CONFLICTS OF INTEREST There are no conflicts of interest.

Purpose: To assess the prognostic and predictive value of selected biomarkers involved in cell-cycle regulation or proliferation in patients with HER2-positive early breast cancer. Experimental Design: Protein expression of TOP2A, Ki67, cyclin D1, and p27 was immunohistochemically determined in tissue microarrays of surgical specimens from 862 patients randomized to trastuzumab (1 or 2 years; N = 561) and observation ( N = 301) arms of the HERA trial. The primary analysis endpoint was disease-free survival (DFS). Biomarkers were examined as continuous or categorical variables (predefined cutoffs). Interaction terms between biomarkers and treatment were assessed in multivariate Cox models adjusted for variables of clinical interest. Results: A significant interaction was detected between p27 and treatment (adjusted P = 0.0049). Trastuzumab effect was significant in the p27-low subgroup (≤70% p27-positive tumor cells; N = 318). HR Comb Trast vs. Obs 0.44, 95% CI, 0.29-0.65 ( P < 0.001). No trastuzumab effect was observed in the p27-high subgroup N = 435; HR Comb Trast vs. Obs 0.97, 95% CI, 0.66-1.44, P = 0.89), indicating that these patients derived little or no benefit from trastuzumab treatment. A prognostic effect of p27 on DFS was observed, with p27-high patients experiencing half the hazard of a DFS event compared with low ones (HR p27 High vs. Low 0.49, 95% CI, 0.32-0.75). TOP2A, Ki67, and cyclin D1, as categorical variables were not predictive, whereas cyclin D1 as continuous variable was predictive of trastuzumab benefit. Conclusions: In TransHERA, patients with HER2-positive early breast cancer with low p27 expression in their tumors benefited from trastuzumab treatment, whereas patients with high p27 expression did not. Clin Cancer Res; 24(13); 3079-86. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Purpose: Genomic profiling of tumor biopsies from advanced gastrointestinal and anal cancers is increasingly used to inform treatment. In some cases, tissue biopsy can be prohibitive, and we sought to investigate whether analysis of blood-derived circulating tumor DNA (ctDNA) may provide a minimally invasive alternative. Experimental Design: Hybrid capture-based genomic profiling of 62 genes was performed on blood-based ctDNA from 417 patients with gastrointestinal carcinomas to assess the presence of genomic alterations (GA) and compare with matched tissue samples. Results: Evidence of ctDNA was detected in 344 of 417 samples (82%), and of these, ≥1 reportable GA was detected in 89% (306/344) of samples. Frequently altered genes were TP53 (72%), KRAS (35%), PIK3CA (14%), BRAF (8%), and EGFR (7%). In temporally matched ctDNA and tissue samples available from 25 patients, 86% of alterations detected in tissue were also detected in ctDNA, including 95% of short variants, but only 50% of amplifications. Conversely, 63% of alterations detected in ctDNA were also detected in matched tissue. Examples demonstrating clinical utility are presented. Conclusions: Genomic profiling of ctDNA detected potentially clinically relevant GAs in a significant subset of patients with gastrointestinal carcinomas. In these tumor types, most alterations detected in matched tissue were also detected in ctDNA, and with the exception of amplifications, ctDNA sequencing routinely detected additional alterations not found in matched tissue, consistent with tumor heterogeneity. These results suggest feasibility and utility of ctDNA testing in advanced gastrointestinal cancers as a complementary approach to tissue testing, and further investigation is warranted. Clin Cancer Res; 24(8); 1881-90. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Purpose: Identification of single-gene biomarkers that are prognostic of outcome can shed new insights on the molecular mechanisms that drive breast cancer and other cancers., Methods: Exploratory analysis of 20,464 single-gene messenger RNAs (mRNAs) in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) discovery cohort indicates that low expression of FGD3 mRNA is prognostic for poor outcome. Prognostic significance of faciogenital dysplasia 3 (FGD3), SUSD3, and other single-gene proliferation markers was evaluated in breast cancer and The Cancer Genome Atlas (TCGA) cohorts., Results: A meta-analysis of Cox regression of FGD3 mRNA as a continuous variable for overall survival of estrogen receptor (ER)-positive samples in METABRIC discovery, METABRIC validation, TCGA breast cancer, and Combination Chemotherapy in Treating Women With Breast Cancer (E2197) cohorts resulted in a combined hazard ratio (HR) of 0.69 (95% CI, 0.63 to 0.75), indicating better outcome with high expression. In the ER-negative samples, the combined meta-analysis HR was 0.72 (95% CI, 0.63 to 0.82), suggesting that FGD3 is prognostic regardless of ER status. The potential of FGD3 as a biomarker for freedom from recurrence was evaluated in the Breast International Group 1-98 (BIG 1-98; Letrozole or Tamoxifen in Treating Postmenopausal Women With Breast Cancer) study (HR, 0.85; 95% CI, 0.76 to 0.93) for breast cancer-free interval. In the Hungarian Academy of Science (HAS) breast cancer cohort, splitting on the median had an HR of 0.49 (95% CI, 0.42 to 0.58) for recurrence-free survival. A comparison of the Stouffer P value in five ER-positive cohorts showed that FGD3 ( P = 3.8 E-14 ) outperformed MKI67 ( P = 1.06 E-8 ) and AURKA ( P = 2.61 E-5 ). A comparison of the Stouffer P value in four ER-negative cohorts showed that FGD3 ( P = 3.88 E-5 ) outperformed MKI67 ( P = .477) and AURKA ( P = .820)., Conclusion: FGD3 was previously shown to inhibit cell migration. FGD3 mRNA is regulated by ESR1 and is associated with favorable outcome in six distinct breast cancer cohorts and four TCGA cancer cohorts. This suggests that FGD3 is an important clinical biomarker., Competing Interests: The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or po.ascopubs.org/site/ifc. Scooter WillisNo relationship to discloseYuliang SunNo relationship to discloseMark AbramovitzNo relationship to discloseTeng FeiNo relationship to discloseBrandon YoungNo relationship to discloseXiaoqian LinNo relationship to discloseMin NiNo relationship to discloseJustin AchuaEmployment: Avera Mckennan HospitalMeredith M. ReganConsulting or Advisory Role: Merck, Ipsen (Inst) Research Funding: Veridex (Inst), OncoGenex (Inst), Pfizer (Inst), Ipsen (Inst), Novartis (Inst), Merck (Inst), Ferring (Inst), Celgene (Inst), AstraZeneca (Inst), Pierre Fabre (Inst), Ipsen (Inst)Kathryn P. GrayStock and Other Ownership Interests: MDGNRobert GrayResearch Funding: Abbott Molecular, Agios, Amgen, AstraZeneca, Bristol-Myers Squibb, Boehringer Ingelheim, Celgene, Genentech, Genomic Health, Genzyme, GlaxoSmithKline, ImClone Systems, Janssen-Ortho, Kanisa, Millennium, Nodality, Onyx, OSI Pharmaceuticals, Pfizer, Sanofi, Sequenta, Syndax, NovartisVictoria WangNo relationship to discloseBradley LongNo relationship to discloseRoswitha KammlerNo relationship to discloseJoseph A. SparanoStock and Other Ownership Interests: Metastat Consulting or Advisory Role: Genentech, Novartis, AstraZeneca, Celgene, Eli Lilly, Celldex, Pfizer, Prescient Therapeutics, Juno Therapeutics, Merrimack Research Funding: Prescient Therapeutics (Inst), Deciphera (Inst), Genentech (Inst), Merck (Inst), Novartis (Inst), Novartis (Inst), Merrimack (Inst)Casey WilliamsResearch Funding: Takeda (Inst), Tesaro (Inst)Lori J. GoldsteinHonoraria: Genentech, Roche Pharma AG, Puma Biotechnology, Pfizer, Glenmark Consulting or Advisory Role: Genentech, Dompé Farmaceutici, Roche Pharma AG, Puma Biotechnology, Pfizer, Merck, AstraZeneca Research Funding: Merck (Inst), Genentech (Inst) Other Relationship: Roche Pharma AGRoberto SalgadoTravel, Accommodations, Expenses: RocheSherene LoiResearch Funding: Genentech (Inst), Pfizer (Inst), Novartis (Inst), Merck (Inst), Puma Biotechnology (Inst), Bristol-Myers Squibb (Inst) Patents, Royalties, Other Intellectual Property: PI3K pathway gene signature granted by the European and US patent offices (Inst)Giancarlo PruneriNo relationship to discloseGiuseppe VialeHonoraria: MSD Oncology Consulting or Advisory Role: Dako, Genentech, AstraZeneca, Bristol-Myers Squibb, Astellas Pharma Travel, Accommodations, Expenses: Roche, CelgeneMyles BrownConsulting or Advisory Role: Novartis, GTx Research Funding: Novartis Patents, Royalties, Other Intellectual Property:: As part of my work at the Dana-Farber Cancer Institute I have made invention disclosures and the institute is filing patents on technology related to endocrine resistance in breast cancer and improved CRISPR dual sgRNA library design. Travel, Accommodations, Expenses: GTxBrian Leyland-JonesStock and Other Ownership Interests: Catalyst Pharmaceuticals, Progenix, Puma Biotechnology, Sucampo Pharmaceuticals, ARIAD, Zogenix Consulting or Advisory Role: GlaxoSmithKline, Amgen Speakers’ Bureau: Genentech, Exelixis Research Funding: Takeda, Tesaro Expert Testimony: Amgen, (© 2017 by American Society of Clinical Oncology.)

Carcinoma of unknown primary (CUP) is a rare and difficult-to-treat malignancy, the management of which might be improved by the identification of actionable driver mutations. We interrogated 54 to 70 genes in 442 patients with CUP using targeted clinical-grade, next-generation sequencing of circulating tumor DNA (ctDNA). Overall, 80% of patients exhibited ctDNA alterations; 66% (290/442) ≥1 characterized alteration(s), excluding variants of unknown significance. TP53 -associated genes were most commonly altered [37.8% (167/442)], followed by genes involved in the MAPK pathway [31.2% (138/442)], PI3K signaling [18.1% (80/442)], and the cell-cycle machinery [10.4% (46/442)]. Among 290 patients harboring characterized alterations, distinct genomic profiles were observed in 87.9% (255/290) of CUP cases, with 99.7% (289/290) exhibiting potentially targetable alterations. An illustrative patient with dynamic changes in ctDNA content during therapy and a responder given a checkpoint inhibitor-based regimen because of a mismatch repair gene anomaly are presented. Our results demonstrate that ctDNA evaluation is feasible in CUP and that most patients harbor a unique somatic profile with pharmacologically actionable alterations, justifying the inclusion of noninvasive liquid biopsies in next-generation clinical trials. Cancer Res; 77(16); 4238-46. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Purpose: Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K-AKT-mTOR pathway is associated with specific sites of breast cancer metastasis. Experimental Design: Next-generation sequencing-based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K-AKT-mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K-AKT-mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions. Results: PIK3CA mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions ( P < 0.01, P = 0.056, and P = 0.053, respectively). However, PIK3CA mutations alone were insufficient in predicting protein activation ( P = 0.32 and P = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); P < 0.01, P = 0.02, and P = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) P < 0.01, mTOR (S2448) P < 0.01, 4EBP1 (S65) P = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined. Conclusions: Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of PIK3CA mutations and activation of the PI3K-AKT-mTOR signaling network. Clin Cancer Res; 23(16); 4919-28. ©2017 AACR ., (©2017 American Association for Cancer Research.)

A triple-negative breast cancer patient had no hereditary BRCA1 , BRCA2 , or TP53 risk variants. After exhaustion of standard treatments, she underwent experimental treatments and whole-exome sequencing of tumor, blood, and a metastasis. Well-tolerated experimental bortezomib monotherapy was administered for a progression-free period of 11 mo. After progression, treatments were changed and the exome data were evaluated, expanded with RNA and exome sequencing of a late-stage metastasis. In the final stage, eribulin alone and in combination with anthracyclines were administered. While suffering from grade 3 adverse events, skin metastases progressed. She lived 51 mo after initial diagnosis.Toxicity from anthracyclines and cisplatin may have been due to associated germline variants CBR3 C4Y and V224M and GSTP1 I105V, respectively. Somatic mutations predicted or reported as pathogenic were detected in 38 genes in tumor tissues. All tumor samples harbored the heterozygous TP53 Y220C variant, known to destabilize p53 and down-regulate p53-mediated apoptosis. The success of bortezomib may be explained by the previously reported up-regulation of caspase-mediated apoptosis, which is p53-independent. Phylogenetic analysis of blood, primary tumor, and two metastases inferred an ancestral tumor cell with 12 expressed tumor mutations from which all three tumors may have evolved.Although our first urgent analysis could only include 40 genes, postmortem analysis uncovered the aggressiveness and suggested experimental therapies including 16 actionable targets, partly validated by immunohistochemistry. Exome and transcriptome analyses yielded comprehensive therapy-relevant information and should be considered for patients at first diagnosis., (© 2017 Meißner et al.; Published by Cold Spring Harbor Laboratory Press.)

Breast cancer (BC) is the most common women cancer and second most common cause of cancer death in women. A woman living in the United States has 12.3% lifetime risk of being diagnosed with BC. From the genomics point of view, the most common three subtypes of BC encountered in clinics are HR+, HER2+, and TNBC or basal-like BC. Estrogen receptor (ER) status or HER2 amplification or chemotherapy is not sufficient to understand the underlying mechanisms of disease progression and resistance (de novo or acquire). Although hormonal therapy and HER2-directed therapies have produced a considerable positive outcome in HR+ and HER2+ BC respectively, there are no established targeted agents for TNBC and basal-like BC. While PARP inhibitors have shown promising activity in BRCA-related cancers, its value in the treatment of TNBC remains to be demonstrated. The PI3K-AKT-mTOR signaling pathway plays a crucial role in the initiation and progress in tumorigenesis including breast tumorigenesis and regulates critical cellular functions including survival, proliferation, and metabolism. This article aims to understand the role of PI3K-mTORC1/C2 alterations in determining the clinical outcome in the specific breast cancer subtypes. The understanding of the tumor cell signaling will help us in the decision-making the process for obtaining the treatment modalities towards further advancement of the precision medicine. In this review, we will restrict our discussion to a basic understanding of the biology of subtype-specific BC and several targeted agents under development for the treatment of BC., (Copyright © 2017 Elsevier Inc. All rights reserved.)

As a genetic disease [1] cancer dysregulates key oncogenic pathways that influence cell growth, proliferation, survival, angiogenesis, and metastasis. Among the major determinants that enable cancer cells to acquire malignant traits are genomic diversity and instability. In the post human genome project era, cancer-specific genomic maps are redesigning tumor taxonomy. The treatment modalities, as well as the overall management of cancer as a disease in today's clinic, have started depending heavily on the molecular pathology of the individual tumor(s) in addition to the fundamental classification of cancers by histopathology. The enrichment tumor taxonomy by genomic morphology has also opened up the possibilities for genomics-driven drug development. The success of a cancer drug today is fundamentally based on the success in identifying target genes that control tumorigenic pathways. One primary goal of precision cancer medicine is to make clinical decisions based on genomic/proteomic data, which can identify a target or targets for therapy, and subsequent inevitable development of therapeutic resistance to the drug. The ability to exploit tumor genetic information for its full clinical potential has only recently become evident. Over the last decade, the convergence of discovery, technology, and therapeutic development has created an unparalleled opportunity to test the hypothesis that systematic knowledge of genomic and proteomic information from individual tumor(s) may significantly improve clinical outcomes for many patients with unmanageable tumor burden. This review presents the signaling logic behind the ground rules for the rational approach to the genomics-driven precision medicine., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Clinical trials have shown that trastuzumab, a recombinant monoclonal antibody against HER2 receptor, significantly improves overall survival and disease-free survival in women with HER2-positive early breast cancer, but long-term follow-up data are needed. We report the results of comparing observation with two durations of trastuzumab treatment at a median follow-up of 11 years, for patients enrolled in the HERA (HERceptin Adjuvant) trial., Methods: HERA (BIG 1-01) is an international, multicentre, open-label, phase 3 randomised trial of 5102 women with HER2-positive early breast cancer, who were enrolled from hospitals in 39 countries between Dec 7, 2001, and June 20, 2005. After completion of all primary therapy (including, surgery, chemotherapy, and radiotherapy as indicated), patients were randomly assigned (1:1:1) to receive trastuzumab for 1 year (once at 8 mg/kg of bodyweight intravenously, then 6 mg/kg once every 3 weeks) or for 2 years (with the same dose schedule), or to the observation group. Primary endpoint is disease-free survival, and analyses are in the intention-to-treat population. Hazard ratios (HRs) were estimated from Cox models, and survival curves were estimated by the Kaplan-Meier method. Comparison of 2 years versus 1 year of trastuzumab is based on 366-day landmark analyses. This study is registered with ClinicalTrials.gov (NCT00045032)., Findings: Of the 5102 women randomly assigned in the HERA trial, three patients had no evidence of having provided written informed consent to participate. We followed up the intention-to-treat population of 5099 patients (1697 in observation, 1702 in 1-year trastuzumab, and 1700 in 2-years trastuzumab groups). After a median follow-up of 11 years (IQR 10·09-11·53), random assignment to 1 year of trastuzumab significantly reduced the risk of a disease-free survival event (HR 0·76, 95% CI 0·68-0·86) and death (0·74, 0·64-0·86) compared with observation. 2 years of adjuvant trastuzumab did not improve disease free-survival outcomes compared with 1 year of this drug (HR 1·02, 95% CI 0·89-1·17). Estimates of 10-year disease-free survival were 63% for observation, 69% for 1 year of trastuzumab, and 69% for 2 years of trastuzumab. 884 (52%) patients assigned to the observation group selectively crossed over to receive trastuzumab. Cardiac toxicity remained low in all groups and occurred mostly during the treatment phase. The incidence of secondary cardiac endpoints was 122 (7·3%) in the 2-years trastuzumab group, 74 (4·4%) in the 1-year trastuzumab group, and 15 (0·9%) in the observation group., Interpretation: 1 year of adjuvant trastuzumab after chemotherapy for patients with HER2-positive early breast cancer significantly improves long-term disease-free survival, compared with observation. 2 years of trastuzumab had no additional benefit., Funding: F Hoffmann-La Roche (Roche)., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The acquisition of integrin-directed metastasis-associated (ID-MA) phenotypes by Triple-Negative Breast Cancer (TNBC) cells is caused by an upregulation of the Wnt-beta-catenin pathway (WP). We reported that WP is one of the salient genetic features of TNBC. RAC-GTPases, small G-proteins which transduce signals from cell surface proteins including integrins, have been implicated in tumorigenesis and metastasis by their role in essential cellular functions like motility. The collective percentage of alteration(s) in RAC1 in ER+ve BC was lower as compared to ER-ve BC (35% vs 57%) (brca/tcga/pub2015). High expression of RAC1 was associated with poor outcome for RFS with HR=1.48 [CI: 1.15-1.9] p=0.0019 in the Hungarian ER-veBC cohort. Here we examined how WP signals are transduced via RAC1 in the context of ID-MA phenotypes in TNBC. Using pharmacological agents (sulindac sulfide), genetic tools (beta-catenin siRNA), WP modulators (Wnt-C59, XAV939), RAC1 inhibitors (NSC23766, W56) and WP stimulations (LWnt3ACM, Wnt3A recombinant) in a panel of 6-7 TNBC cell lines, we studied fibronectin-directed (1) migration, (2) matrigel invasion, (3) RAC1 and Cdc42 activation, (4) actin dynamics (confocal microscopy) and (5) podia-parameters. An attenuation of WP, which (a) decreased cellular levels of beta-catenin, as well as its nuclear active-form, (b) decreased fibronectin-induced migration, (c) decreased invasion, (d) altered actin dynamics and (e) decreased podia-parameters was successful in blocking fibronectin-mediated RAC1/Cdc42 activity. Both Wnt-antagonists and RAC1 inhibitors blocked fibronectin-induced RAC1 activation and inhibited the fibronectin-induced ID-MA phenotypes following specific WP stimulation by LWnt3ACM as well as Wnt3A recombinant protein. To test a direct involvement of RAC1-activation in WP-mediated ID-MA phenotypes, we stimulated brain-metastasis specific MDA-MB231BR cells with LWnt3ACM. LWnt3ACM-stimulated fibronectin-directed migration was blocked by RAC1 inhibition in MDA-MB231BR cells. In the light of our previous report that WP upregulation causes ID-MA phenotypes in TNBC tumor cells, here we provide the first mechanism based evidence to demonstrate that WP upregulation signals ID-MA tumor cell phenotypes in a RAC1-GTPase dependent manner involving exchange-factors like TIAM1 and VAV2. Our study demonstrates for the first time that beta-catenin-RAC1 cascade signals integrin-directed metastasis-associated tumor cell phenotypes in TNBC.

The dual small molecule tyrosine kinase inhibitor lapatinib blocks both human epidermal growth factor receptor (HER-1) and human epidermal growth factor receptor 2 (HER-2) tyrosine kinase activity by binding reversibly to the ATP-binding site of the receptor's intracellular domain. Lapatinib, in combination with capecitabine, has been approved in 2007 for the treatment of patients with advanced HER-2 + breast cancer upon progressive disease following standard chemotherapy. Approval was also extended to the treatment of postmenopausal women with advanced hormone receptor (HR)-positive and HER-2-positive breast cancer in 2010. More recently, clinical trials that have investigated the efficacy of dual HER-2 blockade in both the metastatic and neoadjuvant breast cancer settings. For example, in 2013 the European Medicines Agency approved the combination of lapatinib and trastuzumab in HER-2 + /HR - patients. We review the efficacy results from dual HER-2 blockade studies and present new post hoc analysis efficacy data according to HR status. We show that dual blockade of HER-2 appears to provide a greater magnitude of benefit in the HR - versus the HR + subgroup of patients. Finally, we examine the potential of molecularly subtyping HER-2 + tumors using the PAM50 test as a predictor of response to treatment with the combination of trastuzumab and lapatinib., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose of Review: In this review, we summarize recent and current biomarkers and assays that are being considered in the selection of suitable patients with estrogen receptor-positive early breast cancer for extended (years 5-10) adjuvant endocrine therapy (AET)., Recent Findings: Women with estrogen receptor-positive early-stage breast cancer (65% of cases) continue to have late risk for distant recurrence extending beyond 5 years from surgery. Recent large trials have consistently demonstrated improvement for prolonging endocrine therapy. However, endocrine therapy can cause women bothersome side effects and can negatively impact quality of life. Determining which patients remain at risk for disease recurrence and predicting which of these patients would derive the most benefit from the addition of extended AET are key issues faced by patients and oncologists today. A number of predictive molecular assays have been developed and are being considered as tools to be used in guiding the implementation of adjuvant systemic therapy., Summary: The future holds much promise and as more information and understanding is acquired, treatment regimens will increasingly incorporate clinically validated biomarker assays in the decision-making process that will be of great benefit to these patients. Proving clinical utility, though, will ultimately decide their implementation.

Importance: The impact of a biomarker-based (personalized) cancer treatment strategy in the setting of phase 1 clinical trials was analyzed., Objective: To compare patient outcomes in phase 1 studies that used a biomarker selection strategy with those that did not., Data Sources: PubMed search of phase 1 cancer drug trials (January 1, 2011, through December 31, 2013)., Study Selection: Studies included trials that evaluated single agents, and reported efficacy end points (at least response rate [RR])., Data Extraction and Synthesis: Data were extracted independently by 2 investigators., Main Outcomes and Measures: Response rate and progression-free survival (PFS) were compared for arms that used a personalized strategy (biomarker selection) vs those that did not. Overall survival was not analyzed owing to insufficient data., Results: A total of 346 studies published in the designated 3-year time period were included in the analysis. Multivariable analysis (meta-regression and weighted multiple regression models) demonstrated that the personalized approach independently correlated with a significantly higher median RR (30.6% [95% CI, 25.0%-36.9%] vs 4.9% [95% CI, 4.2%-5.7%]; P < .001) and a longer median PFS (5.7 [95% CI, 2.6-13.8] vs 2.95 [95% CI, 2.3-3.7] months; P < .001). Targeted therapy arms that used a biomarker-based selection strategy (n = 57 trials) were associated with statistically improved RR compared with targeted therapy arms (n = 177 arms) that did not (31.1% [95% CI, 25.4%-37.4%] vs 5.1% [95% CI, 4.3%-6.0%]; P < .001). Nonpersonalized targeted arms had outcomes comparable with those that tested a cytotoxic agent (median RR, 5.1% [95% CI, 4.3%-6.0%] vs 4.7% [95% CI, 3.6%-6.2%]; P = .63; respectively; median PFS, 3.3 [95% CI, 2.6-4.0] months vs 2.5 [95% CI, 2.0-3.7] months; P = .22). Personalized arms using a "genomic (DNA) biomarker" had higher median RR than those using a "protein biomarker" (42.0% [95% CI, 33.7%-50.9%] vs 22.4% [95% CI, 15.6%-30.9%]; P = .001). The median treatment-related mortality was not statistically different for arms that used a personalized strategy vs not (1.89% [95% CI, 1.36%-2.61%] vs 2.27% [95% CI, 1.97%-2.62%]; P = .31)., Conclusions and Relevance: In this meta-analysis, most phase 1 trials of targeted agents did not use a biomarker-based selection strategy. However, use of a biomarker-based approach was associated with significantly improved outcomes (RR and PFS). Response rates were significantly higher with genomic vs protein biomarkers. Studies that used targeted agents without a biomarker had negligible response rates.

Background: Activating, nonamplification ERBB2 mutations (ERBB2mut) are not detected by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH), but are detected by DNA sequencing and may predict clinical responses to human epidermal growth factor receptor (HER2)-targeted therapy. The authors queried 5605 advanced/metastatic breast cancers (mBC) to uncover the frequency of ERBB2mut genomic alterations. Clinical responses to anti-HER2 therapeutics were identified., Methods: DNA was extracted from 40 µm of formalin-fixed paraffin-embedded (FFPE) sections. Comprehensive genomic profiling (CGP) was used to evaluate up to 315 genes (592× mean coverage depth). Results were analyzed for base substitutions, short indels, copy number changes, and selected rearrangements., Results: Of 5605 cases, 698 (12.5%) featured ERBB2 alterations, including 596 (10.6%) ERBB2 amplifications (ERBB2amp) and 138 (2.4%) ERBB2mut; 38 cases (0.7%) had co-occurring ERBB2amp and ERBB2mut. ERBB2mut predominantly affected the kinase (124 cases; 90%) or extracellular (15 cases; 11%) domains. Both primary BC (52 cases; 38%) and metastatic site biopsies (86 cases; 62%) were found to harbor ERBB2mut, which were distributed across carcinoma not otherwise specified (NOS) (69 cases; 50%), invasive ductal carcinoma (IDC) (40 cases; 29%), invasive lobular carcinoma (ILC) (27 cases; 20%), and mucinous mBC (2 cases; 1%). Genes commonly coaltered with ERBB2 were tumor protein 53 (TP53) (49%); phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) (42%); cadherin 1, type 1 (CDH1) (37%); MYC (17%); and cyclin D1 protein (CCND1) (16%). CDH1 mutations were enriched in ERBB2mut mBC (P<0.0006) and associated with recurrent mBC. Selected patients with ERBB2mut, without ERBB2amp, who responded to anti-HER2 targeted therapies are presented herein., Conclusions: Within this large series, 1.8% of cases harbored ERBB2mut, which are undetectable by standard-of-care IHC or FISH tests. Metastatic BC driven by ERBB2mut respond to anti-HER2 targeted therapies, and expanding clinical trials designed to detect ERBB2mut by CGP and optimize targeted treatments are warranted. Cancer 2016. © 2016 American Cancer Society. Cancer 2016;122:2654-2662. © 2016 American Cancer Society., (© 2016 American Cancer Society.)

Importance: A number of studies suggest that response to antihuman epidermal growth factor receptor-2 (currently known as ERBB2, butreferred to asHER2 in this study) agents differs by estrogen receptor (ER) level status. The clinical relevance of this is unknown., Objective: To determine the magnitude of trastuzumab benefit according to quantitative levels of ER and HER2 in the HERceptin Adjuvant (HERA) trial., Design, Setting, and Participants: The HERA trial was an international, multicenter, randomized trial that included 5099 patients with early-stage HER2-positive breast cancer, randomized between 2001 and 2005 to receive either no trastuzumab or trastuzumab, after adjuvant chemotherapy. This is a secondary analysis of the HERA study. Local ER immunohistochemical (IHC) analyses, HER2 fluorescence in situ hybridization (FISH) ratio, and copy number results were available for 3037 patients (59.6%) randomized to observation and trastuzumab (1 or 2 years) (cohort 1). Transcript levels of ESR1 and HER2 genes were available for 615 patients (12.1%) (cohort 2)., Interventions: Patients were randomized to receive either no trastuzumab or 1 year vs 2 years of trastuzumab. Endocrine therapy was given to patients with hormone receptor-positive disease as per local guidelines., Main Outcomes and Measures: Disease-free survival (DFS) and overall survival (OS) were the primary and secondary end points in the intent-to-treat population (ITT). Analyses adjusting for crossover (censored and inverse probability weighted [IPW]) were also performed. Interactions among treatment, ER status, and HER2 amplification using predefined cutoffs were assessed in Cox proportional hazards regression models., Results: Median follow-up time was 8 years. Levels of FISH and HER2 copy numbers were significantly higher in ER-negative patients (P < .001). In cohort 1, for DFS and OS, a significant treatment effect was found for all ER, IHC, and FISH levels, except for the ER-positive/HER2 low FISH ratio (≥2 to <5) group (DFS: 3-way ITT Pvalue for interaction = .07; censored = .02; IPW = .03; OS ITT Pvalue for interaction = .007; censored = .04; IPW = .03). In cohort 2, consistent with cohort 1, a significant predictive effect of the ESR1 gene for both end points was also observed (DFS Pvalue for interaction = .06; OS = .02), indicating that breast cancers with higher ESR1 levels also derive less benefit from trastuzumab., Conclusions and Relevance: Patients with HER2-positive breast cancers that are ER-positive by IHC analyses with low FISH ratio (≥2 to <5), or with higher ESR1 levels derive significantly less benefit from adjuvant trastuzumab after chemotherapy. These data may explain heterogeneity in response to anti-HER2 agents in HER2-positive, ER-positive breast cancers as some may be more luminal-like than HER2 driven., Trial Registration: clinicaltrials.gov Identifier: NCT00045032.

Tumor cells acquire metastasis-associated (MA) phenotypes following genetic alterations in them which cause deregulation of different signaling pathways. Earlier, we reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the genetic salient features of triple-negative breast cancer (TNBC), and WP signaling is associated with metastasis in TNBC. Using cBioPortal, here we found that collective % of alteration(s) in WP genes, CTNNB1, APC and DVL1 among breast-invasive-carcinomas was 21% as compared to 56% in PAM50 Basal. To understand the functional relevance of WP in the biology of heterogeneous/metastasizing TNBC cells, we undertook this comprehensive study using 15 cell lines in which we examined the role of WP in the context of integrin-dependent MA-phenotypes. Directional movement of tumor cells was observed by confocal immunofluorescence microscopy and quantitative confocal-video-microscopy while matrigel-invasion was studied by MMP7-specific casein-zymography. WntC59, XAV939, sulindac sulfide and beta-catenin siRNA (1) inhibited fibronectin-directed migration, (2) decreased podia-parameters and motility-descriptors, (3) altered filamentous-actin, (4) decreased matrigel-invasion and (5) inhibited cell proliferation as well as 3D clonogenic growth. Sulindac sulfide and beta-catenin siRNA decreased beta-catenin/active-beta-catenin and MMP7. LWnt3ACM-stimulated proliferation, clonogenicity, fibronectin-directed migration and matrigel-invasion were perturbed by WP-modulators, sulindac sulfide and GDC-0941. We studied a direct involvement of WP in metastasis by stimulating brain-metastasis-specific MDA-MB231BR cells to demonstrate that LWnt3ACM-stimulated proliferation, clonogenicity and migration were blocked following sulindac sulfide, GDC-0941 and beta-catenin knockdown. We present the first evidence showing a direct functional relationship between WP activation and integrin-dependent MA-phenotypes. By proving the functional relationship between WP activation and MA-phenotypes, our data mechanistically explains (1) why different components of WP are upregulated in TNBC, (2) how WP activation is associated with metastasis and (3) how integrin-dependent MA-phenotypes can be regulated by mitigating the WP., Competing Interests: The authors declare no conflicts of interest.

Purpose: An open-label, noninferiority study to evaluate the impact of epoetin alfa (EPO) on tumor outcomes when used to treat anemia in patients receiving chemotherapy for metastatic breast cancer., Methods: Women with hemoglobin ≤ 11.0 g/dL, receiving first- or second-line chemotherapy for metastatic breast cancer, were randomly assigned to EPO 40,000 IU subcutaneously once a week or best standard of care. The primary end point was progression-free survival (PFS). Secondary end points included overall survival, time to tumor progression, overall response rate, RBC transfusions, and thrombotic vascular events., Results: In 2,098 patients randomly assigned, median PFS (based on investigator-determined disease progression [PD]) was 7.4 months in both groups (hazard ratio [HR], 1.089; 95% CI, 0.988 to 1.200); upper bound exceeded prespecified noninferiority margin of 1.15. Median PFS per independent review committee-determined PD was 7.6 months in both groups (HR, 1.028; 95% CI, 0.922 to 1.146); upper bound did not exceed prespecified noninferiority margin. Median overall survival at clinical cutoff (1,337 deaths) was 17.2 months in the EPO and 17.4 months in the best standard of care group (HR, 1.057; 95% CI, 0.949 to 1.177), median time to tumor progression was 7.5 months in both groups (HR, 1.094; 95% CI, 0.991 to 1.209), and overall response rate was 50% versus 51% (odds ratio, 0.950; 95% CI, 0.799 to 1.130). RBC transfusions were 5.8% versus 11.4% (P < .001), and thrombotic vascular events were 2.8% versus 1.4% (P = .038), respectively., Conclusion: The primary end point, PFS based on investigator-determined PD, did not meet noninferiority criteria. As a consistency assessment with the primary finding, PFS based on independent review committee-determined PD met noninferiority criteria. Overall, this study did not achieve noninferiority objective in ruling out a 15% increased risk in PD/death. RBC transfusion should be the preferred approach for the management of anemia in this population., (© 2016 by American Society of Clinical Oncology.)

Activation of the PI3K-mTOR pathway via HER2: HER3-mediated signaling in HER2+ breast cancers pose one of the major threats towards the success of trastuzumab. First, trastuzumab cannot perturb survival/proliferative signals following HER2: HER3 heterodimerization in HER2+ tumor cells. Second, trastuzumab treatment has been reported to cause drug-mediated resistance in over 50% of HER2+ breast cancers. We have reported that treatment with an anti-angiogenic drug imparted a significant anti-tumor advantage when combined with trastuzumab plus pertuzumab in the trastuzumab-resistant model of HER2+ breast cancers (PMID: 23959459). The very fact as revealed by our study that an inclusion of anti-angiogenic drug conferred a significant anti-tumor advantage when combined with dual anti-HER2 therapy clearly indicated a critical and indispensable role of angiogenesis in these tumors. Hence, we hypothesized that BEZ235 a dual PI3K/mTOR inhibitor will have an effect on the tumor as well as the angiogenic stromal compartments. In vitro and in vivo efficacy of BEZ235 was determined in HER2+ trastuzumab-sensitive, trastuzumab-resistant and HER2 amplified/PIK3CA mutated cell lines. BEZ235 alone and in combination with trastuzumab was tested on the tumor as well as stromal compartments. AKT-mTOR signal was suppressed following BEZ235 treatment in a concentration and time-dependent manner. AnnexinV, cl-CASPASE3, SURVIVIN and p-FOXO1 indicated that BEZ235-induced cell death occurred predominantly via an apoptotic pathway. Heregulin-induced HIF1α synthesis was also significantly decreased. Oncoprint data (cBioPortal) representing PAM50 Her2 enriched tumors (TCGA, Nature 2012) and Her2-positive breast tumors (TCGA, cell 2015) showed 91.4% genetic alterations and 79.2% genetic alterations in a set of four genes comprised of PIK3CA, ERBB2, VEGFA and HIF1alpha. The co-occurrence of HIF1alpha with VEGFA in PAM50 Her2 enriched tumors (TCGA, Nature 2012) and the co-occurrence of HIF1alpha with VEGFA pair as well as HIF1alpha with PIK3CA pair in Her2-positive breast tumors (TCGA, cell 2015) were found statistically significant. In xenograft models, BEZ235 blocked tumor growth and decreased Ki67, CD31, p-AKT, p-S6RP, p-4EBP1 IHC-expressions. These decreases were more pronounced when BEZ235 was combined with trastuzumab in HER2+/trastuzumab-sensitive, trastuzumab-resistant and HER2+/PIK3CA mutated models. We demonstrated that combined targeting of HER2 and the PI3K-AKT-mTOR pathway is superior to HER2-directed therapy alone. Mechanistically the inhibition of tumor-induced angiogenesis by BEZ235 caused by the down-regulation of PI3K-mTOR-HIF1alpha signaling irrespective of the trastuzumab-sensitivity status of HER2+ breast cancers proving evidence for the first time that the inhibition of angiogenesis is an important component of the anti-tumor efficacy of BEZ235 in HER2 defined breast cancers.

Purpose: To discover novel prognostic biomarkers in ovarian serous carcinomas., Methods: A meta-analysis of all single genes probes in the TCGA and HAS ovarian cohorts was performed to identify possible biomarkers using Cox regression as a continuous variable for overall survival. Genes were ranked by p-value using Stouffer's method and selected for statistical significance with a false discovery rate (FDR) <.05 using the Benjamini-Hochberg method., Results: Twelve genes with high mRNA expression were prognostic of poor outcome with an FDR <.05 (AXL, APC, RAB11FIP5, C19orf2, CYBRD1, PINK1, LRRN3, AQP1, DES, XRCC4, BCHE, and ASAP3). Twenty genes with low mRNA expression were prognostic of poor outcome with an FDR <.05 (LRIG1, SLC33A1, NUCB2, POLD3, ESR2, GOLPH3, XBP1, PAXIP1, CYB561, POLA2, CDH1, GMNN, SLC37A4, FAM174B, AGR2, SDR39U1, MAGT1, GJB1, SDF2L1, and C9orf82)., Conclusion: A meta-analysis of all single genes identified thirty-two candidate biomarkers for their possible role in ovarian serous carcinoma. These genes can provide insight into the drivers or regulators of ovarian cancer and should be evaluated in future studies. Genes with high expression indicating poor outcome are possible therapeutic targets with known antagonists or inhibitors. Additionally, the genes could be combined into a prognostic multi-gene signature and tested in future ovarian cohorts.

Importance: The Genomic Grade Index (GGI) was previously developed, evaluated on frozen tissue, and shown to be prognostic in early breast cancer. To test the GGI in formalin-fixed, paraffin-embedded breast cancer tumors, a quantitative reverse transcriptase polymerase chain reaction assay was developed and named the Genomic Grade (GG). The GG assay has the potential to increase the clinical application of the GGI, but robust demonstration of the clinical validity of the GG assay is required., Objective: To evaluate the prognostic ability of the GG assay to detect breast cancer recurrence compared with centrally reviewed immunohistochemical testing of Ki67 antigen proliferation., Design, Setting, and Participants: This is an internationally collaborative substudy of a large phase 3 4-arm adjuvant trial. Patients had endocrine receptor-positive, node-positive, or node-negative nonmetastatic primary breast cancer. Patients included in this study had available formalin-fixed, paraffin-embedded samples of their primary tumors and were randomized to either a 5-year tamoxifen monotherapy arm or a 5-year letrozole monotherapy arm. Associations between either GG assay results or log2-transformed Ki67 data and survival end points were evaluated using Cox regression models stratified for chemotherapy use; the 2 vs 4 arm randomization option; and endocrine therapy assignment with and without adjustment for clinicopathological parameters, including centrally reviewed histological grade, hormone receptors, and ERBB2 (formerly HER2 or HER2/neu). The likelihood ratio statistic was used to assess the added prognostic value., Interventions: Central evaluation and comparison, blinded for clinical information, of the GG assay, breast cancer histological grade, and Ki67., Main Outcomes and Measures: Distant recurrence-free interval (DRFI)., Results: Genomic Grade assay data were obtained in 883 breast cancer samples (62%). At a median follow-up of 8.1 years, 84 (10%) had distant recurrences. Increasing GG or Ki67 were both significantly associated with lower DRFI and added independent prognostic information to the clinicopathological prognostic factors. In patients with early node-negative breast cancer who were endocrine-only treated, 38% were GG1 with a 10-year DRFI of 99% (95% CI, 97%-100%), and 18% were histological grade 1 with a 10-year DRFI of 100% (95% CI, 100%-100%). For GG equivocal patients, the 10-year DRFI was 94% (95% CI, 90%-98%), and for GG3 patients, the 10-year DRFI was 87% (95% CI, 80%-94%)., Conclusions and Relevance: Either the GG assay or centrally reviewed Ki67 significantly improves clinicopathological models to determine distant recurrence of breast cancer. Compared with the histological grade, the GG assay can identify a higher proportion of endocrine-only treated patients with very low risk of distant recurrence at 10 years., Trial Registration: clinicaltrials.gov Identifiers: NCT00004205 and NCT00004205.

Estrogen receptor 1 (ESR1) and ESR2 gene polymorphisms have been associated with endocrine-mediated physiological mechanisms, and inconsistently with breast cancer risk and outcomes, bone mineral density changes, and hot flushes/night sweats. DNA was isolated and genotyped for six ESR1 and two ESR2 single-nucleotide polymorphisms (SNPs) from tumor specimens from 3691 postmenopausal women with hormone receptor-positive breast cancer enrolled in the BIG 1-98 trial to receive tamoxifen and/or letrozole for 5 years. Associations with recurrence and adverse events (AEs) were assessed using Cox proportional hazards models. 3401 samples were successfully genotyped for five SNPs. ESR1 rs9340799(XbaI) (T>C) variants CC or TC were associated with reduced breast cancer risk (HR = 0.82,95% CI = 0.67-1.0), and ESR1 rs2077647 (T>C) variants CC or TC was associated with reduced distant recurrence risk (HR = 0.69, 95% CI = 0.53-0.90), both regardless of the treatments. No differential treatment effects (letrozole vs. tamoxifen) were observed for the association of outcome with any of the SNPs. Letrozole-treated patients with rs2077647 (T>C) variants CC and TC had a reduced risk of bone AE (HR = 0.75, 95% CI = 0.58-0.98, P interaction = 0.08), whereas patients with rs4986938 (G>A) genotype variants AA and AG had an increased risk of bone AE (HR = 1.37, 95% CI = 1.01-1.84, P interaction = 0.07). We observed that (1) rare ESR1 homozygous polymorphisms were associated with lower recurrence, and (2) ESR1 and ESR2 SNPs were associated with bone AEs in letrozole-treated patients. Genes that are involved in estrogen signaling and synthesis have the potential to affect both breast cancer recurrence and side effects, suggesting that individual treatment strategies can incorporate not only oncogenic drivers but also SNPs related to estrogen activity.

The CIP2A gene is an oncogene associated with solid and hematologic malignancies [1]. CIP2A protein is an oncoprotein and a potential cancer therapy target [2]. Literature shows that CIP2A inhibits the tumor suppressor protein PP2A [3] which downregulates phophorylation of AKT, a hallmark of cancers [4] and stabilizes the proto-oncogene, c-MYC in tumor cells [5], the comprehensive action of CIP2A and its functional interaction(s) with other oncoproteins and tumor suppressors is not clearly established. Recently we tried to put forward a contextual mode-of-action of CIP2A protein in a review which proposed that CIP2A influences oncogenesis via an "oncogenic nexus" [1]. In this review we critically evaluated the potential relevance of the mode-of-action of the "oncogenic nexus" of CIP2A in breast carcinogenesis and appraised the role of this nexus in different PAM50 luminal A, PAM50 luminal B, PAM50 HER2-enriched and PAM50 basal BC. This review has a novel approach. Here we have not only compiled and discussed the latest developments in this field but also presented data obtained from c-BioPortal and STRING10 in order to substantiate our view regarding the mode-of-action of the "oncogenic nexus" of CIP2A. We functionally correlated alterations of genes pertaining to the "oncogenic nexus" of CIP2A with protein-protein interactions between the different components of the nexus including (1) subunits of PP2A, (2) multiple transcription factors including MYC oncogene and (3) components of the PI3K-mTOR and the MAPK-ERK oncogenic pathways. Using these proteins as "input" to STRING10 we studied the association, Action view, at the highest Confidence level. OncoPrints (c-BioPortal) showed alterations (%) of regulatory subunits genes of PP2A (PPP2R1A and PPP2R1B) along with alterations of CIP2A in breast invasive carcinoma (TCGA, Nature 2012 & TCGA, Provisional). Similar genetic alterations of PP2A were also observed in samples of breast tumors at our Avera Research Institute, SD. In an attempt to critically evaluate the role of "oncogenic nexus" of CIP2A in subtypes of BC, we used PPP2R1A and PPP2R1B as "inputs" into the STRING10 and obtained their predicted association (Action view) in respect to CIP2A. The outcome of this exercise has been discussed in the light of the literature in the BC research in the context of "oncogenic nexus" of CIP2A. In summary, herein we review the progress in our understanding of how CIP2A regulates oncogenic transformations of breast cells via PP2A-CIP2A "oncogenic nexus" and how we can prospect the clinical relevance of CIP2A in the context of BC.

The many improvements in breast cancer therapy in recent years have so lowered rates of recurrence that it is now difficult or impossible to conduct adequately powered adjuvant clinical trials. Given the many new drugs and potential synergistic combinations, the neoadjuvant approach has been used to test benefit of drug combinations in clinical trials of primary breast cancer. A recent FDA-led meta-analysis showed that pathologic complete response (pCR) predicts disease-free survival (DFS) within patients who have specific breast cancer subtypes. This meta-analysis motivated the FDA's draft guidance for using pCR as a surrogate endpoint in accelerated drug approval. Using pCR as a registration endpoint was challenged at ASCO 2014 Annual Meeting with the presentation of ALTTO, an adjuvant trial in HER2-positive breast cancer that showed a nonsignificant reduction in DFS hazard rate for adding lapatinib, a HER-family tyrosine kinase inhibitor, to trastuzumab and chemotherapy. This conclusion seemed to be inconsistent with the results of NeoALTTO, a neoadjuvant trial that found a statistical improvement in pCR rate for the identical lapatinib-containing regimen. We address differences in the two trials that may account for discordant conclusions. However, we use the FDA meta-analysis to show that there is no discordance at all between the observed pCR difference in NeoALTTO and the observed HR in ALTTO. This underscores the importance of appropriately modeling the two endpoints when designing clinical trials. The I-SPY 2/3 neoadjuvant trials exemplify this approach., (©2015 American Association for Cancer Research.)

To determine whether CYP19A1 polymorphisms are associated with abnormal activity of aromatase and with musculoskeletal and bone side effects of aromatase inhibitors. DNA was isolated from tumor specimens of 4861 postmenopausal women with hormone receptor-positive breast cancer enrolled in the BIG 1-98 trial to receive tamoxifen and/or letrozole for 5 years. Tumors were genotyped for six CYP19A1 polymorphisms using PCR-based methods. Associations with breast cancer-free interval (BCFI), distant recurrence-free interval (DRFI), musculoskeletal and bone adverse events (AEs) were assessed using Cox proportional hazards models. All statistical tests were two-sided. No association between the CYP19A1 genotypes and BCFI or DRFI was observed overall. A reduced risk of a breast cancer event for tamoxifen-treated patients with rs700518 variants was observed (BCFI CC/TC vs. TT: HR 0.53, 95 % CI 0.34-0.82, interaction P = 0.08), but not observed for letrozole-treated patients. There was an increased risk of musculoskeletal AEs for patients with rs700518 variants CC/TC versus TT (HR 1.22, 95 % CI 1.03-1.45, P = 0.02), regardless of treatment. Tamoxifen-treated patients with rs4646 variants had a reduced risk of bone AEs (AA/CA vs. CC: HR 0.76, 95 % CI 0.59-0.98), whereas an increase of minor allele (C) of rs10046 was associated with an increased risk of bone AEs (HR 1.28, 95 % CI 1.07-1.52). rs936308 variants were associated with a reduced risk of bone AEs in letrozole-treated patients (GG/GC vs. CC: HR 0.73, 95 % CI 0.54-0.99), different from in tamoxifen-treated patients (GG/GC vs. CC: HR 1.32, 95 % CI 0.92-1.90, interaction P = 0.01). CYP19A1 rs700518 variants showed associations with BCFI, DRFI, in tamoxifen treated patients and musculoskeletal AEs regardless of treatment. SNPs rs4646, rs10046, and rs936308 were associated with bone AEs.

ERBB3/HER3 is the most intriguing RTK by virtue of its ability to transduce multiple cytosolic signals for the proliferation and growth of tumor cells in spite of being a "kinase dead" receptor that binds to its true ligand, heregulin. Although other members of the HER3 family like EGFR and HER2 have long been recognized to be associated with breast tumorigenesis and studied because of their predictive and prognostic value, the significance of HER3 as an irrefutable component of HER family signalosome is a relatively new development. The recent understanding of signals originating from the oncogenic partnership of HER3 with HER2 in the context of HER2 amplification/overexpression showed the critical clinical value for the treatment of HER2+BC. The downstream signaling cascade (included but not limited to the PI3K signaling) associated with signals originating from HER2:HER3 dimers play a vital role in the tumorigenesis, drug-resistance and tumor progression of HER2+BC. The upregulation of HER3 activity provides an alternate "escape route" via which tumor cells bypass either the inhibition of the HER family RTKs or the inhibition of the downstream PI3K-AKT-mTOR signaling pathway. By understanding the signaling that provides this "escape route" for these tumor cells treated with a targeted therapy (HER2 inhibitors or inhibitors of downstream PI3K-AKT-mTOR signaling pathway), we are just beginning to appreciate the prognostic value of HER3 in breast cancer. In this review, we will discuss the relevance of HER3 signaling in the context of, (1) downstream oncogenic signals and (2) therapeutic options in HER2 amplified BC.

Background: New data on erythropoiesis-stimulating agents (ESAs) regarding overall survival and disease progression-related outcomes in patients with breast cancer receiving chemotherapy are presented in a meta-analysis of controlled trials of ESA use (epoetin α, epoetin β, darbepoetin α, biosimilars)., Patients and Methods: A literature search identified reports from January 1997 through March 2014. We used company databases for Amgen, Inc., or Janssen studies and published data for other studies. Random-effects odds ratios (ORs) were calculated to compare results for patients randomized to ESA with those randomized to control., Results: Deaths were reported for 571 of 2346 patients (24%) in the ESA groups and 523 of 2367 patients (22%) in the control groups [OR, 1.20; 95% confidence interval (CI) 1.03-1.40]. Sensitivity analyses were conducted to explore the effects of individual studies and exclusion of one study (BEST) resulted in an OR for death of 1.12 (95% CI 0.94-1.34). In seven studies reporting progression-related end points (N = 4197; ESA n = 2088; control n = 2109), the OR was 1.01 (95% CI 0.87-1.16) for ESA compared with control., Conclusions: After incorporating recent results of ESA use in patients with breast cancer, risks of survival and progression-free survival remain consistent with previously published data., (© The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Approximately 35% of breast cancers exhibit PIK3CA activating mutation. Since PIK3CA hotspot mutation is the most frequently mutated gene in human breast cancers and primarily overlaps in HER2+ as well as ER+ breast cancers, the subset of patients bearing PIK3CA activating mutation does not get fullest benefit from either anti-HER2 or anti-hormonal agents. Literature also suggests that these patients may have chemotherapy resistance. Indeed, multiple clinical trials are currently evaluating the efficacy of over 30 drugs targeting different nodal points in the PI3K-AKT-mTOR pathway in breast and other cancers. However, to date, responses of solid tumors to PI3K pathway inhibitor monotherapy remains modest with an accompanied rapid emergences of drug resistance. MYC elevation represents one of the potential modes of actions by which breast tumors develop resistance to the PI3K pathway-specific targeted therapies. As products of oncogenes, both MYC and PIK3CA are well-established onco-proteins which contribute to breast oncogenesis. However, their similarities out number their dissimilarities in the context of their specific oncogenic cellular signals. In this review we will describe the specific cellular signals initiated following alteration in the MYC gene and PIK3CA gene in breast cancers. We will interrogate how MYC gene alterations influence the action of PI3K pathway targeted drugs in the context of PIK3CA mutation towards the development PI3K inhibitor induced drug-resistance in breast cancers.

Recent observations connected DNA cytosine deaminase APOBEC3B to the genetic evolution of breast cancer. We addressed whether APOBEC3B is associated with breast cancer clinical outcomes. APOBEC3B messenger RNA (mRNA) levels were related in 1,491 primary breast cancers to disease-free (DFS), metastasis-free (MFS), and overall survival (OS). For independent validation, APOBEC3B mRNA expression was associated with patient outcome data in five additional cohorts (over 3,500 breast cancer cases). In univariate Cox regression analysis, increasing APOBEC3B expression as a continuous variable was associated with worse DFS, MFS, and OS (hazard ratio [HR] = 1.20, 1.21, and 1.24, respectively; all P < .001). Also, in untreated ER-positive (ER+), but not in ER-, lymph-node-negative patients, high APOBEC3B levels were associated with a poor DFS (continuous variable: HR = 1.29, P = .001; dichotomized at the median level, HR = 1.66, P = .0002). This implies that APOBEC3B is a marker of pure prognosis in ER + disease. These findings were confirmed in the analyses of five independent patient sets. In these analyses, APOBEC3B expression dichotomized at the median level was associated with adverse outcomes (METABRIC discovery and validation, 788 and 706 ER + cases, disease-specific survival (DSS), HR = 1.77 and HR = 1.77, respectively, both P < .001; Affymetrix dataset, 754 ER + cases, DFS, HR = 1.57, P = 2.46E-04; NKI295, 181 ER + cases, DFS, HR = 1.72, P = .054; and BIG 1-98, 1,219 ER + cases, breast-cancer-free interval (BCFI), HR = 1.42, P = 0.0079). APOBEC3B is a marker of pure prognosis and poor outcomes for ER + breast cancer, which strongly suggests that genetic aberrations induced by APOBEC3B contribute to breast cancer progression.

Purpose: Increased circulating tumor cells (CTCs; five or more CTCs per 7.5 mL of whole blood) are associated with poor prognosis in metastatic breast cancer (MBC). A randomized trial of patients with persistent increase in CTCs tested whether changing chemotherapy after one cycle of first-line chemotherapy would improve the primary outcome of overall survival (OS)., Patients and Methods: Patients with MBC who did not have increased CTCs at baseline remained on initial therapy until progression (arm A). Patients with initially increased CTCs that decreased after 21 days of therapy remained on initial therapy (arm B). Patients with persistently increased CTCs after 21 days of therapy were randomly assigned to continue initial therapy (arm C1) or change to an alternative chemotherapy (arm C2)., Results: Of 595 eligible and evaluable patients, 276 (46%) did not have increased CTCs (arm A). Of those with initially increased CTCs, 31 (10%) were not retested, 165 were assigned to arm B, and 123 were randomly assigned to arm C1 or C2. No difference in median OS was observed between arm C1 and C2 (10.7 and 12.5 months, respectively; P = .98). CTCs were strongly prognostic. Median OS for arms A, B, and C (C1 and C2 combined) were 35 months, 23 months, and 13 months, respectively (P < .001)., Conclusion: This study confirms the prognostic significance of CTCs in patients with MBC receiving first-line chemotherapy. For patients with persistently increased CTCs after 21 days of first-line chemotherapy, early switching to an alternate cytotoxic therapy was not effective in prolonging OS. For this population, there is a need for more effective treatment than standard chemotherapy., (© 2014 by American Society of Clinical Oncology.)

Oncoprotein CIP2A a Cancerous Inhibitor of PP2A forms an "oncogenic nexus" by virtue of its control on PP2A and MYC stabilization in cancer cells. The expression and prognostic function of CIP2A in different solid tumors including colorectal carcinoma, head and neck cancers, gastric cancers, lung carcinoma, cholangiocarcinoma, esophageal cancers, pancreatic carcinoma, brain cancers, breast carcinoma, bladder cancers, ovarian carcinoma, renal cell carcinomas, tongue cancers, cervical carcinoma, prostate cancers, and oral carcinoma as well as a number of hematological malignancies are just beginning to emerge. Herein, we reviewed the recent progress in our understanding of (1) how an "oncogenic nexus" of CIP2A participates in the tumorigenic transformation of cells and (2) how we can prospect/view the clinical relevance of CIP2A in the context of cancer therapy. The review will try to understand the role of CIP2A (a) as a biomarker in cancers and evaluate the prognostic value of CIP2A in different cancers (b) as a therapeutic target in cancers and (c) in drug response and developing chemo-resistance in cancers.

Purpose: To document the rate and outcome of trastuzumab-associated cardiac dysfunction in patients following 1 or 2 years of adjuvant therapy., Patients and Methods: The Herceptin Adjuvant (HERA) trial is a three-arm, randomized trial comparing 2 years or 1 year of trastuzumab with observation in 5,102 patients with human epidermal growth factor receptor 2 (HER2) -positive early-stage breast cancer. Cardiac function was closely monitored. Eligible patients had left ventricular ejection fraction (LVEF) ≥ 55% at study entry following neoadjuvant chemotherapy with or without radiotherapy. This 8-year median follow-up analysis considered patients randomly assigned to 2 years or 1 year of trastuzumab or observation., Results: The as-treated safety population for 2 years of trastuzumab (n = 1,673), 1 year of trastuzumab (n = 1,682), and observation (n = 1,744) is reported. Cardiac adverse events leading to discontinuation of trastuzumab occurred in 9.4% of patients in the 2-year arm and 5.2% of patients in the 1-year arm. Cardiac death, severe congestive heart failure (CHF), and confirmed significant LVEF decrease remained low in all three arms. The incidence of severe CHF (0.8%, 0.8%, and 0.0%, respectively) and confirmed significant LVEF decrease (7.2%, 4.1%, and 0.9%, respectively) was significantly higher in the 2-year and 1-year trastuzumab arms compared with the observation arm. Severe CHF was the same for 2-year and 1-year trastuzumab. Of patients with confirmed LVEF decrease receiving 2-year trastuzumab, 87.5% reached acute recovery. Of patients with confirmed LVEF decrease receiving 1-year trastuzumab, 81.2% reached acute recovery., Conclusion: Long-term assessment at 8-year median follow-up confirms the low incidence of cardiac events for trastuzumab given sequentially after chemotherapy and radiotherapy, and cardiac events were reversible in the vast majority of patients., (© 2014 by American Society of Clinical Oncology.)

Trastuzumab-containing therapy is a standard of care for human epidermal growth factor receptor-2 (HER2)-positive breast cancer. In pre-clinical models, a wide range of molecular mechanisms have been associated with reduced sensitivity to trastuzumab in vitro. These include expression of the truncated HER2 receptor fragment p95HER2, activating mutation of the gene encoding the class 1A catalytic subunit of phosphatidylinositol 3-kinase (PIK3CA), loss of phosphatase and tensin homolog (PTEN), activation of other downstream signal transducers, prevention of cell cycle arrest, increased signaling through alternative (HER or non-HER) tyrosine kinase receptors, and resistance to antibody-dependent cellular cytotoxicity. However, the clinical significance of these mechanisms as determinants of trastuzumab efficacy in vivo has been unclear. Here, we review clinical studies of potential predictive biomarkers of trastuzumab efficacy in HER2-positive breast cancer and consider whether evaluation of such markers might inform patient selection for therapy. We find that clinical evidence relating to potential predictive biomarkers is mostly limited to small, retrospective studies, many of which have yielded conflicting findings. Some trends are evident in the retrospective data and in biomarker analyses from randomized clinical trials, particularly relating to activation of the phosphatidylinositol 3-kinase pathway, but none is sufficiently strong to form a basis for patient selection. This may be explained by the fact that multiple mechanisms of action determine the clinical efficacy of trastuzumab. In the absence of novel, validated biomarkers of efficacy, trastuzumab eligibility should continue to be based on evaluation of HER2 status according to standard methods., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Mutations of genes in tumor cells of Triple Negative subset of Breast Cancer (TNBC) deregulate pathways of signal transduction. The loss of tumor suppressor gene PTEN is the most common first event associated with basal-like subtype (Martins, De, Almendro, Gonen, and Park, 2012). Here we report for the first time that the functional upregulation of secreted-MMP7, a transcriptional target of Wnt-β-catenin signature pathway in TNBC is associated to the loss of PTEN. We identified differential expression of mRNAs in several key-components genes, and transcriptional target genes of the Wnt-β-catenin pathway (WP), including beta-catenin, FZD7, DVL1, MMP7, c-MYC, BIRC5, CD44, PPARD, c-MET, and NOTCH1 in FFPE tumors samples from TNBC patients of two independent cohorts. A similar differential upregulation of mRNA/protein for beta-catenin, the functional readout of WP, and for MMP7, a transcriptional target gene of beta-catenin was observed in TNBC cell line models. Genetic or pharmacological attenuation of beta-catenin by SiRNA or WP modulators (XAV939 and sulindac sulfide) and pharmacological mimicking of PTEN following LY294002 treatment downregulated MMP7 levels as well as enzymatic function of the secreted MMP7 in MMP7 positive PTEN-null TNBC cells. Patient data revealed that MMP7 mRNA was high in only a subpopulation of TNBC, and this subpopulation was characterized by a concurrent low expression of PTEN mRNA. In cell lines, a high expression of casein-zymograph-positive MMP7 was distinguished by an absence of functional PTEN. A similar inverse relationship between MMP7 and PTEN mRNA levels was observed in the PAM50 data set (a correlation coefficient of -0.54). The PAM50 subtype and outcome data revealed that the high MMP7 group had low pCR (25%) and High Rd (74%) in clinical stage T3 pathologic response in contrast to the high pCR (40%) and low residual disease (RD) (60%) of the low MMP7 group.

Purpose: We investigated antitumor activity of trastuzumab (T)/T-DM1 + pertuzumab (P) + bevacizumab (B) in T-sensitive (BT474) and T-resistant (BT474HerR) BC models in order to test whether or not the addition of an anti-angiogenic drug can provide a supplementary advantage to the antitumor activity of double HER2–mAB combination., Methods: In addition to the antitumor activity (xenograft model), we tested antiproliferative effect, and HER2-mediated signals of different antibodies (T or P or T-DM1) in HER2-amplified T-sensitive, T-resistant and HER2-amplified/PIK3CA mutated (HCC1954) BT cell lines by 3D ON-TOP clonogenic growth assay and Western blots., Results: Data show (1) T, T-DM1 or P blocked p-AKT (>60 %), p-ERK (>50 %) following heregulin in only T-sensitive cells, (2) T/T-DM1 + P, T/T-DM1 + B, and P + B reduced tumor growth as compared to any single-agent treatment, (3) T + P + B achieved almost complete regression of tumor growth, decreased cell proliferation, and inhibited tumor-induced angiogenesis, in both models, (4) antitumor activity of T + P + B was associated with the pharmacodynamic knockdown of p-AKT, and (5) T-DM1 + P caused complete regression of tumor volume in both models., Conclusions: Our data demonstrate that B imparts a significant advantage when combined with T + P in the resistant model, in contrast to T-DM1 + P, as the triple combination of T-DM1 + P + B and the double combination of T-DM1 + P showed a comparable antitumor activity. Our study reveals the preclinical evidence in favor of the inclusion of B when combined with T + P in T-resistant BC patients.

Background: Trastuzumab has established efficacy against breast cancer with overexpression or amplification of the HER2 oncogene. The standard of care is 1 year of adjuvant trastuzumab, but the optimum duration of treatment is unknown. We compared 2 years of treatment with trastuzumab with 1 year of treatment, and updated the comparison of 1 year of trastuzumab versus observation at a median follow-up of 8 years, for patients enrolled in the HERceptin Adjuvant (HERA) trial., Methods: The HERA trial is an international, multicentre, randomised, open-label, phase 3 trial comparing treatment with trastuzumab for 1 and 2 years with observation after standard neoadjuvant chemotherapy, adjuvant chemotherapy, or both in 5102 patients with HER2-positive early breast cancer. The primary endpoint was disease-free survival. The comparison of 2 years versus 1 year of trastuzumab treatment involved a landmark analysis of 3105 patients who were disease-free 12 months after randomisation to one of the trastuzumab groups, and was planned after observing at least 725 disease-free survival events. The updated intention-to-treat comparison of 1 year trastuzumab treatment versus observation alone in 3399 patients at a median follow-up of 8 years (range 0-10) is also reported. This study is registered with ClinicalTrials.gov, number NCT00045032., Findings: We recorded 367 events of disease-free survival in 1552 patients in the 1 year group and 367 events in 1553 patients in the 2 year group (hazard ratio [HR] 0·99, 95% CI 0·85-1·14, p=0·86). Grade 3-4 adverse events and decreases in left ventricular ejection fraction during treatment were reported more frequently in the 2 year treatment group than in the 1 year group (342 [20·4%] vs 275 [16·3%] grade 3-4 adverse events, and 120 [7·2%] vs 69 [4·1%] decreases in left ventricular ejection fraction, respectively). HRs for a comparison of 1 year of trastuzumab treatment versus observation were 0·76 (95% CI 0·67-0·86, p<0·0001) for disease-free survival and 0·76 (0·65-0·88, p=0·0005) for overall survival, despite crossover of 884 (52%) patients from the observation group to trastuzumab therapy., Interpretation: 2 years of adjuvant trastuzumab is not more effective than is 1 year of treatment for patients with HER2-positive early breast cancer. 1 year of treatment provides a significant disease-free and overall survival benefit compared with observation and remains the standard of care., Funding: F Hoffmann-La Roche (Roche)., (Copyright © 2013 Elsevier Ltd. All rights reserved.)