Ryan Bender

Molecular Profiling of Infiltrating Urothelial Carcinoma of Bladder and Nonbladder Origin

BACKGROUND Infiltrating UC represents the second most common genitourinary malignancy. Advanced UC has a poor prognosis and new treatments are needed. Molecular profiling of UC might identify biomarkers associated with targeted therapies or chemotherapeutics, providing physicians with new treatment options. MATERIALS AND METHODS Five hundred thirty-seven cases of locally advanced or metastatic UC of the bladder, 74 nonbladder, and 55 nonurothelial bladder cancers were profiled using mutation analysis, in situ hybridization, and immunohistochemistry assays for biomarkers predictive of therapy response. RESULTS Molecular profiling of UC showed high overexpression of topoisomerase 2α, common phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha and/or phosophatase and tensin homolog (PTEN) alterations in nonbladder (27%) and bladder UC (21%), and rare gene mutations across subtypes. Compared with nonbladder, bladder UC consistently exhibited more frequent abnormal protein expression, including HER2 (10% vs. 3%; P = .04), tyrosine protein c-Kit receptor kinases (11% vs. 5%), c-Met proto-oncogene, receptor tyrosine kinases (25% vs. 8%), androgen receptor (16% vs. 6%), O(6)-methylguanine-methyltransferase (63% vs. 43%), ribonucleotide reductase M1 (32% vs. 11%), Serum protein acidic and rich in cysteine (SPARC) (69% vs. 33%), and topoisomerase 1 (63% vs. 39%). Bladder UC also exhibited increased amplification of HER2 (12% vs. 2%; P = .06). CONCLUSION Comprehensive molecular profiling of UC identified a large number of biomarkers aberrations that might direct treatment in conventional chemotherapies and targeted therapies, not currently recommended in this population. As a group, bladder UC exhibited higher levels of actionable biomarkers, suggesting that UC from different primary sites and non-UC are driven by different molecular pathways. These differences could have clinical implications resulting in different treatment regimens depending on the site of origin of UC.

Molecular Profiling of Clear Cell Ovarian Cancers: Identifying Potential Treatment Targets for Clinical Trials

Background Advanced stage/recurrent clear cell ovarian cancers (CCOCs) are characterized by a low response to chemotherapy and a poor prognosis. There is growing interest in investigating novel/molecular targeted therapies in patients with CCOC in histotype-specific trials. However, CCOCs are not a uniform entity and comprise a number of molecular subtypes and it is unlikely that a single approach to treatment will be appropriate for all patients. The aim of this study was to analyze the results of a multiplatform profiling panel in CCOCs to identify potential therapeutic targets. Patients and Methods Tumor profiling was performed on 521 CCOCs. They were grouped into pure (n = 422) and mixed (n = 99) CCOC for analysis. Testing included a combination of DNA sequencing (including next-generation sequencing) using a 46-gene panel, immunohistochemistry, fluorescent or chromogenic in situ hybridization, and RNA fragment analysis. Results The most common findings were in the PIK3CA/Akt/mTOR pathway, with 61% of all CCOCs showing a molecular alteration in one of these pathway components. Next-generation sequencing revealed PIK3CA mutations in 50% of pure CCOCs. Significant differences were observed between pure and mixed CCOCs with respect to hormone receptor expression (9% vs 34.7% for ER, 13.45 vs 26.4% for PR), cMET (24.1% vs 11.6%), PD-1 tumor infiltrating lymphocytes (48.1% vs 100%), expression of PD-L1 (7.4% vs 25%), and TOPO1 (41% vs 27.1%) on immunohistochemistry, whereas next-generation sequencing revealed significant differences in mutation frequency in PIK3CA (50% vs 18.5%), TP53 (18.1% vs 57.7%), KRAS (12.4% vs 3.7%), and cMET (1.9% vs 11.1%). Conclusions This large study confirms that the PIK3CA/Akt/mTOR pathway is commonly altered in CCOCs, and highlights the significant differences between pure and mixed CCOCs. Clear cell ovarian cancers are molecularly heterogeneous and there are a number of potential therapeutic targets which could be tested in clinical trials.

Mutations in the Kinase Domain of the HER2/ERBB2 Gene Identified in a Wide Variety of Human Cancers

The HER2 (official name ERBB2) gene encodes a membrane receptor in the epidermal growth factor receptor family amplified and overexpressed in adenocarcinoma. Activating mutations also occur in several cancers. We report mutation analyses of the HER2 kinase domain in 7497 histologically diverse cancers. Forty-five genes, including the kinase domain of HER2 with HER2 IHC and dual in situ hybridization, were analyzed in tumors from 7497 patients with cancer, including 850 breast, 770 colorectal, 910 non-small cell lung, 823 uterine or cervical, 1372 ovarian, and 297 pancreatic cancers, as well as 323 melanomas and 2152 other solid tumors. Sixty-nine HER2 kinase domain mutations were identified in tumors from 68 patients (approximately 1% of all cases, ranging from absent in sarcomas to 4% in urothelial cancers), which included previously published activating mutations and 13 novel mutations. Fourteen cases with coexisting HER2 mutation and amplification and/or overexpression were identified. Fifty-two of 68 patients had additional mutations in other analyzed genes, whereas 16 patients (23%) had HER2 mutations identified as the sole driver mutation. HER2 mutations coexisted with HER2 gene amplification and overexpression and with mutations in other functionally important genes. HER2 mutations were identified as the only driver mutation in a significant proportion of solid cancers. Evaluation of anti-HER2 therapies in nonamplified, HER2-mutated cancers is warranted.

Juxtaglomerular cell tumor: A morphological, immunohistochemical and genetic study of six cases

Juxtaglomerular cell tumors (JGCTs) are rare tumors characterized by renin synthesis, hyperaldosteronism and hypertension. A curious immunohistochemical overlap between JGCT and gastrointestinal stromal tumor (GIST) including the expression of vimentin, CD34, CD117, α-smooth muscle actin was previously reported, prompting us to further investigate JGCT and its phenotypic and molecular genetic characteristics. Virtual karyotyping showed gain of chromosomes 3, 4, 10, 13, 17 and 18 in one JGCT, and fluorescence in situ hybridization (FISH) study confirmed this multiple gain pattern. Additionally, loss of chromosome 9 was observed in four of six cases analyzed with FISH. A whole genome expression analysis revealed 415 up-regulated (including renin, and CD117) and 325 down-regulated genes between the 2 cases. The study confirmed earlier reports on the gain of chromosomes 4 and 10, and provided further evidence of up-regulation of the genes located on these 2 chromosomes. For the first time our study indicated the importance of the loss of chromosome 9 and loss of expression of several tumor suppressor genes located on this chromosome as possible pathogenetic events important in development of JGCT.

PD-L1 Status in Refractory Lymphomas.

Targeted immunotherapy based on PD-1/PD-L1 suppression has revolutionized the treatment of various solid tumors. A remarkable improvement has also been observed in the treatment of patients with refractory/relapsing classical Hodgkin lymphoma (cHL). We investigated PD-L1 status in a variety of treatment resistant lymphomas. Tumor samples from 78 patients with therapy resistant lymphomas were immunohistochemically (IHC) investigated for the expression of PD-L1 using two antibody clones (SP142 and SP263, Ventana). Thirteen PD-L1+ cases were further analyzed for gene copy number variations (CNV) by NGS and for PD-L1/JAK2/PD-L2 co-amplification using fluorescent in-situ hybridization assay (FISH). PD-L1 positivity (≥5% positive cancer cells, IHC) was present in 32/77 (42%) and 33/71 cases (46%) using SP142 and SP263 antibodies, respectively. Concordance between the two anti-PD-L1 clones was high with only three (4%) discrepant cases. The strongest and consistent (10/11 cases) expression was observed in cHL and primary mediastinal B-cell lymphomas (3/3). Diffuse large B-cell lymphomas (DLBCL) were frequently positive (13/26) irrespective of subtype. Follicular (1/8), peripheral T-cell (3/11) and mantle cell (1/8) lymphomas were rarely positive, while small lymphocytic lymphoma/CLL and marginal zone lymphomas were consistently negative (3/3). Co-amplification/CNVs of PD-L1/JAK2/PD-L2 were observed in 3 cases of DLBCL and cHL, respectively. Of note, all three cHL-amplified cases were positive by FISH, but not by NGS. Since only a fraction of the IHC positive lymphoma cases were positive by FISH and NGS assays, other mechanisms are involved in PD-L1 upregulation, especially in DLBCL. FISH assay may be more suitable than NGS assay for determination of PD-L1 alterations in cHL.

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.

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.

Oncocytoma-Like Renal Tumor With Transformation Toward High-Grade Oncocytic Carcinoma A Unique Case With Morphologic, Immunohistochemical, and Genomic Characterization

AbstractRenal oncocytoma is a benign tumor with characteristic histologic findings. We describe an oncocytoma-like renal tumor with progression to high-grade oncocytic carcinoma and metastasis.A 74-year-old man with no family history of cancer presented with hematuria. Computed tomography showed an 11 cm heterogeneous multilobulated mass in the right kidney lower pole, enlarged aortocaval lymph nodes, and multiple lung nodules. In the nephrectomy specimen, approximately one third of the renal tumor histologically showed regions classic for benign oncocytoma transitioning to regions of high-grade carcinoma without sharp demarcation.With extensive genomic investigation using single nucleotide polymorphism-based array virtual karyotyping, multiregion sequencing, and expression array analysis, we were able to show a common lineage between the benign oncocytoma and high-grade oncocytic carcinoma regions in the tumor. We were also able to show karyotypic differences underlying this progression. The benign oncocytoma showed no chromosomal aberrations, whereas the high-grade oncocytic carcinoma showed loss of the 17p region housing FLCN (folliculin [Birt–Hogg–Dubé protein]), loss of 8p, and gain of 8q. Gene expression patterns supported dysregulation and activation of phosphoinositide 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (Akt), mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK), and mechanistic target of rapamycin (serine/threonine kinase) (mTOR) pathways in the high-grade oncocytic carcinoma regions. This was partly attributable to FLCN underexpression but further accentuated by overexpression of numerous genes on 8q. In the high-grade oncocytic carcinoma region, vascular endothelial growth factor A along with metalloproteinases matrix metallopeptidase 9 and matrix metallopeptidase 12 were overexpressed, facilitating angiogenesis and invasiveness.Genetic molecular testing provided evidence for the development of an aggressive oncocytic carcinoma from an oncocytoma, leading to aggressive targeted treatment but eventual death 39 months after the diagnosis.

2771 BRCA1 and BRCA2 mutations in 1691 epithelial ovarian tumors identify subgroups with distinct molecular characteristics

• In epithelial ovarian cancer, BRCA mutations are seen in 14.3% of tumors. While serous histology shows the highest mutation rate, endometrioid, clear cell and carcinosarcoma all harbor various rates of BRCA mutations. Mucinous histology, however, rarely shows BRCA mutations. • While BRCA-mutated patients carry a survival advantage and may benefit more from DNA-damaging agents including platinum agents and PARP inhibitors, BRCA-wild type tumors are more likely to carry PIK3CA, KRAS, CTNNB1 mutations and cMET overexpression that may serve as therapeutic targets. • Even though clear cell ovarian cancer has been conventionally considered a hormone-independent histological subtype, our data suggest that in the rare event when deleterious BRCA mutations are found in clear cell ovarian tumors, estrogen receptor expression is high, suggesting a combination of hormonal therapies with either platinum or PARP inhibitors as a logical approach for clinical trial design. • Biomarker frequencies indicate that the predicted benefit rate of commonly used chemotherapies including platinums, anthracyclines, gemcitabine and topotecan are similar in BRCA-mutated and wild type cohorts; on the other hand, fluoropyrimidines are more likely to benefit the BRCA-wild type cohort while taxanes are moderately but significantly more likely to benefit the BRCA-mutated cohort. • BRCA-mutated and, therefore, homologous recombination deficient tumors, are more likely to harbor PD-1 positive tumor-infiltrating lymphocytes and can be further explored with immune checkpoint inhibitors. BRCA1 and BRCA2 mutations in 1691 epithelial ovarian tumors identify subgroups with distinct molecular characteristics Thomas Herzog1, Joanne Xiu2, Ryan Bender2, Zoran Gatalica2, Sandeep Reddy2 1University of Cincinnati, College of Medicine, Cincinnati, USA. 2Caris Life Sciences, Phoenix, USA.

213 BRAF mutations are potentially targetable alterations in a wide variety of solid cancers

updated) Background: The BRAF gene mutations are potentially targetable with several treatment modalities approved in a limited number of cancer types. The present study explored the spectrum and frequencies of BRAF mutations in a wide variety of solid tumors. Materials and Methods: 36,312 solid tumors were profiled using different gene sequencing assays (Sanger and Next-generation sequencing, Caris Life Sciences, Phoenix, AZ) and immunohistochemistry. Results: Overall, 5% of all solid tumors harbored BRAF mutations (77% V600E and 23% non-V600E). As expected, BRAF mutations were detected in 38% of thyroid cancers (4% non-V600E), 35% malignant melanomas (13% non-V600E), 9% colorectal (10% non-V600E), 6% small intestinal cancers (92% non-V600E) and 4% NSCLC (65% non-V600E). Non-V600E mutations with functional impact on kinase activity included (% variant/non-V600E): V600K (24%), D594G (7%), G469A (9%), G466V (6%), and G469V (5%). Some other cancer types also harbored BRAF mutations (GBM; 75% V600E and 25% non-V600E, biliary tract cancers; 43% V600E, 57% non-V600E), while esophageal/GEJ, gastric, liver, pancreatic and head and neck cancers were mostly devoid of BRAF mutations. The most common concurrent alterations associated with potential resistance to BRAF inhibitors, and with implications for dual-targeting approaches, included mutations of PIK3CA (7%), PTEN (6%), KRAS (5%), NRAS (2%), AKT1 and NRAS (1%, respectively), and over-expression of EGFR (53%). Of interest, overexpression of EGFR in BRAFmutated tumors was highest in the tumor type with poor clinical response to BRAF inhibitors (colorectal cancer with 80%), and lowest in melanoma (6%) where monotherapy has been more successful. Conclusions: Activating BRAF mutations (both V600E and non-V600E) are potentially targetable in a substantial proportion of various solid malignancies. A subset of BRAF-mutated cancers harbors additional genetic alterations and/or over-express EGFR which may require expanded/dual targeting treatment to overcome resistance. Background The BRAF gene, located on chromosome 7q34, is a constitutive part of the MAPK/ERK signaling pathway involved in cancer initiation and progression. BRAF is one of the most frequently mutated genes in human cancer (~7%) (1). Since 2002 when Davies et al. described BRAF mutations in a subset of human neoplasms (2) there have been numerous studies exploring BRAF status in human neoplasms (e.g. melanoma, thyroid, colorectal, lung, ovarian cancer, hairy cell leukemia, multiple myeloma, histiocytoses) (3-4). The BRAF gene (V600E and non-V600E) mutations are clinically relevant due to the targeted treatment modalities (e.g. vemurafenib, dabrafenib, tramatenib) that have been approved in a limited number of cancers so far (3-4). In the present study we explored the spectrum and frequencies of BRAF mutations in a large cohort of solid tumors and analyzed other potentially targetable biomarkers co-existing with BRAF mutations. Results Methods Tissue samples A cohort of 36,312 solid tumors were profiled at Caris Life Sciences, (Phoenix, AZ) using Sanger and NGS assays. Next-generation sequencing (NGS) and Sanger sequencing: NGS was performed on enriched tumor genomic DNA isolated from formalin-fixed paraffin embedded samples using the Illumina MiSeq platform. Specific regions of the genome were amplified using the Illumina TruSeq Amplicon Cancer Panel. The NGS panel included 45 different genes listed here: http://www.carismolecularintelligence.com/next-generationsequencing-profile). All variants were detected with >99% confidence based on the frequency of the mutation present and the amplicon coverage using a mutation frequency threshold of 10% (4, 5). All regions that were sequenced achieved a minimum of 100x coverage and overall samples had an average coverage of >500x. Sanger Sequencing was used to explore selected regions of BRAF, KRAS, c-KIT, EGFR, and PIK3CA. Testing also included one or more of the following: protein expression (IHC), gene amplification (C/FISH), microsatellite instability testing by fragment analysis (FA). Figure 1. BRAF mutation spectrum across solid tumors. Of 36,312 solid tumors tested, 1867 harbor BRAF mutation, for a mutation frequency of 5%. Frequency displayed is % of BRAF mutations among total tested, across tumor types. Blue and Red bars indicate the distribution of V600and non-V600 mutations across tumor types, respectively. Conclusions • Activating BRAF mutations (both V600E and non-V600E) are potentially targetable in a substantial proportion of various solid malignancies. • The V600E mutations were more prevalent (77%) although a substantial proportion of non-V600E mutations with functional impact on kinase activity was also detected: V600K (24%), D594G (7%), G469A (9%) G466V (6%) and G469V (5%). • A subset of BRAF-mutated cancers harbors additional genetic alterations (KRAS, PIK3CA, PTEN, AKT1, NRAS) and/or over-express EGFR, which may require expanded/dual targeting treatment to overcome a potential resistance mechanism. References 1. Jabbar KJ et al. Am J Surg Pathol 2015;39:454-61. 2. Davies H et al. Nature 2002;417:949-54. 3. Hall RD, Kudchadkar RR. Cancer Control 2014;21:221-30. 4. Hyman, DH et al. NEJM 2015;373:726-736. 5. Gatalica Z et al. Oncotarget 2015;6:19819-25. Tumor Type (BRAF-mutated/n, n=total tested tumor type) Prevalence of BRAF mutations, %