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TMB, TFB and CRPC scores across individual samples and disease stages and crosscorrelation analyses

Prostate cancer is the second leading cause of cancer-related death in men. Despite having a relatively lower tumor mutational burden than most tumor types, multiple gene fusions such as TMPRSS2:ERG have been characterized and linked to more aggressive disease. Individual tumor samples have been found to contain multiple fusions, and it remains unknown whether these fusions increase tumor immunogenicity. Here, we investigated the role of fusion burden on the prevalence and expression of key molecular and immune effectors in prostate cancer tissue specimens that represented the different stages of disease progression and androgen sensitivity, including hormone-sensitive and castration-resistant prostate cancer. We found that tumor fusion burden was inversely correlated with tumor mutational burden and not associated with disease stage. High fusion burden correlated with high immune infiltration, PD-L1 expression on immune cells, and immune signatures, representing activation of T cells and M1 macrophages. High fusion burden inversely correlated with immune-suppressive signatures. Our findings suggest that high tumor fusion burden may be a more appropriate biomarker than tumor mutational burden in prostate cancer, as it more closely associates with immunogenicity, and suggests that tumors with high fusion burden could be potential candidates for immunotherapeutic agents.

Supplementary Figure S7: MCF-7 DOX-inducible ER-alpha overexpressing cells without DOX induction respond similarly to taselisib and/or fulvestrant as MCF-7 parental cells.

The identification of early breast cancer patients who may benefit from adjuvant chemotherapy has evolved to include assessment of clinicopathologic features such as tumor size and nodal status, as well as several gene-expression profiles for ER-positive, HER2-negative cancers. However, these tools do not reliably identify patients at the greatest risk of recurrence. The mutation and copy-number landscape of triple-negative breast cancer (TNBC) subtypes defined by gene expression is also largely unknown, and elucidation of this landscape may shed light on novel therapeutic opportunities. The USO01062 phase III clinical trial of standard chemotherapy (with or without capecitabine) enrolled a cohort of putatively high-risk patients based on clinical features, yet only observed a 5-year disease-free survival event rate of 11.6%. In order to uncover genomic aberrations associated with recurrence, a targeted next-generation sequencing panel was used to compare tumor specimens from patients who had a recurrence event with a matched set who did not. The somatic mutation and copy-number alteration landscapes of high-risk early breast cancer patients were characterized and alterations associated with relapse were identified. Tumor mutational burden was evaluated but was not prognostic in this study, nor did it correlate with PDL1 or CD8 gene expression. However, TNBC subtypes had substantial genomic heterogeneity with a distinct pattern of genomic alterations and putative underlying driver mutations.Implications:The present study uncovers a compendium of genomic alterations with utility to more precisely identify high-risk patients for adjuvant trials of novel therapeutic agents.

Supplemental Methods describing NGS and gene expression studies.

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PDF file - 22K, Changes in shed Met do not correlate with changes in cHGF over the duration of the first cycle of onartuzumab administration in the Phase 1A study.

FoundationOne sequencing data for n=399 breast cancer population. Each sheet contains a distinct type of alteration. The clinical covariates used in our analysis are included.

PDF file - 24K, cHGF levels are stable over time and are not variable between screening (any time during visit -24 hours) and predose (Day 0).

The objective of this study was to evaluate circulating hepatocyte growth factor (cHGF) as a pharmacodynamic biomarker of Met inhibition for onartuzumab (MetMAb, OA5D5v2) in a phase I trial in patients with advanced cancers and a phase II trial in non–small cell lung cancer (NSCLC). The phase I study was a dose escalation trial with onartuzumab administered i.v. once every three weeks. The phase II study was a randomized two-arm trial in which onartuzumab or placebo was administered in combination with erlotinib in 137 patients with second and third line (2/3L) NSCLC. cHGF levels were evaluated by ELISA at multiple time points over the treatment period. Onartuzumab administration resulted in an acute and sustained rise in cHGF in both the phase I and phase II studies. Elevation in cHGF was independent of dose or drug exposure and was restricted to onartuzumab treatment. Neither higher baseline nor elevated change in cHGF levels upon treatment could simply be attributed to tumor burden or number of liver metastasis. We have shown that elevated cHGF can consistently and reproducibly be measured as a pharmacodynamic biomarker of onartuzumab activity. The elevation in cHGF is independent of tumor type, dose administered, or dose duration. Although these studies were not powered to directly address the contribution of cHGF as a predictive, on-treatment, circulating biomarker, these data suggest that measurement of cHGF in future expanded studies is warranted. Mol Cancer Ther; 12(6); 1122–30. ©2013 AACR.

Supplementary Table S1 from Genetic Alterations and Oncogenic Pathways Associated with Breast Cancer Subtypes

Enriched Pathway Analysis.

Supplemental figures 1,2,4-8 as supporting figures to the manuscript.

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutation of the PIK3CA gene, loss or mutation of phosphatase and tensin homolog (PTEN), and deregulation of mammalian target of rapamycin (mTOR) complexes. The dysregulation of this pathway has been implicated in tumor initiation, cell growth and survival, invasion and angiogenesis, thus, PI3K and mTOR are promising therapeutic targets for cancer. We discovered GDC-0980, a selective, potent, orally bioavailable inhibitor of Class I PI3 kinase and mTOR kinase (TORC1/2) with excellent pharmacokinetic and pharmaceutical properties. GDC-0980 potently inhibits signal transduction downstream of both PI3K and mTOR, as measured by pharmacodynamic (PD) biomarkers, thereby acting upon two key pathway nodes to produce the strongest attainable inhibition of signaling in the pathway. Correspondingly, GDC-0980 was potent across a broad panel of cancer cell lines, with the greatest potency in breast, prostate, and lung cancers and less activity in melanoma and pancreatic cancers, consistent with KRAS and BRAF acting as resistance markers. Treatment of cancer cell lines with GDC-0980 resulted in G1 cell-cycle arrest, and in contrast to mTOR inhibitors, GDC-0980 induced apoptosis in certain cancer cell lines, including those with direct pathway activation via PI3K and PTEN. Low doses of GDC-0980 potently inhibited tumor growth in xenograft models including those with activated PI3K, loss of LKB1 or PTEN, and elicited an exposure-related decrease in PD biomarkers. These preclinical data show that GDC-0980 is a potent and effective dual PI3K/mTOR inhibitor with promise for the clinic. Mol Cancer Ther; 10(12); 2426–36. ©2011 AACR.

Supplementary Figures S1-S4 from Genetic Alterations and Oncogenic Pathways Associated with Breast Cancer Subtypes

The RAS/RAF/MEK pathway is activated in more than 30% of human cancers, most commonly via mutation in the K-ras oncogene and also via mutations in BRAF. Several allosteric mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitors, aimed at treating tumors with RAS/RAF pathway alterations, are in clinical development. However, acquired resistance to these inhibitors has been documented both in preclinical and clinical samples. To identify strategies to overcome this resistance, we have derived three independent MEK inhibitor–resistant cell lines. Resistance to allosteric MEK inhibitors in these cell lines was consistently linked to acquired mutations in the allosteric binding pocket of MEK. In one cell line, concurrent amplification of mutant K-ras was observed in conjunction with MEK allosteric pocket mutations. Clonal analysis showed that both resistance mechanisms occur in the same cell and contribute to enhanced resistance. Importantly, in all cases the MEK-resistant cell lines retained their addiction to the mitogen-activated protein kinase (MAPK) pathway, as evidenced by their sensitivity to a selective inhibitor of the ERK1/2 kinases. These data suggest that tumors with acquired MEK inhibitor resistance remain dependent on the MAPK pathway and are therefore sensitive to inhibitors that act downstream of the mutated MEK target. Importantly, we show that dual inhibition of MEK and ERK by small molecule inhibitors was synergistic and acted to both inhibit the emergence of resistance, as well as to overcome acquired resistance to MEK inhibitors. Therefore, our data provide a rationale for cotargeting multiple nodes within the MAPK signaling cascade in K-ras mutant tumors to maximize therapeutic benefit for patients. Mol Cancer Ther; 11(5); 1143–54. ©2012 AACR.

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Figure S1: Forest plot of hazard ratios by subgroup in the ITT population (planned analysis) for (A) DFS and (B) OS

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Figure S2: Distribution of central Ki-67 scores in (A) ER-positive/HER2-negative breast cancer (n=824) and (B) TNBC (n=454)

cfDNA tumor fraction dynamics.

Purpose: Elevated levels or increases in circulating tumor cells (CTC) portend poor prognosis in patients with epithelial cancers. Less is known about CTCs as surrogate endpoints or their use for predictive biomarker evaluation. This study investigated the utility of CTC enumeration and characterization using the CellSearch platform, as well as mutation detection in circulating tumor DNA (ctDNA), in patients with advanced non–small cell lung cancer (NSCLC).Experimental Design: Forty-one patients were enrolled in a single-arm phase II clinical trial of erlotinib and pertuzumab. Peripheral blood was analyzed for CTC enumeration, EGFR expression in CTCs, and detection of oncogenic mutations in CTCs and ctDNA. Changes in CTC levels were correlated with 2[18F]fluoro-2-deoxy-d-glucose–positron emission tomographic (FDG-PET) and computed tomographic (CT) imaging and survival endpoints.Results: CTCs were detected (≥1 CTC) at baseline in 78% of patients. Greater sensitivity for mutation detection was observed in ctDNA than in CTCs and detected mutations were strongly concordant with mutation status in matched tumor. Higher baseline CTC counts were associated with response to treatment by Response Evaluation Criteria in Solid Tumors (RECIST, P = 0.009) and decreased CTC counts upon treatment were associated with FDG-PET and RECIST response (P = 0.014 and P = 0.019) and longer progression-free survival (P = 0.050).Conclusion: These data provide evidence of a correlation between decreases in CTC counts and radiographic response by either FDG-PET or RECIST in patients with advanced NSCLC. These findings require prospective validation but suggest a potential role for using CTC decreases as an early indication of response to therapy and ctDNA for real-time assessment of mutation status from blood. Clin Cancer Res; 18(8); 2391–401. ©2012 AACR.

Purpose: Up to one third of ovarian cancer patients are intrinsically resistant to platinum-based treatment. However, predictive and therapeutic strategies are lacking due to a poor understanding of the underlying molecular mechanisms. This study aimed to identify key molecular characteristics that are associated with primary chemoresistance in epithelial ovarian cancers.Experimental Design: Gene expression profiling was performed on a discovery set of 85 ovarian tumors with clinically well-defined response to chemotherapies as well as on an independent validation dataset containing 138 ovarian patients from the chemotreatment arm of the ICON7 trial.Results: We identified a distinct “reactive stroma” gene signature that is specifically associated with primary chemoresistant tumors and was further upregulated in posttreatment recurrent tumors. Immunohistochemistry (IHC) and RNA in situ hybridization (RNA ISH) analyses on three of the highest-ranked signature genes (POSTN, LOX, and FAP) confirmed that modulation of the reactive stroma signature genes within the peritumoral stromal compartments was specifically associated with the clinical chemoresistance. Consistent with these findings, chemosensitive ovarian cells grown in the presence of recombinant POSTN promoted resistance to carboplatin and paclitaxel treatment in vitro. Finally, we validated the reactive stroma signature in an independent dataset and demonstrated that a high POSTN expression level predicts shorter progression-free survival following first-line chemotherapy.Conclusions: Our findings highlight the important interplay between cancer and the tumor microenvironment in ovarian cancer biology and treatment. The identified reactive stromal components in this study provide a molecular basis to the further development of novel diagnostic and therapeutic strategies for overcoming chemoresistance in ovarian cancer. Clin Cancer Res; 21(13); 2941–51. ©2015 AACR.

Supplementary Fig. S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Purpose: Class 1 phosphatidylinositol 3-kinase (PI3K) plays a major role in cell proliferation and survival in a wide variety of human cancers. Here, we investigated biomarker strategies for PI3K pathway inhibitors in non–small-cell lung cancer (NSCLC).Experimental Design: Molecular profiling for candidate PI3K predictive biomarkers was conducted on a collection of NSCLC tumor samples. Assays included comparative genomic hybridization, reverse-transcription polymerase chain reaction gene expression, mutation detection for PIK3CA and other oncogenes, PTEN immunohistochemistry, and FISH for PIK3CA copy number. In addition, a panel of NSCLC cell lines characterized for alterations in the PI3K pathway was screened with PI3K and dual PI3K/mTOR inhibitors to assess the preclinical predictive value of candidate biomarkers.Results: PIK3CA amplification was detected in 37% of squamous tumors and 5% of adenocarcinomas, whereas PIK3CA mutations were found in 9% of squamous and 0% of adenocarcinomas. Total loss of PTEN immunostaining was found in 21% of squamous tumors and 4% of adenocarcinomas. Cell lines harboring pathway alterations (receptor tyrosine kinase activation, PI3K mutation or amplification, and PTEN loss) were exquisitely sensitive to the PI3K inhibitor GDC-0941. A dual PI3K/mTOR inhibitor had broader activity across the cell line panel and in tumor xenografts. The combination of GDC-0941 with paclitaxel, erlotinib, or a mitogen-activated protein–extracellular signal-regulated kinase inhibitor had greater effects on cell viability than PI3K inhibition alone.Conclusions: Candidate biomarkers for PI3K inhibitors have predictive value in preclinical models and show histology-specific alterations in primary tumors, suggesting that distinct biomarker strategies may be required in squamous compared with nonsquamous NSCLC patient populations. Clin Cancer Res; 18(24); 6771–83. ©2012 AACR.

PDF file 155K, Principal component analysis (PCA) of the ten biomarkers provides a biomarker profile for each cell line

XLSX file - 602KB, Supplementary Table 5. Cancer cell lines analyzed in this study. Supplementary Table 6. Tumor tissues analyzed in this study. Supplementary Table 7. List of 340 well characterized hotspot mutations interrogated by the amplicon libraries in this study. Variants also assayed by asPCR are indicated. Supplementary Table 8. Variants detected in 66 cancer cell lines, after applying post-processing filters. Note that indels and predicted deleterious SNVs are reported for all interrogated genes and transcripts. Supplementary Table 9. Variants detected in 73 FFPE endometrial tumor tissues, after applying post-processing filters. Note that indels and predicted deleterious SNVs are reported for all interrogated genes and transcripts. Supplementary Table 10. For cell line data, concordance with COSMIC somatic mutation annotation.

Supplementary Table 1. Additional pharmacokinetic parameters of pictilisib

Purpose: To investigate the clinical relevance of PTEN in HER2-amplified and HER2-nonamplified disease.Experimental Design: We assessed PTEN status in two large adjuvant breast cancer trials (BCIRG-006 and BCIRG-005) using a PTEN immunohistochemical (IHC) assay that was previously validated in a panel of 33 breast cancer cell lines and prostate cancer tissues with known PTEN gene deletion.Results: In the HER2-positive patient population, absence of tumor cell PTEN staining occurred at a rate of 5.4% and was independent of ER/PR status. In contrast, 15.9% of HER2-negative patients exhibited absence of PTEN staining with the highest frequency seen in triple-negative breast cancer (TNBC) subgroup versus ER/PR-positive patients (35.1% vs. 10.9%). Complete absence of PTEN staining in tumor cells was associated with poor clinical outcome in HER2-positive disease. Those patients whose cancers demonstrated absent PTEN staining had a significant decrease in disease-free survival (DFS) and overall survival (OS) compared with patients with tumors exhibiting any PTEN staining patterns (low, moderate, or high). Trastuzumab appeared to provide clinical benefit even for patients lacking PTEN staining. In the HER2-negative population, there were no statistically significant differences in clinical outcome based on PTEN status.Conclusions: This study is the largest to date examining PTEN status in breast cancer and the data suggest that the rate and significance of PTEN status differ between HER2-positive and HER2-negative disease. Furthermore, the data clearly suggest that HER2-positive patients with PTEN loss still benefit from trastuzumab. Clin Cancer Res; 21(9); 2065–74. ©2015 AACR.

Purpose:We developed a method to monitor copy number variations (CNV) in plasma cell-free DNA (cfDNA) from patients with metastatic squamous non–small cell lung cancer (NSCLC). We aimed to explore the association between tumor-derived cfDNA and clinical outcomes, and sought CNVs that may suggest potential resistance mechanisms.Experimental Design:Sensitivity and specificity of low-pass whole-genome sequencing (LP-WGS) were first determined using cell line DNA and cfDNA. LP-WGS was performed on baseline and longitudinal cfDNA of 152 patients with squamous NSCLC treated with chemotherapy, or in combination with pictilisib, a pan-PI3K inhibitor. cfDNA tumor fraction and detected CNVs were analyzed in association with clinical outcomes.Results:LP-WGS successfully detected CNVs in cfDNA with tumor fraction ≥10%, which represented approximately 30% of the first-line NSCLC patients in this study. The most frequent CNVs were gains in chromosome 3q, which harbors the PIK3CA and SOX2 oncogenes. The CNV landscape in cfDNA with a high tumor fraction generally matched that of corresponding tumor tissue. Tumor fraction in cfDNA was dynamic during treatment, and increases in tumor fraction and corresponding CNVs could be detected before radiographic progression in 7 of 12 patients. Recurrent CNVs, such as MYC amplification, were enriched in cfDNA from posttreatment samples compared with the baseline, suggesting a potential resistance mechanism to pictilisib.Conclusions:LP-WGS offers an unbiased and high-throughput way to investigate CNVs and tumor fraction in cfDNA of patients with cancer. It may also be valuable for monitoring treatment response, detecting disease progression early, and identifying emergent clones associated with therapeutic resistance.

PDF file 285K, Biomarker validation by Western blot

Supplementary figure 3 shows H3K27 acetylation ChIPseq QC analyses.

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PDF file 252K, In vivo pharmacodynamic IHC-based biomarker dose-response quantifications

Commentary on this Article from Molecular predictors of response to a humanized anti–insulin-like growth factor-I receptor monoclonal antibody in breast and colorectal cancer

Regulatory T cells (Treg) are immunosuppressive and negatively impact response to cancer immunotherapies. CREB-binding protein (CBP) and p300 are closely related acetyltransferases and transcriptional coactivators. Here, we evaluate the mechanisms by which CBP/p300 regulate Treg differentiation and the consequences of CBP/p300 loss-of-function mutations in follicular lymphoma. Transcriptional and epigenetic profiling identified a cascade of transcription factors essential for Treg differentiation. Mass spectrometry analysis showed that CBP/p300 acetylates prostacyclin synthase, which regulates Treg differentiation by altering proinflammatory cytokine secretion by T and B cells. Reduced Treg presence in tissues harboring CBP/p300 loss-of-function mutations was observed in follicular lymphoma. Our findings provide novel insights into the regulation of Treg differentiation by CBP/p300, with potential clinical implications on alteration of the immune landscape.Significance:This study provides insights into the dynamic role of CBP/p300 in the differentiation of Tregs, with potential clinical implications in the alteration of the immune landscape in follicular lymphoma.

The insulin-like growth factor-I receptor (IGF-IR) pathway is required for the maintenance of the transformed phenotype in neoplastic cells and hence has been the subject of intensive drug discovery efforts. A key aspect of successful clinical development of targeted therapies directed against IGF-IR will be identification of responsive patient populations. Toward that end, we have endeavored to identify predictive biomarkers of response to an anti-IGF-IR-targeting monoclonal antibody in preclinical models of breast and colorectal cancer. We find that levels of the IGF-IR itself may have predictive value in these tumor types and identify other gene expression predictors of in vitro response. Studies in breast cancer models suggest that IGF-IR expression is both correlated and functionally linked with estrogen receptor signaling and provide a basis for both patient stratification and rational combination therapy with antiestrogen-targeting agents. In addition, we find that levels of other components of the signaling pathway such as the adaptor proteins IRS1 and IRS2, as well as the ligand IGF-II, have predictive value and report on the development of a pathway-focused panel of diagnostic biomarkers that could be used to test these hypotheses during clinical development of IGF-IR-targeting therapies. [Mol Cancer Ther 2009;8(8):2110–21]

Supplementary Table S3 from Predictive Biomarkers of Sensitivity to the Phosphatidylinositol 3′ Kinase Inhibitor GDC-0941 in Breast Cancer Preclinical Models

PDF file 64K, Unsupervised clustering of the core biomarker modulation in cell lines, xenograft tumors, and in tumors from three patients in the 100mg cohort

Supplementary Table S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Although breast cancer molecular subtypes have been extensively defined by means of gene expression profiling over the past decade, little is known, at the proteomic level, as to how signaling pathways are differentially activated and serve to control proliferation in different breast cancer subtypes. We used reverse-phase protein arrays to examine phosphorylation status of 100 proteins in a panel of 30 breast cancer cell lines and showed distinct pathway activation differences between different subtypes that are not obvious from previous gene expression studies. We also show that basal levels of phosphorylation of key signaling nodes may have diagnostic utility in predicting response to selective inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. Finally, we show that reverse-phase protein arrays allow the parallel analysis of multiple pharmacodynamic biomarkers of response to targeted kinase inhibitors and that inhibitors of epidermal growth factor receptor and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase result in compensatory up-regulation of the phosphatidylinositol 3-kinase/Akt signaling pathway. [Mol Cancer Ther 2008;7(12):3695–706]

Supplementary Figure 2. Waterfall plot of changes in18F-FDG-PET SUVmax grouped according to pictilisib AUC as assessed by an independent review facility.

Supplementary Table from Proteomic analysis of breast cancer molecular subtypes and biomarkers of response to targeted kinase inhibitors using reverse-phase protein microarrays

Figure S3: Forest plots for DFS comparing patients with central Ki-67 high versus low based on quartile cutoffs (exploratory analysis) in (A) ER-positive/HER2-negative breast cancer (n=824) and (B) TNBC (n=454)

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Supplementary Figure 4. Association between severity of treatment-related rash and steady-state exposure (day 15 AUC0-24) to pictilisib. (Gr=Grade).