Generation of HER2-specific antibody immunity during trastuzumab adjuvant therapy associates with reduced relapse in resected HER2 breast cancer.

BACKGROUND: Resected HER2 breast cancer patients treated with adjuvant trastuzumab and chemotherapy have superior survival compared to patients treated with chemotherapy alone. We previously showed that trastuzumab and chemotherapy induce HER2-specific antibodies which correlate with improved survival in HER2 metastatic breast cancer patients. It remains unclear whether the generation of immunity required trastuzumab and whether endogenous antibody immunity is associated with improved disease-free survival in the adjuvant setting. In this study, we addressed this question by analyzing serum anti-HER2 antibodies from a subset of patients enrolled in the NCCTG trial N9831, which includes an arm (Arm A) in which trastuzumab was not used. Arms B and C received trastuzumab sequentially or concurrently to chemotherapy, respectively. METHODS: Pre-and post-treatment initiation sera were obtained from 50 women enrolled in N9831. Lambda IgG antibodies (to avoid detection of trastuzumab) to HER2 were measured and compared between arms and with disease-free survival. RESULTS: Prior to therapy, across all three arms, N9831 patients had similar mean anti-HER2 IgG levels. Following treatment, the mean levels of antibodies increased in the trastuzumab arms but not the chemotherapy-only arm. The proportion of patients who demonstrated antibodies increased by 4% in Arm A and by 43% in the Arms B and C combined (p = 0.003). Cox modeling demonstrated that larger increases in antibodies were associated with improved disease-free survival in all patients (HR = 0.23; p = 0.04). CONCLUSIONS: These results show that the increased endogenous antibody immunity observed in adjuvant patients treated with combination trastuzumab and chemotherapy is clinically significant, in view of its correlation with improved disease-free survival. The findings may have important implications for predicting treatment outcomes in patients treated with trastuzumab in the adjuvant setting. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00005970 . Registered on July 5, 2000.

Chromoanasynthesis is a common mechanism that leads to ERBB2 amplifications in a cohort of early stage HER2+ breast cancer samples.

BACKGROUND: HER2 positive (HER2+) breast cancers involve chromosomal structural alterations that act as oncogenic driver events. METHODS: We interrogated the genomic structure of 18 clinically-defined HER2+ breast tumors through integrated analysis of whole genome and transcriptome sequencing, coupled with clinical information. RESULTS: ERBB2 overexpression in 15 of these tumors was associated with ERBB2 amplification due to chromoanasynthesis with six of them containing single events and the other nine exhibiting multiple events. Two of the more complex cases had adverse clinical outcomes. Chromosomes 8 was commonly involved in the same chromoanasynthesis with 17. In ten cases where chromosome 8 was involved we observed NRG1 fusions (two cases), NRG1 amplification (one case), FGFR1 amplification and ADAM32 or ADAM5 fusions. ERBB3 over-expression was associated with NRG1 fusions and EGFR and ERBB3 expressions were anti-correlated. Of the remaining three cases, one had a small duplication fully encompassing ERBB2 and was accompanied with a pathogenic mutation. CONCLUSION: Chromoanasynthesis involving chromosome 17 can lead to ERBB2 amplifications in HER2+ breast cancer. However, additional large genomic alterations contribute to a high level of genomic complexity, generating the hypothesis that worse outcome could be associated with multiple chromoanasynthetic events.

Genome-wide association study of cardiotoxicity in the NCCTG N9831 (Alliance) adjuvant trastuzumab trial.

OBJECTIVES: The major clinical side effect of the ERBB2-targeted breast cancer therapy, trastuzumab, is a decline in the left ventricular ejection fraction (LVEF). Improved markers are needed to better identify patients susceptible to cardiotoxicity. METHODS: The NCCTG N9831 trial compared adjuvant doxorubicin and cyclophosphamide followed by either weekly paclitaxel (arm A); paclitaxel then trastuzumab (arm B); or concurrent paclitaxel and trastuzumab (arm C) in patients with HER2-positive breast cancer. A genome-wide association study was performed on all patients with available DNA (N=1446). We used linear regression to identify single nucleotide polymorphisms (SNPs) associated with decline in LVEF, adjusting for age, baseline LVEF, antihypertensive medications, and the first two principle components. RESULTS: In total, 618 863 SNPs passed quality control and DNA from 1191 patients passed genotyping quality control and were identified as Whites of non-Hispanic origin. SNPs at six loci were associated with a decline in LVEF (P=7.73×10 to 8.93×10), LDB2, BRINP1, chr6 intergenic, RAB22A, TRPC6, and LINC01060, in patients who received chemotherapy plus trastuzumab (arms BC, N=800). None of these loci were significant in patients who received chemotherapy only (arm A, N=391) and did not increase in significance in the combined analysis of all patients. We did not observe association, P<0.05, with SNPs previously associated with trastuzumab-induced cardiotoxicity at ERBB2, I655V, and P1170A. We replicated association, P<0.05, with SNPs previously associated with anthracycline-induced cardiotoxicity at CBR3 and ABCB1. CONCLUSION: Our study identified six putative novel cardiotoxicity loci in patients treated with combination chemotherapy and trastuzumab that require further investigation and confirmed known associations of anthracycline-induced cardiotoxicity.

Higher and Sustained Cell-Mediated Immune Responses After 3 Doses of mRNA COVID-19 Vaccine in Patients With Inflammatory Bowel Disease on Anti-Tumor Necrosis Factor Therapy.

INTRODUCTION: Studies suggest that the generation of durable T-cell immunity following coronavirus disease 2019 (COVID-19) vaccination protects against severe disease. The aim of this study was to measure cell-mediated immune response (CMIR) 1-2 months and 6 months after a third dose of a COVID-19 mRNA vaccine. METHODS: This prospective study (HumoRal and CellULar initial and Sustained immunogenicity in patients with inflammatory bowel disease [IBD]) evaluated CMIR at 28-65 days (t 1 ) after dose 2, 28-65 days (t 2 ) (n = 183) and 6 months (±45 days) (t 3 ) (n = 167) after a third dose of an mRNA COVID-19 vaccine. A small cohort had blood sample available 28-65 days (t 4 ) (n = 55) after a fourth dose. Primary outcomes were CMIR at (t 2 ) and (t 3 ). Secondary outcomes included the effect of immunosuppressing IBD medications on CMIR and response at (t 4 ). RESULTS: All patients had measurable CMIR at all time points. CMIR increased at t 2 compared with that at t 1 (median 1,467 responding cells per million (interquartile range [IQR] 410-5,971) vs 313 (94-960) P < 0.001). There was no significant waning in t 2 vs t 3 or significant boosting at t 4 . Those on anti-tumor necrosis factor monotherapy had a higher CMIR compared with those not on this therapy at t 2 (4,132 [IQR 1,136-8,795] vs 869 [IQR 343-3,221] P < 0.001) and t 3 (2,843 [IQR 596-6,459] vs 654 [IQR 143-2,067] P < 0.001). In univariable analysis, anti-tumor necrosis factor monotherapy was associated with a higher CMIR at t 2 ( P < 0.001) and t 3 ( P < 0.001) and confirmed in a multivariable model ( P < 0.001). DISCUSSION: A third dose of a COVID-19 vaccine boosts CMIR, and the response is sustained in patients with IBD.

A novel bioinformatics pipeline for identification and characterization of fusion transcripts in breast cancer and normal cell lines.

SnowShoes-FTD, developed for fusion transcript detection in paired-end mRNA-Seq data, employs multiple steps of false positive filtering to nominate fusion transcripts with near 100% confidence. Unique features include: (i) identification of multiple fusion isoforms from two gene partners; (ii) prediction of genomic rearrangements; (iii) identification of exon fusion boundaries; (iv) generation of a 5'-3' fusion spanning sequence for PCR validation; and (v) prediction of the protein sequences, including frame shift and amino acid insertions. We applied SnowShoes-FTD to identify 50 fusion candidates in 22 breast cancer and 9 non-transformed cell lines. Five additional fusion candidates with two isoforms were confirmed. In all, 30 of 55 fusion candidates had in-frame protein products. No fusion transcripts were detected in non-transformed cells. Consideration of the possible functions of a subset of predicted fusion proteins suggests several potentially important functions in transformation, including a possible new mechanism for overexpression of ERBB2 in a HER-positive cell line. The source code of SnowShoes-FTD is provided in two formats: one configured to run on the Sun Grid Engine for parallelization, and the other formatted to run on a single LINUX node. Executables in PERL are available for download from our web site: http://mayoresearch.mayo.edu/mayo/research/biostat/stand-alone-packages.cfm.

Th17-inducing dendritic cell vaccines stimulate effective CD4 T cell-dependent antitumor immunity in ovarian cancer that overcomes resistance to immune checkpoint blockade.

BACKGROUND: Ovarian cancer (OC), a highly lethal cancer in women, has a 48% 5-year overall survival rate. Prior studies link the presence of IL-17 and Th17 T cells in the tumor microenvironment to improved survival in OC patients. To determine if Th17-inducing vaccines are therapeutically effective in OC, we created a murine model of Th17-inducing dendritic cell (DC) (Th17-DC) vaccination generated by stimulating IL-15 while blocking p38 MAPK in bone marrow-derived DCs, followed by antigen pulsing. METHODS: ID8 tumor cells were injected intraperitoneally into mice. Mice were treated with Th17-DC or conventional DC (cDC) vaccine alone or with immune checkpoint blockade (ICB). Systemic immunity, tumor associated immunity, tumor size and survival were examined using a variety of experimental strategies. RESULTS: Th17-DC vaccines increased Th17 T cells in the tumor microenvironment, reshaped the myeloid microenvironment, and improved mouse survival compared with cDC vaccines. ICB had limited efficacy in OC, but Th17-inducing DC vaccination sensitized it to anti-PD-1 ICB, resulting in durable progression-free survival by overcoming IL-10-mediated resistance. Th17-DC vaccine efficacy, alone or with ICB, was mediated by CD4 T cells, but not CD8 T cells. CONCLUSIONS: These findings emphasize using biologically relevant immune modifiers, like Th17-DC vaccines, in OC treatment to reshape the tumor microenvironment and enhance clinical responses to ICB therapy.

Higher Cell-Mediated Immune Responses in Patients With Inflammatory Bowel Disease on Anti-TNF Therapy After COVID-19 Vaccination.

BACKGROUND: Some patients with inflammatory bowel disease (IBD) on immunosuppressive therapies may have a blunted response to certain vaccines, including the messenger RNA (mRNA) coronavirus disease 2019 (COVID-19) vaccines. However, few studies have evaluated the cell-mediated immune response (CMIR), which is critical to host defense after COVID-19 infection. The aim of this study was to evaluate the humoral immune response and CMIR after mRNA COVID-19 vaccination in patients with IBD. METHODS: This prospective study (HERCULES [HumoRal and CellULar initial and Sustained immunogenicity in patients with IBD] study) evaluated humoral immune response and CMIR after completion of 2 doses of mRNA COVID-19 vaccines in 158 IBD patients and 20 healthy control (HC) subjects. The primary outcome was the CMIR to mRNA COVID-19 vaccines in patients with IBD. The secondary outcomes were a comparison of (1) the CMIR in patients with IBD and HC subjects, (2) CMIR and humoral immune response in all participants, and (3) correlation between CMIR and humoral immune response. RESULTS: The majority (89%) of patients with IBD developed a CMIR, which was not different vs HC subjects (94%) (P = .6667). There was no significant difference (P = .5488) in CMIR between immunocompetent (median 255 [interquartile range, 146-958] spike T cells per million peripheral blood mononuclear cells) and immunosuppressed patients (median 377 [interquartile range, 123-1440]). There was no correlation between humoral and cell-mediated immunity after vaccination (P = .5215). In univariable analysis, anti-tumor necrosis factor therapy was associated with a higher CMIRs (P = .02) and confirmed in a multivariable model (P = .02). No other variables were associated with CMIR. CONCLUSIONS: Most patients with IBD achieved CMIR to a COVID-19 vaccine. Future studies are needed evaluating sustained CMIR and clinical outcomes.

Antibody and T cell responses to coronavirus disease 2019 vaccines in patients with inflammatory bowel disease do not correlate. Most patients with inflammatory bowel disease mount a T cell response despite being on biologic therapies, those on anti-tumor necrosis factor may have a higher T cell response. Anti-tumor necrosis factor therapy has been associated with a lower antibody response to coronavirus disease 2019 vaccines, but the T cell response is augmented.

TRPC6 N338S is a gain-of-function mutant identified in patient with doxorubicin-induced cardiotoxicity.

The canonical transient receptor potential 6 gene, TRPC6, has been implicated as a putative risk gene for chemotherapy-induced congestive heart failure, but knowledge of specific risk variants is lacking. Following our genome-wide association study and subsequent fine-mapping, a rare missense mutant of TRPC6 N338S, was identified in a breast cancer patient who received anthracycline-containing chemotherapy regiments and developed congestive heart failure. However, the function of N338S mutant has not been examined. Using intracellular Ca2+ imaging, patch clamp recording and molecular docking techniques, we assessed the function of N338S mutant heterologously expressed in HEK293 cells and HL-1 cardiac cells. We found that expression of TRPC6 N338S significantly increased intracellular Ca2+ levels ([Ca2+]i) and current densities in response to 50 µM 1-oleoyl 2-acetyl-sn-glycerol (OAG), an activator of TRPC6 channels, compared to those of TRPC6 WT. A 24-h pretreatment with 0.5 µM doxorubicin (DOX) further potentiated the OAG effects on TRPC6 N338S current densities and [Ca2+]i, and these effects were abolished by 1 µM BI-749327, a highly selective TRPC6 inhibitor. Moreover, DOX treatment significantly upregulated the mRNA and protein expressions of TRPC6 N338S, compared to those of TRPC6 WT. Molecular docking and dynamics simulation showed that OAG binds to the pocket constituted by the pore-helix, S5 and S6 domains of TRPC6. However, the N338S mutation strengthened the interaction with OAG, therefore stabilizing the OAG-TRPC6 N338S complex and enhancing OAG binding affinity. Our results indicate that TRPC6 N338S is a gain-of-function mutant that may contribute to DOX-induced cardiotoxicity by increasing Ca2+ influx and [Ca2+]i in cardiomyocytes.

Trpc6 Promotes Doxorubicin-Induced Cardiomyopathy in Male Mice With Pleiotropic Differences Between Males and Females.

Background: Doxorubicin is a widely used and effective chemotherapy, but the major limiting side effect is cardiomyopathy which in some patients leads to congestive heart failure. Genetic variants in TRPC6 have been associated with the development of doxorubicin-induced cardiotoxicity, suggesting that TRPC6 may be a therapeutic target for cardioprotection in cancer patients. Methods: Assessment of Trpc6 deficiency to prevent doxorubicin-induced cardiac damage and function was conducted in male and female B6.129 and Trpc6 knock-out mice. Mice were treated with doxorubicin intraperitoneally every other day for a total of 6 injections (4 mg/kg/dose, cumulative dose 24 mg/kg). Cardiac damage was measured in heart sections by quantification of vacuolation and fibrosis, and in heart tissue by gene expression of Tnni3 and Myh7. Cardiac function was determined by echocardiography. Results: When treated with doxorubicin, male Trpc6-deficient mice showed improvement in markers of cardiac damage with significantly reduced vacuolation, fibrosis and Myh7 expression and increased Tnni3 expression in the heart compared to wild-type controls. Similarly, male Trpc6-deficient mice treated with doxorubicin had improved LVEF, fractional shortening, cardiac output and stroke volume. Female mice were less susceptible to doxorubicin-induced cardiac damage and functional changes than males, but Trpc6-deficient females had improved vacuolation with doxorubicin treatment. Sex differences were observed in wild-type and Trpc6-deficient mice in body-weight and expression of Trpc1, Trpc3 and Rcan1 in response to doxorubicin. Conclusions: Trpc6 promotes cardiac damage following treatment with doxorubicin resulting in cardiomyopathy in male mice. Female mice are less susceptible to cardiotoxicity with more robust ability to modulate other Trpc channels and Rcan1 expression.

Association of Genetic Variants at TRPC6 With Chemotherapy-Related Heart Failure.

Background: Our previous GWAS identified genetic variants at six novel loci that were associated with a decline in left ventricular ejection fraction (LVEF), p < 1 × 10-5 in 1,191 early breast cancer patients from the N9831 clinical trial of chemotherapy plus trastuzumab. In this study we sought replication of these loci. Methods: We tested the top loci from the GWAS for association with chemotherapy-related heart failure (CRHF) using 26 CRHF cases from N9831 and 984 patients from the Mayo Clinic Biobank which included CRHF cases (N = 12) and control groups of patients treated with anthracycline +/- trastuzumab without HF (N = 282) and patients with HF that were never treated with anthracycline or trastuzumab (N = 690). We further examined associated loci in the context of gene expression and rare coding variants using a TWAS approach in heart left ventricle and Sanger sequencing, respectively. Doxorubicin-induced apoptosis and cardiomyopathy was modeled in human iPSC-derived cardiomyocytes and endothelial cells and a mouse model, respectively, that were pre-treated with GsMTx-4, an inhibitor of TRPC6. Results: TRPC6 5' flanking variant rs57242572-T was significantly more frequent in cases compared to controls, p = 0.031, and rs61918162-T showed a trend for association, p = 0.065. The rs61918162 T-allele was associated with higher TRPC6 expression in the heart left ventricle. We identified a single TRPC6 rare missense variant (rs767086724, N338S, prevalence 0.0025% in GnomAD) in one of 38 patients (2.6%) with CRHF. Pre-treatment of cardiomyocytes and endothelial cells with GsMTx4 significantly reduced doxorubicin-induced apoptosis. Similarly, mice treated with GsMTx4 had significantly improved doxorubicin-induced cardiac dysfunction. Conclusions: Genetic variants that are associated with increased TRPC6 expression in the heart and rare TRPC6 missense variants may be clinically useful as risk factors for CRHF. GsMTx-4 may be a cardioprotective agent in patients with TRPC6 risk variants. Replication of the genetic associations in larger well-characterized samples and functional studies are required.

Folate receptor alpha expression associates with improved disease-free survival in triple negative breast cancer patients.

Triple negative breast cancer (TNBC) comprises 15-20% of all invasive breast cancer and is associated with a poor prognosis. As therapy options are limited for this subtype, there is a significant need to identify new targeted approaches for TNBC patient management. The expression of the folate receptor alpha (FRα) is significantly increased in patients with TNBC and is therefore a potential biomarker and therapeutic target. We optimized and validated a FRα immunohistochemistry method, specific to TNBC, to measure FRα expression in a centrally confirmed cohort of 384 patients with TNBC in order to determine if expression of the protein is associated with invasive disease-free survival (IDFS) and overall survival (OS). The FRα IHC demonstrated exceptional performance characteristics with low intra- and interassay variability as well as minimal lot-to-lot variation. FRα expression, which varied widely from sample to sample, was detected in 274 (71%) of the TNBC lesions. In a multivariable model adjusted for baseline characteristics, FRα expression was associated with improved IDFS (HR = 0.63, p = 0.01) but not with OS. The results demonstrate the potential of targeting the FRα in the majority of TNBC patients and suggest that variable expression may point to a need to stratify on FRα expression in clinical studies.

Toll-like receptor 4 mediates cross-talk between peroxisome proliferator-activated receptor gamma and nuclear factor-kappaB in macrophages.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in macrophages and plays an important role in suppressing the inflammatory response. Lipopolysaccharides (LPS), which activate Toll-like receptor 4 (TLR4), reduced PPARgamma expression and function in peritoneal macrophages and macrophage cell lines. Moreover, pretreatment with the synthetic PPARgamma ligand, rosiglitazone did not prevent LPS-mediated downregulation of PPARgamma. Inhibition of PPARgamma expression was not blocked by cycloheximide, indicating that de novo protein synthesis is not required for LPS-mediated suppression of PPARgamma. Destabilization of PPARgamma messenger RNA (mRNA) was not observed in LPS-stimulated macrophages, suggesting that LPS regulates the synthesis of PPARgamma mRNA. LPS had no effect on PPARgamma expression in macrophages from TLR4 knockout mice, whereas LPS inhibited PPARgamma expression in cells that had been reconstituted to express functional TLR4. Targeting the TLR4 pathway with inhibitors of MEK1/2, p38, JNK and AP-1 had no effect on PPARgamma downregulation by LPS. However, inhibitors that target NEMO, IkappaB and NF-kappaB abolished LPS-mediated downregulation of PPARgamma in LPS-stimulated macrophages. Our data indicate that activation of TLR4 inhibits PPARgamma mRNA synthesis by an NF-kappaB-dependent mechanism. Low-density genomic profiling of macrophage-specific PPARgamma knockout cells indicated that PPARgamma suppresses inflammation under basal conditions, and that loss of PPARgamma expression is sufficient to induce a proinflammatory state. Our data reveal a regulatory feedback loop in which PPARgamma represses NF-kappaB-mediated inflammatory signalling in unstimulated macrophages; however, upon activation of TLR4, NF-kappaB drives down PPARgamma expression and thereby obviates any potential anti-inflammatory effects of PPARgamma in LPS-stimulated macrophages.

The high affinity peroxisome proliferator-activated receptor-gamma agonist RS5444 inhibits both initiation and progression of colon tumors in azoxymethane-treated mice.

We evaluated RS5444, a thiazolidinedione high affinity PPARgamma agonist, for the ability to inhibit colon carcinogenesis in azoxymethane (AOM)-treated mice. In our initial experiment, mice were treated with RS5444 during AOM treatment, and the drug was withdrawn 12 weeks after the last injection of AOM. RS5444 significantly inhibited aberrant crypt focus formation under these circumstances. Furthermore, exposure to RS5444 during the course of AOM treatment effectively blocked colon tumor formation after withdrawal of the agonist. PPARgamma expression and nuclear localization were reduced in adenomas. RS5444 did not inhibit DNA synthesis in tumor cells, suggesting that PPARgamma activity was impaired in adenomas. To test this hypothesis, pre-existing adenomas were treated with RS5444 for 16 weeks. We observed a slight, albeit not statistically significant, reduction in tumor incidence in RS5444-treated mice. However, histological examination revealed that tumors from RS5444-treated mice were of significantly lower grade, as evaluated by the extent of dysplasia. Furthermore, carcinoma in situ was observed in about one-third of control tumors, but was never observed in RS5444-treated tumors. We conclude that RS5444 inhibits both initiation and progression of colon tumors in the AOM model of sporadic colon carcinogenesis.

Differential expression, distribution, and function of PPAR-gamma in the proximal and distal colon.

Suppression of colon carcinogenesis by peroxisome proliferator-activated receptor (PPAR)-gamma is likely due to some effect of PPAR-gamma on normal colonic epithelial cells. However, our understanding of the effects of PPAR-gamma in such cells is limited. We analyzed the abundance, distribution, and function of PPAR-gamma in epithelial cells isolated from the murine proximal and distal colon. Marked differences in PPAR-gamma abundance and distribution were observed, suggesting tissue-specific responses. Analysis of PPAR-gamma effects on DNA synthesis, formation of preneoplastic lesions, and activation of MAPK signaling in proximal and distal colonic epithelial cells in vivo indicates that PPAR-gamma regulates both tissue-specific and common responses within the proximal and distal colon. Three major functional cohorts of PPAR-gamma target genes were identified by genomic profiling of isolated colonic epithelial cells: genes that are involved in metabolism, in signaling, and in cellular adhesion and motility. Two subsets of PPAR-gamma target genes were differentially expressed in the proximal and distal epithelium. Proximal target genes were primarily involved in metabolic activities, whereas signal transduction, adhesion, and motility targets were more pronounced in the distal colon. Remarkably, those target genes that are differentially expressed in the proximal colon were all induced on activation of PPAR-gamma, whereas all target genes that are preferentially expressed in the distal colon were repressed. Our data indicate that PPAR-gamma exerts both common and tissue-specific effects in the colon and challenge the general conclusions that PPAR-gamma is induced on differentiation of colonic epithelial cells and that this receptor stimulates differentiated function in epithelial cells throughout the colon.

The antineoplastic drug, trastuzumab, dysregulates metabolism in iPSC-derived cardiomyocytes.

BACKGROUND: The targeted ERBB2 therapy, trastuzumab, has had a tremendous impact on management of patients with HER2+ breast cancer, leading to development and increased use of further HER2 targeted therapies. The major clinical side effect is cardiotoxicity but the mechanism is largely unknown. On the basis that gene expression is known to be altered in multiple models of heart failure, we examined differential gene expression of iPSC-derived cardiomyocytes treated at day 11 with the ERBB2 targeted monoclonal antibody, trastuzumab for 48 h and the small molecule tyrosine kinase inhibitor of EGFR and ERBB2. RESULTS: Transcriptome sequencing was performed on four replicates from each group (48 h untreated, 48 h trastuzumab and 48 h lapatinib) and differential gene expression analyses were performed on each treatment group relative to untreated cardiomyocytes. 517 and 1358 genes were differentially expressed, p < 0.05, respectively in cardiomyocytes treated with trastuzumab and lapatinib. Gene ontology analyses revealed in cardiomyocytes treated with trastuzumab, significant down-regulation of genes involved in small molecule metabolism (p = 3.22 × 10-9) and cholesterol (p = 0.01) and sterol (p = 0.03) processing. We next measured glucose uptake and lactate production in iPSC-derived cardiomyocytes 13 days post-plating, treated with trastuzumab up to 96 h. We observed significantly decreased glucose uptake from the media of iPSC-derived cardiomyocytes treated with trastuzumab as early as 24 h (p = 0.001) and consistently up to 96 h (p = 0.03). CONCLUSIONS: Our study suggests dysregulation of cardiac gene expression and metabolism as key elements of ERBB2 signaling that could potentially be early biomarkers of cardiotoxicity.

Breast Cancer Clinical Trial of Chemotherapy and Trastuzumab: Potential Tool to Identify Cardiac Modifying Variants of Dilated Cardiomyopathy.

Doxorubicin and the ERBB2 targeted therapy, trastuzumab, are routinely used in the treatment of HER2+ breast cancer. In mouse models, doxorubicin is known to cause cardiomyopathy and conditional cardiac knock out of Erbb2 results in dilated cardiomyopathy and increased sensitivity to doxorubicin-induced cell death. In humans, these drugs also result in cardiac phenotypes, but severity and reversibility is highly variable. We examined the association of decline in left ventricular ejection fraction (LVEF) at 15,204 single nucleotide polymorphisms (SNPs) spanning 72 cardiomyopathy genes, in 800 breast cancer patients who received doxorubicin and trastuzumab. For 7033 common SNPs (minor allele frequency (MAF) > 0.01) we performed single marker linear regression. For all SNPs, we performed gene-based testing with SNP-set (Sequence) Kernel Association Tests: SKAT, SKAT-O and SKAT-common/rare under rare variant non-burden; rare variant optimized burden and non-burden tests; and a combination of rare and common variants respectively. Single marker analyses identified seven missense variants in OBSCN (p = 0.0045-0.0009, MAF = 0.18-0.50) and two in TTN (both p = 0.04, MAF = 0.22). Gene-based rare variant analyses, SKAT and SKAT-O, performed very similarly (ILK, TCAP, DSC2, VCL, FXN, DSP and KCNQ1, p = 0.042-0.006). Gene-based tests of rare/common variants were significant at the nominal 5% level for OBSCN as well as TCAP, DSC2, VCL, NEXN, KCNJ2 and DMD (p = 0.044-0.008). Our results suggest that rare and common variants in OBSCN, as well as in other genes, could have modifying effects in cardiomyopathy.

Corrigendum: Recurrent hormone-binding domain truncated ESR1 amplifications in primary endometrial cancers suggest their implication in hormone independent growth.

This corrects the article DOI: 10.1038/srep25521.

Intrinsic Subtype and Therapeutic Response Among HER2-Positive Breaty st Tumors from the NCCTG (Alliance) N9831 Trial.

Background: Genomic data from human epidermal growth factor receptor 2-positive (HER2+) tumors were analyzed to assess the association between intrinsic subtype and clinical outcome in a large, well-annotated patient cohort. Methods: Samples from the NCCTG (Alliance) N9831 trial were analyzed using the Prosigna algorithm on the NanoString platform to define intrinsic subtype, risk of recurrence scores, and risk categories for 1392 HER2+ tumors. Subtypes were evaluated for recurrence-free survival (RFS) using Kaplan-Meier and Cox model analysis following adjuvant chemotherapy (n = 484) or chemotherapy plus trastuzumab (n = 908). All statistical tests were two-sided. Results: Patients with HER2+ tumors from N9831 were primarily scored as HER2-enriched (72.1%). These individuals received statistically significant benefit from trastuzumab (hazard ratio [HR] = 0.68, 95% confidence interval [CI] = 0.52 to 0.89, P = .005), as did the patients (291 of 1392) with luminal-type tumors (HR = 0.52, 95% CI = 0.32 to 0.85, P = .01). Patients with basal-like tumors (97 of 1392) did not have statistically significantly better RFS when treated with trastuzumab and chemotherapy compared with chemotherapy alone (HR = 1.06, 95% CI = 0.53 to 2.13, P = .87). Conclusions: The majority of clinically defined HER2-positive tumors were classified as HER2-enriched or luminal using the Prosigna algorithm. Intrinsic subtype alone cannot replace conventional histopathological evaluation of HER2 status because many tumors that are classified as luminal A or luminal B will benefit from adjuvant trastuzumab if that subtype is accompanied by HER2 overexpression. However, among tumors that overexpress HER2, we speculate that assessment of intrinsic subtype may influence treatment, particularly with respect to evaluating alternative therapeutic approaches for that subset of HER2-positive tumors of the basal-like subtype.

Comprehensive Genomic Profiling of a Rare Thyroid Follicular Dendritic Cell Sarcoma.

We previously reported an extremely rare case of follicular dendritic cell sarcoma (FDCS) presented as a thyroid mass. Given the rarity of this disease, there are no personalized and molecularly targeted treatment options due to the lack of knowledge in the genomic makeup of the tumor. A 44-year-old white woman was diagnosed with an extranodal FDCS in thyroid. The patient underwent a total thyroidectomy, central compartment dissection, parathyroid re-implantation, and adjuvant radiation therapy. Tumor DNA sequencing of 236 genes by FoundationOne panel found truncating mutations in PTEN and missense mutations in RET and TP53. However, patient-matched germline DNA was not sequenced which is critical for identification of true somatic mutations. Furthermore, the FoundationOne panel doesn't measure genomic rearrangements which have been shown to be abundant in sarcomas and are associated with sarcoma tumorigenesis and progression. In the current study, we carried out comprehensive genomic sequencing of the tumor, adjacent normal tissues, and patient-matched blood, in an effort to understand the genomic makeup of this rare extranodal FDCS and to identify potential therapeutic targets. Eighty-one somatic point mutations were identified in tumor but not in adjacent normal tissues or blood. A clonal truncating mutation in the CLTCL1 gene, which stabilizes the mitotic spindle, was likely a driver mutation of tumorigenesis and could explain the extensive copy number aberrations (CNAs) and genomic rearrangements in the tumor including a chr15/chr17 local chromothripsis resulted in 6 expressed fusion genes. The fusion gene HDGFRP3→SHC4 led to a 200-fold increase in the expression of oncogene SHC4 which is a potential target of the commercial drug Dasatinib. Missense mutations in ATM and splice-site mutation in VEGFR1 were also detected in addition to the TP53 missense mutation reported by FoundationOne.

RS5444, a novel PPARgamma agonist, regulates aspects of the differentiated phenotype in nontransformed intestinal epithelial cells.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is expressed in the intestinal epithelium, yet little is known about the physiological role of PPARgamma in the small bowel or the effects of PPARgamma on small intestinal epithelial cells. The present studies investigate cellular and genomic effects of PPARgamma in nontransformed rat intestinal epithelial cells (RIE). These cells were engineered to express mouse PPARgamma1, and thereby to model the molecular phenotype that obtains upon induction of PPARgamma at the crypt/villus junction in the small intestine. In these studies, we have used a novel third generation thiazolidinedione derivative, RS5444, which activates PPARgamma with an EC50 about 1/50th that of rosiglitazone and has no effect on RIE cells that do not express PPARgamma. We used Affymetrix oligonucleotide microarrays to identify potential PPARgamma-regulated processes in RIE cells, including lipid metabolism, cell proliferation and differentiation, remodeling of the extracellular matrix, cell morphology, cell-cell adhesion, and motility. The genomic profile reflects cellular events that occur following PPARgamma activation: RS5444 inhibited culture growth and caused irreversible G1 arrest, but did not induce apoptosis. In addition, RS5444 caused dramatic changes in cellular morphology which were associated with increased motility and diminished cellular adherence, but no increase in the ability of such cells to digest and invade Matrigel. Inhibition of proliferation, cell cycle arrest, increased motility, and altered adherence are aspects of the differentiated phenotype of villus epithelial cells, which withdraw from the cell cycle at the crypt/villus interface, migrate to the villus tips, and are subsequently shed by loss of contact with the epithelium and the underlying extracellular matrix. Our results are consistent with the hypothesis that PPARgamma regulates critical aspects of differentiation in the small intestinal epithelium. Many nuclear receptors regulate differentiation. However, our results point to novel effects of PPARgamma on cell-cell and cell-matrix interactions, which are not typical of other nuclear receptors.