Lajos Pusztai

S1-7: Molecular Tumor Characteristics Influence Adjuvant Endocrine Treatment Outcome.

Background Used upfront or after 2 to 3 years (yrs) of tamoxifen (TAM) or as extended treatment after 5 yrs of TAM, aromatase inhibitors (AIs) are associated with less recurrences than with 5 yrs of TAM. However, up front AIs were not superior to TAM→AI sequence in node negative (N-) tumors (TEAM study, SABCS 2009; BIG1-98 trial, NEJM 2009). The observation is consistent with the hypothesis that early relapses may be more frequently due to intrinsic endocrine resistance irrespective of the use of TAM or AIs whereas late relapses depend on acquired resistance. Methods Affymetrix HGU133A-based gene expression profiles from two adjuvant datasets (n=556) of patients treated with 5 yrs TAM were evaluated and included 285 N-, 247 N+ and 24 Nx estrogen receptor-positive cases. A proliferation score based on the expression of 12 mitotic kinases (MKS) (Bianchini, Cancer Res 2010) and a 4-gene estrogen-related score (ERS) adopted from the Oncotype DX Recurrence Score were assessed. Median cut-off points were used for both biomarkers. Pattern of relapse according to marker status were evaluated in three distinct time cohorts (0–2.5 yrs, 2.5–5 yrs, 5–10 yrs). Outcome was assessed according to distant relapse rates. Results A violation of the proportional hazards assumption was found for both scores indicating time-dependent effects. The table shows distant relapse rates according to biomarker group within each time cohort. Among low proliferation tumors (lowMKS), the highERS group had low and steadily increasing recurrence rates, while the lowERS group showed a higher risk of relapse that increased continuously over the time. Among the highly proliferative tumors (highMKS), the high endocrine-sensitive group (highERS) had a lower risk of relapse within the 0–2.5 yrs compared to lowERS cancers. The difference is not persisting at the longer follow up of 2.5–5 yrs, and at 5–10 yrs the highERS group has higher risk of relapse. The pattern of relapse was similar for N- and N+ cancers. However, in the 0–2.5 yrs interval all recurrences occurred in the highMKS/lowERS among N- cancers, while only 57% of recurrences did occur in N+ cases in the same biomarkers group (p=0.012). Discussion Highly proliferative, high endocrine-sensitive (highERS) cancers are at the greatest risk of late relapse and extended endocrine therapy beyond 5 yrs may be useful. Low proliferation, low ERS cancers also remain at substantial risk for late recurrences. Between 0 to 2.5 yrs all recurrences in N- tumors and close to 60% in N+ were in high proliferative/low endocrine-sensitive tumors. This difference could explain the beneficial trend for up-front AIs vs. sequential TAM→AIs in the N+, but not in N- subgroups of the TEAM and BIG1-98 trials. It also is consistent with the hypothesis that the highly proliferative low ERS group includes tumors intrinsically resistant to both AIs and TAM. Risk stratification by these two simple metrics may improve the readout of future adjuvant clinical trials with endocrine drugs, and may influence their design. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S1-7.

TQuest, A Web-Based Platform to Enable Precision Medicine by Linking a Tumor’s Genetic Defects to Therapeutic Options

PURPOSE Currently, there are only a few software tools designed to assist physicians to translate molecular abnormalities in the cancer genome into potential treatment options. There is a pressing need to develop software to reliably identify known targeted therapies and experimental treatments for patients on the basis of the results of tumor DNA sequencing. METHODS The TQuest platform includes a data layer, data acquisition layer, search engine, and user interface. It identifies associations between one or more molecular targets and therapeutic options. The data layer consists of indexed interventional clinical trials and an expert-curated database of clinically or experimentally validated associations between mutations and drug response. The data acquisition layer includes an information-harvesting module that keeps an up-to-date full-text index of clinical trials by crawling clinicaltrials.gov and combining it with US Food and Drug Administration label data. The user interface is a Web-based module that allows users to upload genomic variants, tumor morphology, and diagnosis. The search results are qualified and ranked by a relevance score. RESULTS We have manually curated information for 368 distinct genomic variants of 162 gene targets corresponding to 863 drug and target interactions. The platform currently contains a full-text index of approximately 80,000 interventional clinical trials. We applied TQuest to molecular data from 73 metastatic breast cancers. TQuest identified a total of 276 drugs as potential therapeutic options, ranging from one to 103 per patient. CONCLUSION TQuest correctly identified all US Food and Drug Administration-approved drugs and routine indications for all cases and also identified many additional drugs that were used in the context of a given molecular abnormality in various clinical trials. The prototype Web application is available at www.tquest.us , and the source code is open and available on GitHub.

Abstract S1-07: Immune sculpting of the triple negative breast cancer genome

Background: Tumors with infiltrating lymphocytes (TIL) demonstrate a better prognosis particularly in TNBC and HER2 positive breast cancer. Two competing hypothesis predict contrasting relationships of TILs and genomic heterogeneity. On one hand, a strong immune response may lead to “pruning” of intratumor heterogeneity by eliminating immunogenic clones resulting in a near equilibrium, hence better prognosis, while cancers that escape the surveillance may evolve towards greater clonal heterogeneity and genomic complexity. In some cancers, the predicted neoantigens are less frequent than expected by chance also suggesting immune mediated elimination of neoplastic clones (Rooney et al. 2015). Studies also showed an inverse association between immune cell infiltration and intratumor clonal heterogeneity (Morris et al. 2016). On the other hand, cancers with greater genomic instability and mutational burden will have larger clonal heterogeneity and therefore more neoantigens and greater immune infiltration. Indeed, a positive correlation between overall mutation load and immune activity in the tumor microenvironment was observed in pooled data across a broad range of cancer types (Brown et al. 2014, Rooney et al. 2015, Schumacher and Schreiber 2015). Methods: We assessed these two competing hypothesis and examined the relationship between genomic complexity and immune gene expression in different breast cancer subtypes. We used previously described immune metagene expression (DNA microarray n=655) as measures of immune infiltration in the TCGA data set (RNA-Seq n=1215). We compared somatic mutations, mutation count, neoantigen load, clonal heterogeneity metrics and the distribution of mutations in 119 canonical cancer genes and 12 cancer pathways between good and poor prognosis TNBC (n=208) corresponding to high and low immune infiltration. Results:A positive but weak correlation between mutation count and immune metagene expression was observed when all breast cancer subtypes were analyzed together (P=0.08). This was driven by the generally higher mutation count and immune infiltration in TNBC. When TNBC was analyzed separately, good prognosis TNBC with high immune infiltration had lower total mutation count (P=0.021) and predicted neo-antigen count (P=0.035). Clonal heterogeneity was also lower in good prognosis TNBC (P=0.001). There was a strong inverse relationship of dispersion in mutation variant allele frequencies and immune metagene expression. CASP8 was the top enriched mutation in TNBC with high immune infiltration (P=0.007 with no adjustment for multiple testing). Conclusions:High immune infiltration is associated with reduced intratumor heterogeneity in TNBC suggesting immune sculpting of the tumor and a near equilibrium between the cancer and immune surveillance. Surgical resection of the primary tumor may tilt the balance towards the immune system resulting in the better prognosis of high-TIL TNBC. TNBC with low immune infiltration has greater clonal heterogeneity and mutation load and may represent the consequence of escape from immune surveillance. Mutation of CASP8 may be one way to evade tumor cell killing in high-TIL TNBC as previously noted. Citation Format: Karn T, Jiang T, Hatzis C, Sanger N, El-Balat A, Holtrich U, Becker S, Bianchini G, Pusztai L. Immune sculpting of the triple negative breast cancer genome [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S1-07.

Abstract P4-07-01: DNA repair deficiency enhances immune response and correlates with excellent clinical outcome in triple negative breast cancer

Background: Mutations or epigenetic silencing of BRCA1/2 genes result in DNA repair deficiency in a large proportion of TNBC cases. Yet it is unclear whether this deficiency is associated with increased chemosensitivity and improved benefit from standard-of-care chemotherapy. We systematically evaluated BRCA deficiency in TNBC using integrated DNA and RNA sequencing data and its association clinical outcome, exploring the potential role of tumor immune response. Patients and Methods: Whole-exome, DNA methylation, copy number variation, and RNA sequencing data from 102 stage I-III TNBCs were retrieved from The Cancer Genome Atlas (TCGA). Almost all patients received adjuvant taxane-anthracycline-cyclophosphamide (T-AC) chemotherapy and had >30 days of follow-up. The number of predicted neoantigens and an estimate of the level of immune cell activity for 77 of these tumors were previously published. Deleterious germline or somatic BRCA1/2 mutations were identified by majority voting on predictions of 5 variant scoring algorithms. Definition of BRCA1/2 deficiency (BRCA-D) included carrier of deleterious BRCA1/2 mutations or BRACA1/2 normal (BRCA-N) with wild type BRCA1/2 expression less than the maximum observed in mutation carriers. Normalized genomic mutation rate and mutant allele tumor heterogeneity (MATH) were computed as broad measures of genomic instability. Characteristics of BRCA-D vs N tumors were compared using the Wilcoxon rank test. Results: Twenty tumors (19.6%) had mutations in BRCA1, 6 (5.8%) in BRCA2, and 2 (1.9%) in both. Based on the expanded definition, 39 cases (38%) were characterized as BRCA1 deficient, 5 (4.9%) as BRCA2 deficient and 4 (3.9%) as deficient in both. BRCA-D tumors (47%) were associated with a significantly higher mutation rate (P=8x10-4) but had similar clonal heterogeneity (P=0.55) as BRCA-N tumors. BRCA-D tumors had excellent 4-year overall survival (100%) compared to 79.5% (95%CI: 66.6- 94.9) for BRCA-N tumors (log-rank P=0.02). BRCA-D tumors also presented a significantly higher number of predicted neoantigents (P=0.003), which resulted in increased level of immune cell activity. In contrast, low immunogenic TNBC tumors were underrepresented in BRCA-D (p=0.05) and showed potential signs of immunoediting (observed/expected number of predicted neoantigens Conclusions: Deleterious mutations in BRCA1/2 genes occur in 25% of TNBC tumors, but parallel quantification of wild-type BRCA1/2 expression identifies 47% of TNBC samples with double strand break DNA repair deficiency. These BRCA-D TNBC tumors are characterized by a significantly higher mutation rate and present a significantly greater number of neoantigens that result in increased immune cell activity. Our analysis suggested that enhanced immune activation could explain to a large extent the excellent clinical outcome in patients with BRCA-D tumors treated with standard-of-care T-AC chemotherapy. Citation Format: Jiang T, Safonov A, Bianchini G, Shi W, Wali VB, Pusztai L, Hatzis C. DNA repair deficiency enhances immune response and correlates with excellent clinical outcome in triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-07-01.

Abstract P4-04-20: Subtype specific differential expression and immunogenicity of endogenous retrovirus elements in breast cancer

Background: Endogenous retroviruses (ERVs) are germline encoded DNA sequences that entered the human genome millions of years ago. While they are mostly inactivated due to accumulated termination codons and deletions, previous studies have demonstrated overexpression and antibody-targeted immunotherapeutic potential, of ERV-related env proteins in breast cancer. We sought to elucidate subtype specificity, immunogenicity, and correlation with innate immunity related gene signatures of ERVs in breast cancer. Methods: We utilized publically available RNASeq gene expression data of breast cancer samples along and corresponding matched normals from TCGA. The dataset included 191 ER-/HER2- (TN), 197 HER2+, and 627 ER+/HER2- (Luminal) breast cancers. ERV expression was obtained by mapping bowtie2-aligned reads of recently annotated to be transcriptionally active to the RNAseq bam files (Rooney et al 2015, Mayer et al 2011). ERVs preferentially expressed in tumors compared normal tissue were identified as those for which the 5th percentile of ERV expression in the tumors exceeded the 95th percentile of ERV expression in the normal samples. A gene signature involving GZMB, PRF1, CXCL13, IRF1, IKZF1, and HLA-E was used as a measure of immune activity. To assess the immunogenic potential of the tumor-specific ERVs, we compared the expression level of the ERV within the lower and higher immune signature tertiles using the Wilcoxon signed-rank test. To elucidate mechanism of potential immune response, ERVs found to be significantly associated with immune response at a false discovery rate of Results: Out of the 66 original annotated ERVs, 47 were found to be expressed at significantly higher levels in breast cancer compared to normal tissue and 22 were immunogenic. Examples include members of the ERV-K family, as have also been previously detected by flow cytometry and IHC. Subtype-specific immunogenic potential was demonstrated in 4 ERVs in TNBC (ERVK10, ERVK17, ERVFRD.1, ERVPABLB.1) and in 7 ERVs in the luminal subtype (ERV3.1, ERVE.4, ERVFRD.2, ERVK.15, ERVK.19, ERVK.20, ERVK.25, ERVW.3). Twelve of the 22 immunogenic ERVs were significantly correlated with expression of all ten TLR evaluated, while four ERVs showed more specific correlation patterns with TLRs. High ERV3.1 expression was associated with high TLR3, TLR8, TLR9 that specifically target double stranded or single stranded RNA, suggesting a potential mechanism for mediation of ERV related immune response. Conclusion: Our results suggest breast cancer subtype specific ERV dysregulation and immunogenicity. The potentially immunogenic ERVs were generally not self-correlated or located in the same amplicon as HLA genes, suggesting an independent immune response pathway. Furthermore, ERV expression correlates with specific endosomal nucleic-acid recognizing toll-like receptors, which may prompt further investigation into subtype-specific TLR-targeted therapy. Citation Format: Safonov A, Bianchini G, Jiang T, Pusztai L, Hatzis C. Subtype specific differential expression and immunogenicity of endogenous retrovirus elements in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-20.

Abstract P2-11-04: Expression of immune checkpoint related genes are prognostic in untreated breast cancer

Background The prognostic and predictive relevance of different components of the adaptive immune system in HER2-positive, TN and high proliferation ER+/HER2- subtypes have been shown. In this scenario, the remarkable therapeutic efficacy of new immunomodulatory drugs targeting the immune checkpoints (i.e. CTLA4) provides attractive opportunities. We aimed to investigate the prognostic value of genes related to CD28 and CTLA-4 signaling pathways in early breast cancer patients. Methods We assessed the affymetrix gene expression data of 789 untreated, node-negative, early breast cancer patients. Expression of CTLA4, CD28 and the two corresponding natural ligands CD80 (B7-1) and CD86 (B7-2) were evaluated. The sum of these two ligands was combined as a unique marker (CDligs). The prognostic value of the three biomarkers and their interaction were assessed independently in each molecular subtype (ER-/HER2-, HER2+ and high proliferation ER+/HER2- [ER+/HER2- HP]). Multivariate analysis was adjusted for PGR, proliferation and other immune related genes (Tcell, IGG, MHC II, MHC I, Interferon and STAT1) (Gianni L, SABCS 2012). Distant event free survival was the outcome of interest. Results A significant moderately positive correlation was observed between each biomarker. Expression of CTLA4 and CDligs was higher in ER-/HER2- and HER2+ compared to luminal subtypes. High expression of CTLA4 and CD28 were associated with lower risk of relapse in HER2+ (p = 0.008 and p = 0.03, respectively) and ER+/HER2-HP (p = 0.03 and p = 0.008, respectively). High expression of CDligs was also associated with lower risk of distant event in HER2+ group only (p = 0.01). No interactions were observed between CD28 and CTLA4 in any subtype. In both TN and ER+/HER2- HP subtypes, a significant interaction between CDligs and CTLA4 (interaction term p = 0.009 and p = 0.025, respectively), CDligs and CD28 (interaction term p = 0.028 and p = 0.008, respectively) and CDligs and Tcell metagene (interaction term p = 0.006 and p = 0.019, respectively) was observed. For instance, in high-CTLA4 group (above median cut point), higher expression of CDligs was associated with significantly lower risk of distant relapse in TN [HR 0.41 (0.21-0.82); p = 0.012], HER2+ [HR 0.25 (0.08-0.72); p = 0.011) and ER+/HER2- HP [HR 0.40 (0.29-0.85); p = 0.01] subtypes. Instead, in the low-CTLA4 group, CDligs was not associated with distant relapse in any subtype [TN, HR 1.80 (0.28-3.96) p = 0.141; HER2+, HR 0.69 (0.24-1.99) p = 0.495; ER+/HER2- HP, HR 1.28 (0.70-2.35) p = 0.425]. These interactions retain statistical significance in multivariate analysis including PGR, proliferation scores and other immune metagenes. Conclusions Genes related to the CD28/CTLA4 immune checkpoint are associated with risk of distant relapse independently of other biomarkers and immune related metagenes. A significant interaction between expression of ligands (CD80/CD86) and the two corresponding receptors CD28 and CTLA4 was found in TN and ER+/HER2- HP subtypes. These data confirm the complex interactions involved in the adaptive immune system regulation which may not be fully appreciated without consideration of biomarker interactions. They also provide additional evidence for exploring the use of immunomodulatory drugs in breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-04.

P4-03-04: Identification of Molecular Targets for Cancer-Initiating Cells Using a Triple-Negative Breast Cancer Mouse Model.

Background Triple-negative breast cancer (TNBC), which is negative for estrogen receptor, progesterone receptor, and HER2, cannot be targeted with hormonal or anti-HER2 agents and frequently relapses. Thus, innovative molecular targeted therapies for TNBC are needed. Cancer-initiating cells (CICs) play a crucial role in tumor recurrences. TNBC has a much higher proportion of CICs compared with the other breast cancer subtypes, reflecting TNBC9s clinical aggressiveness. However, qualitative or molecular traits of TNBC CICs have not been fully elucidated. We established a TNBC mouse model that successfully recapitulated human-like TNBC in vivo (SABCS 2010 abstract P1-03-02). We here report the phenotypes and molecular features of CICs in our TNBC mouse model. Methods : We created this animal model by transferring HRAS(G12V) into Ink4a/Arf -knockout (KO) mouse mammary epithelial cells (MECs) in vitro and inducing tumors in mammary fat pads of recipient mice. The induced TNBCs were digested, and single suspended cells were reacted with antibodies to candidate CIC markers CD29, CD24, CD44, Sca1, CD61, and CD49f. Expression patterns of the tumor cells were analyzed by FACS. Tumor cells were sorted according to their expression levels and inoculated into recipient mouse mammary fat pads to determine tumor-initiating activity. To analyze the cell-cycle status of tumor cells, we stained them with Hoechst 33342 (for DNA staining) and pyronin Y (for RNA staining) and analyzed them by FACS. The CICs’ genome-wide molecular profiles were determined by mouse microarray expression analysis. Results : In FACS and the in vivo tumor-initiating assay, the CD49f expression level was the most significant for defining tumor-initiating activity. That is, CD49f − tumor cells had much higher tumor-initiating activity than did CD49f + cells at 100-cell inoculations (CD49f − , 5 tumors/8 mice; CD49f + , 0/8). We hypothesized that tumor initiation by CD49f − cells depends on their quiescent profile, which is a trait of CICs. To test this hypothesis, we further fractionated CD49f − cells into CD49f − quiescent (CD49f − q) cells and CD49f − dividing cells and inoculated these fractions into recipient mice. CD49f − q cells had higher tumor-initiating activity than that of CD49f − dividing cells. We thus determined that CD49f − q cells were CICs in our model. Genome-wide expression patterns for CD49f − q cells and CD49f + cells were compared by paired t test, and over- and underexpressed genes in CD49f − q cells were functionally annotated by Ingenuity pathway analysis. The top-ranked bioprocess of overexpressed genes in CD49f − q cells was hepatic satellite cell activation (HSCA), followed by pantothenale and Coenzyme A biosynthesis, and HER2 signaling in breast cancer. HSCA is known to result in liver fibrosis due to collagen deposits in response to liver damage. In CD49f − q cells in our model, collagen family proteins and the upstream endothelin-A receptor were overexpressed and significantly correlated with HSCA. Conclusion: We identified CICs in a mouse TNBC model and revealed their molecular profile. We plan to analyze expression of HSCA-associated proteins in human TNBC. Our first step is to determine whether endothelin-A receptor is a therapeutic target in human TNBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-03-04.