Joanne Xiu

Mutational burden, immune checkpoint expression, and mismatch repair in glioma: Implications for immune checkpoint immunotherapy

Background Despite a multiplicity of clinical trials testing immune checkpoint inhibitors, the frequency of expression of potential predictive biomarkers is unknown in glioma. Methods In this study, we profiled the frequency of shared biomarker phenotypes. To clarify the relationships among tumor mutational load (TML), mismatch repair (MMR), and immune checkpoint expression, we profiled patients with glioma (n = 327), including glioblastoma (GBM) (n = 198), whose samples had been submitted for analysis from 2009 to 2016. The calculation algorithm for TML included nonsynonymous mutation counts per tumor, with germline mutations filtered out. Immunohistochemical analysis and next-generation sequencing were used to determine tumor-infiltrating lymphocyte expression positive for programmed cell death protein 1 (PD-1), PD ligand 1 (PD-L1) expression on tumor cells, MMR (MLH1, MSH2, MSH6, and PMS2) protein expression and mutations, and DNA polymerase epsilon (POLE) mutations. Results High TML was only found in 3.5% of GBM patients (7 of 198) and was associated with the absence of protein expression of mutL homolog 1 (MLH1) (P = .0345), mutS homolog 2 (MSH2) (P = .0099), MSH6 (P = .0022), and postmeiotic segregation increased 2 (PMS2) (P = .0345) and the presence of DNA MMR mutations. High and moderate TML GBMs did not have an enriched influx of CD8+ T cells, PD-1+ T cells, or tumor-expressed PD-L1. IDH1 mutant gliomas were not enriched for high TML, PD-1+ T cells, or PD-L1 expression. Conclusions To clarify the relationships among TML, MMR, and immune checkpoint expression, we profiled the frequency of shared biomarker phenotypes. On the basis of a variety of potential biomarkers of response to immune checkpoints, only small subsets of glioma patients are likely to benefit from monotherapy immune checkpoint inhibition.

Immune checkpoint blockade as a potential therapeutic target: surveying CNS malignancies

BACKGROUND Expression of programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) across glioma grades is undocumented, and their interactions with commonly expressed genetic and epigenetic alterations are undefined but nonetheless highly relevant to combinatorial treatments. METHODS Patients with CNS malignancies were profiled by Caris Life Sciences from 2009 to 2016. Immunohistochemistry findings for PD-1 on tumor-infiltrating lymphocytes (TIL) and PD-L1 on tumor cells were available for 347 cases. Next-generation sequencing, pyrosequencing, immunohistochemistry, fragment analysis, and fluorescence in situ hybridization were used to determine isocitrate dehydrogenase 1 (IDH1), phosphatase and tensin homolog (PTEN), and tumor protein 53 mutational status, O(6)-DNA methylguanine-methyltransferase promoter methylation (MGMT-Me) status, PTEN expression, plus epidermal growth factor receptor variant III and 1p/19q codeletion status. RESULTS PD-1+ TIL expression and grade IV gliomas were significantly positively correlated (odds ratio [OR]: 6.363; 95% CI: 1.263, 96.236)-especially in gliosarcomas compared with glioblastoma multiforme (P = .014). PD-L1 expression was significantly correlated with tumor grade with all PD-L1+ cases (n = 21) being associated with grade IV gliomas. PD-1+ TIL expression and PD-L1 expression were significantly correlated (OR: 5.209; 95% CI: 1.555, 20.144). Mutations of PTEN, tumor protein 53, BRAF, IDH1, and epidermal growth factor receptor or MGMT-Me did not associate with increased intratumoral expression of either PD-1+ TIL or PD-L1 in glioblastoma multiforme even before false discovery rate correction for multiple comparison. CONCLUSIONS Targeting immune checkpoints in combination with other therapeutics based on positive biomarker selection will require screening of large patient cohorts.

Comparative molecular analyses of left-sided colon, right-sided colon, and rectal cancers

Tumor sidedness has emerged as an important prognostic and predictive factor in the treatment of colorectal cancer. Recent studies demonstrate that patients with advanced right-sided colon cancers have a worse prognosis than those with left-sided colon or rectal cancers, and these patient subgroups respond differently to biological therapies. Historically, management of patients with metastatic colon and rectal cancers has been similar, and colon and rectal cancer patients have been grouped together in large clinical trials. Clearly, the differences in molecular biology among right-sided colon, left-sided colon, and rectal cancers should be further studied in order to account for disparities in clinical outcomes. We profiled 10,570 colorectal tumors (of which 2,413 were identified as arising from the left colon, right colon, or rectum) using next-generation sequencing, immunohistochemistry, chromogenic in-situ hybridization, and fragment analysis (Caris Life Sciences, Phoenix, AZ). Right-sided colon cancers had higher rates of microsatellite instability, more frequent aberrant activation of the EGFR pathway including higher BRAF and PIK3CA mutation rates, and increased mutational burden compared to left-sided colon and rectal cancers. Rectal cancers had higher rates of TOPO1 expression and Her2/neu amplification compared to both left- and right-sided colon cancers. Molecular variations among right-sided colon, left-sided colon, and rectal tumors may contribute to differences in clinical behavior. The site of tumor origin (left colon, right colon, or rectum) should certainly be considered when selecting treatment regimens and stratifying patients for future clinical trials.

Molecular profiling of 6,892 colorectal cancer samples suggests different possible treatment options specific to metastatic sites

Metastatic colorectal cancer (mCRC) carries a poor prognosis with an overall 5-year survival of 13.1%. Therapies guided by tumor profiling have suggested benefit in advanced cancer. We used a multiplatform molecular profiling (MP) approach to identify key molecular changes that may provide therapeutic options not typically considered in mCRC. We evaluated 6892 mCRC referred to Caris Life Sciences by MP including sequencing (Sanger/NGS), immunohistochemistry (IHC) and in-situ hybridization (ISH). mCRC metastases to liver, brain, ovary or lung (n = 1507) showed differential expression of markers including high protein expression of TOPO1 (52%) and/or low RRM1 (57%), TS (71%) and MGMT (39%), suggesting possible benefit from irinotecan, gemcitabine, 5FU/capecitabine and temozolomide, respectively. Lung metastases harbored a higher Her2 protein expression than the primary colon tumors (4% vs. 1.8%, p = 0.028). Brain and lung metastases had higher KRAS mutations than other sites (65% vs 59% vs 47%, respectively, p = 0.07, <0.01), suggesting poor response to anti-EGFR therapies. BRAF-mutated CRC (n = 455) showed coincident high protein expression of RRM1 (56%), TS (53%) and low PDGFR (22%) as compared with BRAF wild-type tumors. KRAS-mutated mCRC had higher protein expression of c-MET (47% vs. 36%) and lower MGMT (56% vs. 63%), suggesting consideration of c-MET inhibitors and temozolomide. KRAS-mutated CRC had high TUBB3 (42% vs. 33%) and low Her2 by IHC (0.5%) and HER2 by FISH (3%, p <0.05). CRC primaries had a lower incidence of PIK3CA and BRAF mutations in rectal cancer versus colon cancer (10% and 3.3%, respectively). MP of 6892 CRCs identified significant differences between primary and metastatic sites and among BRAF/KRAS sub-types. Our findings are hypothesis generating and need to be examined in prospective studies. Specific therapies may be considered for different actionable targets in mCRC as revealed by MP.

Multiplatform molecular profiling identifies potentially targetable biomarkers in malignant phyllodes tumors of the breast.

Malignant phyllodes tumor is a rare breast malignancy with sarcomatous overgrowth and with limited effective treatment options for recurrent and metastatic cases. Recent clinical trials indicated a potential for anti-angiogenic, anti-EGFR and immunotherapeutic approaches for patients with sarcomas, which led us to investigate these and other targetable pathways in malignant phyllodes tumor of the breast. Thirty-six malignant phyllodes tumors (including 8 metastatic tumors with two cases having matched primary and metastatic tumors) were profiled using gene sequencing, gene copy number analysis, whole genome expression, and protein expression. Whole genome expression analysis demonstrated consistent over-expression of genes involved in angiogenesis including VEGFA, Angiopoietin-2, VCAM1, PDGFRA, and PTTG1. EGFR protein overexpression was observed in 26/27 (96%) of cases with amplification of the EGFR gene in 8/24 (33%) cases. Two EGFR mutations were identified including EGFRvIII and a presumed pathogenic V774M mutation, respectively. The most common pathogenic mutations included TP53 (50%) and PIK3CA (15%). Cases with matched primary and metastatic tumors harbored identical mutations in both sites (PIK3CA/KRAS and RB1 gene mutations, respectively). Tumor expression of PD-L1 immunoregulatory protein was observed in 3/22 (14%) of cases. Overexpression of molecular biomarkers of increased angiogenesis, EGFR and immune checkpoints provides novel targeted therapy options in malignant phyllodes tumors of the breast.

Comprehensive multiplatform biomarker analysis of 350 hepatocellular carcinomas identifies potential novel therapeutic options

Effective therapies for hepatocellular carcinoma (HCC) are limited. Molecular profiling of HCC was performed to identify novel therapeutic targets.

Distinct molecular landscapes between endometrioid and non‐endometrioid uterine carcinomas

Endometrial carcinoma (EC) is traditionally characterized as endometrioid and nonendometrioid based on histopathologic phenotypes. Molecular‐based classifications have been proposed, but are not widely implemented. Herein we examine molecular profiles between EC histologic subtypes. 3133 ECs were submitted between March 2011 and July 2014: 1634 Type I and 1226 Type II. In situ hybridization and immunohistochemistry were used to assess copy number and protein expression of selected genes. Sequenced variants in 47 genes were analyzed using the Illumina TruSeq Amplicon Cancer Panel. Type II EC included 628 cases of uterine serous cancer (USC), 136 cases of clear cell (CC), 361 cases of carcinosarcoma (CS), 38 cases of mucinous, and 36 cases of squamous cell. PI3K/Akt/mTOR pathway was most frequently dysregulated within Type I and mucinous histologies, least altered in CS and squamous. PD‐L1 expression was highest in mucinous, absent in squamous. ER/PR expression was common in Type II, most frequent in USC, mucinous, and squamous. Receptor tyrosine kinase was frequently dysregulated in Type II disease: HER2 amplification highest in USC and CC, EGFR mutations exclusively seen in mucinous EC, KRAS mutations common in mucinous, squamous, and Type I, and c‐MET overexpression high in CC and mucinous. BRCA1 and BRCA2 were most frequently mutated in CS. Grade 3 EC shares features of G1 tumor and Type II disease, most notably resembling CS. Endometrial carcinomas are a molecularly heterogeneous group of tumors. A histology‐based molecular map can identify rational targets to optimize treatment and guide future clinical trials.

Multi-platform molecular profiling of a large cohort of glioblastomas reveals potential therapeutic strategies.

Glioblastomas (GBM) are the most aggressive and prevalent form of gliomas with abysmal prognosis and limited treatment options. We analyzed clinically relevant molecular aberrations suggestive of response to therapies in 1035 GBM tumors. Our analysis revealed mutations in 39 genes of 48 tested. IHC revealed expression of PD-L1 in 19% and PD-1 in 46%. MGMT-methylation was seen in 43%, EGFRvIII in 19% and 1p19q co-deletion in 2%. TP53 mutation was associated with concurrent mutations, while IDH1 mutation was associated with MGMT-methylation and TP53 mutation and was mutually exclusive of EGFRvIII mutation. Distinct biomarker profiles were seen in GBM compared with WHO grade III astrocytoma, suggesting different biology and potentially different treatment approaches. Analysis of 17 metachronous paired tumors showed frequent biomarker changes, including MGMT-methylation and EGFR aberrations, indicating the need for a re-biopsy for tumor profiling to direct subsequent therapy. MGMT-methylation, PR and TOPO1 appeared as significant prognostic markers in sub-cohorts of GBM defined by age. The current study represents the largest biomarker study on clinical GBM tumors using multiple technologies to detect gene mutation, amplification, protein expression and promoter methylation. These data will inform planning for future personalized biomarker-based clinical trials and identifying effective treatments based on tumor biomarkers.

Identification of potential therapeutic targets by molecular profiling of 628 cases of uterine serous carcinoma

BACKGROUND Therapeutic options are limited for uterine serous carcinoma (USC). TP53, PIK3CA, FBXW7, and ERBB mutations, as well as HER2 and EGFR overexpression have been reported. We aim to evaluate patterns of molecular, genomic and protein changes in 628USC tumors. METHODS 628 consecutive cases of USC submitted to Caris Life Sciences from Mar, 2011 to July, 2014 were reviewed. These were analyzed using the Illumina TruSeq Amplcon Cancer panel to search for sequenced variants in 47 genes commonly implicated in carcinomatosis. In situ hybridization and immunohistochemistry were also used to assess copy number and protein expression, respectively, of selected genes. RESULTS 31 out of 47 genes of interest harbored mutations, including TP53 (76%), PIK3CA (29%), FBXW7 (12%) and KRAS (9.3%). BRCA1 and BRCA2 were mutated in 9.1% and 6.3%, respectively. ERCC1 and MGMT were absent in 81% and 46% of tumors analyzed, respectively, suggesting potential benefit from platinum and alkylating agents. While not traditionally considered hormone-dependent, our cohort showed high ERα (60%), PR (32%), and AR (27%) expression. HER2 overexpression was 10% via IHC, amplification was 17% via CISH/FISH and mutation was 2% via NGS. While low in PTEN mutation frequency (7%), 45% of USC showed PTEN loss on IHC, and 29% harbored PIK3A mutation, suggesting deregulation of P13K/AKT pathway in a subset of patients. 11% expressed PDL1 and 67% expressed PD1. CONCLUSIONS Our findings suggest hormonal receptors, as well as genes implicated in DNA repair, cell proliferation and cell cycle pathways are of interest in USC.

BRCA2, EGFR, and NTRK mutations in mismatch repair-deficient colorectal cancers with MSH2 or MLH1 mutations

Deficient mismatch repair (MMR) and microsatellite instability (MSI) contribute to ~15% of colorectal cancer (CRCs). We hypothesized MSI leads to mutations in DNA repair proteins including BRCA2 and cancer drivers including EGFR. We analyzed mutations among a discovery cohort of 26 MSI-High (MSI-H) and 558 non-MSI-H CRCs profiled at Caris Life Sciences. Caris-profiled MSI-H CRCs had high mutation rates (50% vs 14% in non-MSI-H, P < 0.0001) in BRCA2. Of 1104 profiled CRCs from a second cohort (COSMIC), MSH2/MLH1-mutant CRCs showed higher mutation rates in BRCA2 compared to non-MSH2/MLH1-mutant tumors (38% vs 6%, P < 0.0000001). BRCA2 mutations in MSH2/MLH1-mutant CRCs included 75 unique mutations not known to occur in breast or pancreatic cancer per COSMIC v73. Only 5 deleterious BRCA2 mutations in CRC were previously reported in the BIC database as germ-line mutations in breast cancer. Some BRCA2 mutations were predicted to disrupt interactions with partner proteins DSS1 and RAD51. Some CRCs harbored multiple BRCA2 mutations. EGFR was mutated in 45.5% of MSH2/MLH1-mutant and 6.5% of non-MSH2/MLH1-mutant tumors (P < 0.0000001). Approximately 15% of EGFR mutations found may be actionable through TKI therapy, including N700D, G719D, T725M, T790M, and E884K. NTRK gene mutations were identified in MSH2/MLH1-mutant CRC including NTRK1 I699V, NTRK2 P716S, and NTRK3 R745L. Our findings have clinical relevance regarding therapeutic targeting of BRCA2 vulnerabilities, EGFR mutations or other identified oncogenic drivers such as NTRK in MSH2/MLH1-mutant CRCs or other tumors with mismatch repair deficiency.