Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer.

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.

Comprehensive Genomic Profiling of Advanced Penile Carcinoma Suggests a High Frequency of Clinically Relevant Genomic Alterations.

BACKGROUND: Advanced penile squamous cell carcinoma (PSCC) is associated with poor survival due to the aggressiveness of the disease and lack of effective systemic therapies. Comprehensive genomic profiling (CGP) was performed to identify clinically relevant genomic alterations (CRGAs). MATERIALS AND METHODS: DNA was extracted from 40 µm of formalin-fixed, paraffin-embedded sections in patients with advanced PSCC. CGP was performed on hybridization-captured, adaptor ligation-based libraries to a mean coverage depth of 692× for 3,769 exons of 236 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer. CRGAs were defined as genomic alterations (GAs) linked to targeted therapies on the market or under evaluation in mechanism-driven clinical trials. RESULTS: Twenty male patients with a median age of 60 years (range, 46-87 years) were assessed. Seventeen (85%) cases were stage IV and three cases (15%) were stage III. CGP revealed 109 GAs (5.45 per tumor), 44 of which were CRGAs (2.2 per tumor). At least one CRGA was detected in 19 (95%) cases, and the most common CRGAs were CDKN2A point mutations and homozygous deletion (40%), NOTCH1 point mutations and rearrangements (25%), PIK3CA point mutations and amplification (25%), EGFR amplification (20%), CCND1 amplification (20%), BRCA2 insertions/deletions (10%), RICTOR amplifications (10%), and FBXW7 point mutations (10%). CONCLUSION: CGP identified CRGAs in patients with advanced PSCC, including EGFR amplification and PIK3CA alterations, which can lead to the rational administration of targeted therapy and subsequent benefit for these patients. IMPLICATIONS FOR PRACTICE: Few treatment options exist for patients with advanced penile squamous cell carcinoma (PSCC). Outcomes are dismal with platinum-based chemotherapy, with median survival estimated at 1 year or less across multiple series. Biological studies of patients with PSCC to date have principally focused on human papillomavirus status, but few studies have elucidated molecular drivers of the disease. To this end, comprehensive genomic profiling was performed in a cohort of 20 patients with advanced PSCC. Findings of frequent mutations in CDKN2A, NOTCH1, PIK3CA, and EGFR (all in excess of 20%) point to potential therapeutic avenues. Trials of targeted therapies directed toward these mutations should be explored.

Clinical Actionability of Comprehensive Genomic Profiling for Management of Rare or Refractory Cancers.

BACKGROUND: The frequency with which targeted tumor sequencing results will lead to implemented change in care is unclear. Prospective assessment of the feasibility and limitations of using genomic sequencing is critically important. METHODS: A prospective clinical study was conducted on 100 patients with diverse-histology, rare, or poor-prognosis cancers to evaluate the clinical actionability of a Clinical Laboratory Improvement Amendments (CLIA)-certified, comprehensive genomic profiling assay (FoundationOne), using formalin-fixed, paraffin-embedded tumors. The primary objectives were to assess utility, feasibility, and limitations of genomic sequencing for genomically guided therapy or other clinical purpose in the setting of a multidisciplinary molecular tumor board. RESULTS: Of the tumors from the 92 patients with sufficient tissue, 88 (96%) had at least one genomic alteration (average 3.6, range 0-10). Commonly altered pathways included p53 (46%), RAS/RAF/MAPK (rat sarcoma; rapidly accelerated fibrosarcoma; mitogen-activated protein kinase) (45%), receptor tyrosine kinases/ligand (44%), PI3K/AKT/mTOR (phosphatidylinositol-4,5-bisphosphate 3-kinase; protein kinase B; mammalian target of rapamycin) (35%), transcription factors/regulators (31%), and cell cycle regulators (30%). Many low frequency but potentially actionable alterations were identified in diverse histologies. Use of comprehensive profiling led to implementable clinical action in 35% of tumors with genomic alterations, including genomically guided therapy, diagnostic modification, and trigger for germline genetic testing. CONCLUSION: Use of targeted next-generation sequencing in the setting of an institutional molecular tumor board led to implementable clinical action in more than one third of patients with rare and poor-prognosis cancers. Major barriers to implementation of genomically guided therapy were clinical status of the patient and drug access. Early and serial sequencing in the clinical course and expanded access to genomically guided early-phase clinical trials and targeted agents may increase actionability. IMPLICATIONS FOR PRACTICE: Identification of key factors that facilitate use of genomic tumor testing results and implementation of genomically guided therapy may lead to enhanced benefit for patients with rare or difficult to treat cancers. Clinical use of a targeted next-generation sequencing assay in the setting of an institutional molecular tumor board led to implementable clinical action in over one third of patients with rare and poor prognosis cancers. The major barriers to implementation of genomically guided therapy were clinical status of the patient and drug access both on trial and off label. Approaches to increase actionability include early and serial sequencing in the clinical course and expanded access to genomically guided early phase clinical trials and targeted agents.

Prospective comprehensive genomic profiling of advanced gastric carcinoma cases reveals frequent clinically relevant genomic alterations and new routes for targeted therapies.

BACKGROUND: Gastric cancer (GC) is a major global cancer burden and the second most common cause of global cancer-related deaths. The addition of anti-ERBB2 (HER2) targeted therapy to chemotherapy improves survival for ERBB2-amplified advanced GC patients; however, the majority of GC patients do not harbor this alteration and thus cannot benefit from targeted therapy under current practice paradigms. MATERIALS AND METHODS: Prospective comprehensive genomic profiling of 116 predominantly locally advanced or metastatic (90.0%) gastric cancer cases was performed to identify genomic alterations (GAs) associated with a potential response to targeted therapies approved by the U.S. Food and Drug Administration or targeted therapy-based clinical trials. RESULTS: Overall, 78% of GC cases harbored one clinically relevant GA or more, with the most frequent alterations being found in TP53 (50%), ARID1A (24%), KRAS (16%), CDH1 (15%), CDKN2A (14%), CCND1 (9.5%), ERBB2 (8.5%), PIK3CA (8.6%), MLL2 (6.9%), FGFR2 (6.0%), and MET (6.0%). Receptor tyrosine kinase genomic alterations were detected in 20.6% of cases, primarily ERBB2, FGFR2, and MET amplification, with ERBB2 alterations evenly split between amplifications and base substitutions. Rare BRAF mutations (2.6%) were also observed. One MET-amplified GC patient responded for 5 months to crizotinib, a multitargeted ALK/ROS1/MET inhibitor. CONCLUSION: Comprehensive genomic profiling of GC identifies clinically relevant GAs that suggest benefit from targeted therapy including MET-amplified GC and ERBB2 base substitutions.

Comprehensive genomic profiling of inflammatory breast cancer cases reveals a high frequency of clinically relevant genomic alterations.

Inflammatory breast cancer (IBC) is a distinct clinicopathologic entity that carries a worse prognosis relative to non-IBC breast cancer even when matched for standard biomarkers (ER/PR/HER2). The objective of this study was to identify opportunities for benefit from targeted therapy, which are not currently identifiable in the standard workup for advanced breast cancer. Comprehensive genomic profiling on 53 IBC formalin-fixed paraffin-embedded specimens (mean, 800× + coverage) using the hybrid capture-based FoundationOne assay. Academic and community oncology clinics. From a series of 2208 clinical cases of advanced/refractory invasive breast cancers, 53 cases with IBC were identified. The presence of clinically relevant genomic alterations (CRGA) in IBC and responses to targeted therapies. CRGA were defined as genomic alterations (GA) associated with on label targeted therapies and targeted therapies in mechanism-driven clinical trials. For the 44 IBCs with available biomarker data, 19 (39 %) were ER-/PR-/HER2- (triple-negative breast cancer, TNBC). For patients in which the clinical HER2 status was known, 11 (25 %) were HER2+ with complete (100 %) concordance with ERBB2 (HER2) amplification detected by the CGP assay. The 53 sequenced IBC cases harbored a total of 266 GA with an average of 5.0 GA/tumor (range 1-15). At least one alteration associated with an FDA approved therapy or clinical trial was identified in 51/53 (96 %) of cases with an average of 2.6 CRGA/case. The most frequently altered genes were TP53 (62 %), MYC (32 %), PIK3CA (28 %), ERBB2 (26 %), FGFR1 (17 %), BRCA2 (15 %), and PTEN (15 %). In the TNBC subset of IBC, 8/19 (42 %) showed MYC amplification (median copy number 8X, range 7-20) as compared to 9/32 (28 %) in non-TNBC IBC (median copy number 7X, range 6-21). Comprehensive genomic profiling uncovered a high frequency of GA in IBC with 96 % of cases harboring at least 1 CRGA. The clinical benefit of selected targeted therapies in individual IBC cases suggests that a further study of CGP in IBC is warranted.

Characterization of Clinical Cases of Collecting Duct Carcinoma of the Kidney Assessed by Comprehensive Genomic Profiling.

BACKGROUND: Collecting duct carcinoma (CDC) is a rare type of renal cell carcinoma (RCC) originating from the renal medulla. Clinical outcomes are poor, and there are no consensus guidelines to guide therapy. OBJECTIVE: To determine genomic alterations (GAs) in a series of patients with locally advanced or metastatic CDC for whom genomic profiling was performed during the course of clinical care. DESIGN, SETTING, AND PARTICIPANTS: Formalin-fixed, paraffin-embedded blocks or slides were obtained for 17 patients with CDC. DNA was extracted and comprehensive genomic profiling was performed in a laboratory certified under the Clinical Laboratory Improvement Amendments. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Bayesian algorithms and local alignment algorithms were used to detect substitutions and insertions/deletions, respectively. A comparison to normal control samples was used to detect copy number alterations. Clinically relevant GAs (CRGAs) were defined as those linked to approved or investigational targeted therapies. RESULTS AND LIMITATIONS: The median age in the cohort was 53 yr (range 26-73), and 14 primary tumors and three metastatic sites assessed. A total of 36 GAs were detected in this series of patients, with an average of 2.1 GAs per case. The most common GAs were in NF2 (5/17, 29%), SETD2 (4/17, 24%), SMARCB1 (3/17, 18%), and CDKN2A (2/17, 12%). Of nine cases assessed for FH GAs, two patients had FH homozygous loss. A limitation is that targeted interrogation of genes known to be implicated in other cancers was performed, so mutations outside of these cannot be excluded. CONCLUSIONS: Recurrent CRGAs were detected in this series of CDC cases and suggest a possible benefit from targeted therapy. In particular, mTOR inhibitors may be of interest in patients with NF2 alterations. Alterations in FH and SMARCB1 also occurred in a mutually exclusive manner to NF2 alterations. PATIENT SUMMARY: This report provides important genomic insights into collecting duct carcinoma, a rare type of renal cell carcinoma with a very aggressive course. These insights could further rationalize the use of targeted therapies for rare tumors according to the individual genomic alterations harbored.

A Poorly Differentiated Malignant Neoplasm Lacking Lung Markers Harbors an EML4-ALK Rearrangement and Responds to Crizotinib.

Suspected metastatic site lesions that are poorly differentiated present a diagnostic challenge when morphologic and immunohistochemical profiling cannot establish the primary tumor site. Here we present a patient diagnosed with both a malignant neoplasm in the lung and a right upper extremity (RUE) neoplasm of unclear histogenetic origin. Immunohistochemical staining performed on the latter specimen was inconclusive in determining the site of origin. Although the lung biopsy sample was insufficient for molecular testing, hybrid capture-based comprehensive genomic profiling (FoundationOne) identified an EML4-ALK rearrangement in the RUE lesion. Crizotinib treatment resulted in a major response in both the RUE and the lung lesions. This report illustrates the utility of comprehensive genomic profiling employed at the initial presentation of an unknown primary malignant neoplasm, which resulted in the front-line use of targeted therapy and a significant and sustained antitumor response.

Durable Response to Crizotinib in a MET-Amplified, KRAS-Mutated Carcinoma of Unknown Primary.

BACKGROUND: Carcinoma of unknown primary (CUP) accounts for 3-5% of all adult solid tumors. An extensive search for the anatomic site of origin is often undertaken in an attempt to tailor systemic treatment, but the latter often has limited efficacy - especially in the setting of an initial treatment failure. Molecularly targeted therapy is an emerging approach that may offer greater efficacy and less toxicity but is most likely to be effective when pairing a tumor harboring a sensitizing genomic alteration with an agent directed at the altered gene product. We report a patient with a CUP harboring a MET amplification with a complete metabolic response to crizotinib despite also harboring a KRAS mutation. METHODS: Ge-nomic profiling was performed using a clinical next-generation-sequencing-based assay, FoundationOne(®), in a CAP-accredited laboratory certified by Clinical Laboratory Improvement Amendments (Foundation Medicine, Cambridge, Mass., USA). RESULTS: The CUP harbored both MET amplification (16 copies) and a KRAS G12V mutation. The patient was treated with crizotinib, a MET inhibitor, and has experienced a complete normalization of tumor metabolic activity for more than 19 months. CONCLUSIONS: Genomic profiling of CUP may reveal clinically meaningful genomic alterations that can guide targeted therapy decision-making. The use of this approach should be studied prospectively as a strategy for the effective treatment of CUP patients and for avoiding resource-intensive workups to identify the tumor site of origin.