CDx Test Research Articles

Successful management of metastatic ovarian carcinosarcoma with olaparib plus bevacizumab: a case report

Background: Ovarian carcinosarcoma (OCS) is a rare, highly aggressive, and treatment-resistant tumor. Some important advances in chemotherapy have been reported, including an anti-vascular endothelial growth factor antibody, bevacizumab (Bev)-containing regimen, and a poly-ADP ribose polymerase (PARP) inhibitor, olaparib. Olaparib is specifically a treatment option for OCSs with breast cancer gene (BRCA) mutations, and the clinical study on the combination of olaparib and Bev was reported for homologous recombination deficient epithelial ovarian cancer. However, there are no reports on treating advanced OCS with BRCA mutations by olaparib plus Bev effectively. In this study, to the best of our knowledge, we report the first case of successful management of multiple liver and para-aortic metastases of OCS with a BRCA mutation using olaparib plus Bev. Case: A 75-year-old woman presented to the hospital with complaints of abdominal distension. A staging laparotomy and histological analysis revealed a primary OCS. She received platinum doublet regimens as adjuvant chemotherapy. As myChoice® CDx test of the surgical specimen revealed a BRCA2 mutation, the combination regimen of olaparib (400 mg daily) plus Bev (15 mg/kg every three weeks) was chosen as the maintenance therapy. After four months of this chemotherapy, a computed tomography scan revealed significant shrinkage of both the liver metastases and para-aortic lymph nodes. Progression-free survival has been noted for over 14 months. Conclusions: The combination therapy of olaparib plus Bev could be a potential therapeutic option for advanced OCS.

FDA Cleared Companion Diagnostics (CDx) Tests (IDH1, IDH2 and FLT3) for Acute Myeloid Leukemia (AML) Patient Care

Acute myeloid leukemia (AML) is one of the most lethal blood cancers from which nearly 10,000 people die in the United States each year. While therapies for other blood cancers have made some progress, the standard of care for AML, a combination of toxic chemotherapies, has changed very little over the past four decades. In recent years the US Food and Drug Administration (FDA) has been very active in approving targeted therapeutic drugs for AML patients including Midostaurin (Rydapt; 2017) and Gilteritinib (Xospata;2018) for FLT3 mutations; Enasidenib (Idhifa; 2017) for IDH2 mutations and Ivosidenib (Tibsovo; 2018) for IDH1 mutations. Additionally, the Leukemia and Lymphoma Society's Beat AML Master Clinical Trial has shown that waiting for molecular results prior to treatment decision leads to better outcomes. Versiti Blood Center of Wisconsin Diagnostics laboratory which is certified under the Clinical Laboratory Improvement Amendments (CLIA) and qualified to perform high complexity clinical laboratory testing has performed the verification studies and offers two companion diagnostics tests for IDH1 and IDH2 mutations for AML patients. Also, in collaboration with Invivoscribe Inc., the AML patient can be tested for Leukostrat CDx FLT3 mutations assay so that the same AML patient can get three CDx test results leading to available drug therapy treatment decision making by physicians.IDH1CDX Test: IDH1 CDx is indicated as an aid in identifying AML patients with an IDH1 mutation for treatment with ivosidenib (TIBSOVO®). Mutations in codon R132 of IDH1 can be found in 6% to 10% of AML patients. The IDH1 CDx test detects five IDH1 mutations R132H (CAT), R132C (TGT), R132G (GGT), R132S (AGT), and R132L (CTT) using PCR technology with homogeneous real-time fluorescence detection. The assay sensitivity for these five IDH1 mutations is 100% at variant allele frequencies of 2% and higher and 98% or greater at variant allele frequencies of 1% and higher. This test has been approved by the FDA as companion diagnostic device (PMA number P170041).IDH2CDx: IDH2 CDx is indicated as an aid in identifying AML patients with an IDH2 mutation for treatment with IDHIFA® (enasidenib). Mutations in the R140 and R172 codons of IDH2 8% to 19% of AML patients.The IDH2 CDX test detects nine IDH2 mutations (R140Q, R140L, R140G, R140W, R172K, R172M, R172G, R172S, and R172W) using PCR technology with real-time fluorescent detection. The assay sensitivity for these nine IDH2 mutations is 99.8% or greater at variant allele frequencies of 2% and higher or 93.5% or greater at variant allele frequencies of 1% and higher. This test has been approved by the FDA as companion diagnostic device (PMA number P170005).FLT3CDx: The FLT3 Leukostrat® CDx Assay is the FDA approved (PMA number P160040) predictive test for the efficacy of midostaurin (RYDAPT®) therapy in all AML patients, regardless of cytogenetics and efficacy of gilteritinib (XOSPATA ® ) therapy in relapsed or refractory AML patients. FLT3 is one of the most commonly mutated genes in AML with 30% of patients at the time of diagnosis 1. The most prevalent type of FLT3 mutation is an internal tandem duplication (ITD) in the juxtamembrane domain. The second most common mutation type in the FLT3 gene is a tyrosine kinase domain (TKD) point mutation in the codon for an aspartate (D835) or an isoleucine (I836) residue. The LeukoStrat® CDx FLT3 Mutation Assay is a PCR-based, in vitro diagnostic test designed to detect internal tandem duplication (ITD) mutations and the tyrosine kinase domain (TKD) mutations D835 and I836 in genomic DNA extracted from mononuclear cells obtained from peripheral blood or bone marrow aspirates of patients diagnosed with AML. Versiti Blood Center sends the patient specimens to Invivoscribe Inc. where the LeukoStrat® CDx FLT3 Mutation Assay is performed and the interpretive comments are included in the patient report by Versiti.From our experience pathologists and treating physicians want molecular test results as fast as possible, especially for the actionable gene mutations in IDH1, IDH2 and FLT3. The IDH1 CDx, IDH2 CDx and FLT3 CDx tests are highly sensitive and Versiti provides average turn around time of 3 business days which enable rapid decision making on the recently available drug therapies for AML patients. We strongly recommend that the IDH1 CDx, IDH2 CDx and FLT3 CDx tests should be performed on all AML patients for better care. DisclosuresNo relevant conflicts of interest to declare. OffLabel Disclosure:In recent years the US Food and Drug Administration (FDA) approved targeted therapeutic drugs for AML patients including Midostaurin (Rydapt; 2017) and Gilteritinib (Xospata;2018) for FLT3 mutations; Enasidenib (Idhifa; 2017) for IDH2 mutations and Ivosidenib (Tibsovo; 2018) for IDH1 mutations.

Concordance of BRCA mutation detection in tumor versus blood, and frequency of bi-allelic loss of BRCA in tumors from patients in the phase III SOLO2 trial

ObjectiveMaintenance olaparib provided a progression-free survival benefit in the phase III SOLO2 trial (NCT01874353) in patients with platinum-sensitive relapsed ovarian cancer and a BRCA mutation (BRCAm). However, questions remain regarding tumor versus germline BRCA testing and the impact of heterozygous versus bi-allelic loss of BRCA1 or BRCA2 in the tumor. MethodsBlood and tumor samples were analyzed. A concordance analysis of germline BRCAm status (BRACAnalysis® CLIA test) and tumor BRCAm status (myChoice® CDx test) was conducted (Myriad Genetic Laboratories, Inc.). Bi-allelic loss of BRCA1 and BRCA2 and a genomic instability score (GIS) (myChoice® CDx test) were also determined. Results289 of 295 enrolled patients had a germline BRCAm confirmed centrally and tumor BRCAm status was evaluable in 241 patients. There was 98% and 100% concordance between tumor and germline testing for BRCA1m and BRCA2m, respectively, with discordance found in four cases. Of 210 tumor samples evaluable for BRCA zygosity, 100% of germline BRCA1-mutated tumors (n = 144) and 98% of germline BRCA2-mutated tumors (n = 66) had bi-allelic loss of BRCA. One patient with a heterozygous BRCA2m had a GIS of 53, was progression free for 911 days and remained on olaparib at data cut-off. ConclusionsVery high concordance was demonstrated between tumor and germline BRCA testing, supporting wider implementation of tumor BRCA testing in ovarian cancer. Near 100% rates of bi-allelic loss of BRCA in platinum-sensitive relapsed ovarian tumors suggest routine testing for BRCA zygosity is not required in this population and reflects BRCA loss being a driver of tumorigenesis.

P103 - Germ cell tumors with malignant somatic transformation: Genomic profile by FoundationOne CDx test

P103 - Germ cell tumors with malignant somatic transformation: Genomic profile by FoundationOne CDx test

Analysis of mutation status and homologous recombination deficiency in tumors of patients with germline BRCA1 or BRCA2 mutations and metastatic breast cancer: OlympiAD

Presence of a germline BRCA1 and/or BRCA2 mutation (gBRCAm) may sensitize tumors to poly(ADP-ribose) polymerase (PARP) inhibition via inactivation of the second allele, resulting in gene-specific loss of heterozygosity (gsLOH) and homologous recombination deficiency (HRD). Here we explore whether tissue sample testing provides an additional route to germline testing to inform treatment selection for PARP inhibition. In this prespecified exploratory analysis, BRCA1 and/or BRCA2 mutations in blood samples (gBRCAm) and tumor tissue (tBRCAm) were analyzed from patients with human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer and known gBRCAm, enrolled in the phase III OlympiAD trial. The frequency and nature of tBRCAm, HRD score status [HRD-positive (score ≥42) versus HRD-negative (score <42) using the Myriad myChoice® CDx test] and rates of gsLOH were determined, and their impact on clinical efficacy (objective response rate and progression-free survival) was explored. Tissue samples from 161/302 patients yielded tBRCAm, HRD and gsLOH data for 143 (47%), 129 (43%) and 125 (41%) patients, respectively. Concordance between gBRCAm and tBRCAm was 99%. gsLOH was observed in 118/125 (94%) patients [BRCA1m, 73/76 (96%); BRCA2m, 45/49 (92%)]. A second mutation event was recorded for two of the three BRCA1m patients without gsLOH. The incidence of HRD-negative was 16% (21/129) and was more common for BRCA2m (versus BRCA1m) and/or for hormone receptor-positive (versus triple-negative) disease. Olaparib antitumor activity was observed irrespective of HRD score. gBRCAm identified in patients with HER2-negative metastatic breast cancer by germline testing in blood was also identified by tumor tissue testing. gsLOH was common, indicating a high rate of biallelic inactivation in metastatic breast cancer. Olaparib activity was seen regardless of gsLOH status or HRD score. Thus, additional tumor testing to inform PARP inhibitor treatment selection may not be supported for these patients.

Olaparib treatment (Tx) in patients (pts) with platinum-sensitive relapsed ovarian cancer (PSR OC) by BRCA mutation (BRCAm) and homologous recombination deficiency (HRD) status: Overall survival (OS) results from the phase II LIGHT study.

5515 Background: LIGHT (NCT02983799) evaluated olaparib Tx in pts with PSR OC in cohorts with known BRCAm and HRD status. We report the final OS analyses. Methods: We conducted anopen-label, non-randomized, multicenter study of pts with PSR OC and ≥1 prior line of platinum-based chemotherapy (CTx). Pts were assigned to one of 4 cohorts: germline (g) BRCAm; somatic (s) BRCAm; HRD +ve (non-BRCAm); and HRD -ve. Genomic instability score (GIS) and gBRCAm status were determined by Myriad myChoice and BRACAnalysis CDx tests, respectively. HRD +ve tumors were defined by a GIS ≥42. Pts received olaparib Tx (starting dose 300 mg bid) until disease progression or unacceptable toxicity. OS was a secondary endpoint and was analyzed at 12 months (mo) after the primary analysis and 18 mo after the last pt was enrolled. Safety was assessed in pts who received ≥1 dose. Results: Data cut-off (DCO) was Aug 27, 2020. Of 272 enrolled pts, 271 received olaparib; of these, 270 had measurable disease at baseline and were included in efficacy analyses (Table). At DCO, 40% of pts had died (maturity) with a median follow-up in censored pts of 26.3 mo. Kaplan–Meier 18-mo OS rates were 86%, 88%, 79%, and 60% in the gBRCAm, sBRCAm, HRD +ve (non-BRCAm), and HRD -ve cohorts, respectively. Platinum-based CTx was the most common first subsequent Tx and was received by 39% pts after olaparib discontinuation. At DCO, the median duration of Tx was 7.4 mo and 244 pts had discontinued treatment, mainly due to disease progression (72%); 5% discontinued due to treatment-emergent adverse events (TEAEs). The only TEAE leading to discontinuation in &gt;1 pt was nausea (2 pts). Serious TEAEs were reported in 25% of pts. The most common serious TEAE was small intestinal obstruction (6%). Three adverse events of special interest occurred, each in 1 pt (&lt;1%): acute myeloid leukemia (post discontinuation), pneumonitis, and pulmonary fibrosis. Conclusions: In the final OS analysis, 18-mo OS ranged from 60–88% across the 4 cohorts. Consistent with the primary analysis, the 18-mo OS rate was highest in the BRCAm cohorts (similar OS in g and sBRCAm); among pts without a BRCAm, 18-mo OS was highest in the HRD +ve cohort. No new safety signals were observed compared with the primary analysis and with prior olaparib studies. Clinical trial information: NCT02983799. [Table: see text]

Best practices for the extraction of genomic DNA from formalin-fixed paraffin-embedded tumor tissue for cancer genomic profiling tests.

Recently, two cancer genomic profiling tests have been approved in Japan and implemented in routine clinical practice: the FDA-approved FoundationOne CDx test, and the OncoGuide NCC Oncopanel test. The quality and quantity of DNA significantly affects the sequencing results; therefore, preparing a sufficient amount of high-quality DNA for clinical cancer genomic profiling tests is important. We examined the best practices for the extraction of cancer genomic DNA from formalin-fixed paraffin-embedded (FFPE) tumor tissues of pancreatic, lung and colon cancer specimens. We found that the quality of cancer genomic DNA extracted from 10-μm-thick FFPE samples improved significantly, compared with that from 4-μm-thick FFPE samples, suggesting that 10-μm-thick FFPE samples are preferable for clinical cancer genomic profiling tests. For convenience, we created a quick reference table for calculating the required number of FFPE slides.

Clinical influence of switching companion diagnostic tests for EGFR-TKs from Therascreen to Cobas v2.

BackgroundSeveral companion diagnostic (CDx) tests for epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs) have been approved. In our institute, the CDx test for EGFR‐TKIs was changed from the Therascreen test (Therascreen) to the Cobas EGFR v2 test (Cobas) because only Cobas was approved for the use of osimertinib in patients with EGFR‐mutated non‐small cell lung cancer (NSCLC) with T790M mutations. The clinical influence of switching the CDx test has not yet been examined comprehensively.MethodsAll serial patients with lung cancer tested for EGFR mutations with CDx tests between February 2014 and February 2016 at the Cancer Institute Hospital of the Japanese Foundation for Cancer Research (JFCR) were enrolled in this analysis.ResultsTherascreen was used as a CDx test for EGFR‐TKI therapy in 607 patients between February 2014 and January 2015, and Cobas was used in 621 patients between February 2015 and February 2016. EGFR mutations were detected in 218 patients (35.9%) and 244 patients (39.3%) tested with Therascreen and Cobas, respectively. At the initial diagnosis, 400 and 459 patients were tested with Therascreen and Cobas, respectively. EGFR mutation subtypes, including del19, L858R, and others, were detected in 13.0%, 17.0%, and 2.5% of patients using Therascreen and 17.4%, 14.4%, and 1.5% of patients using Cobas, respectively.ConclusionsNo significant impact of switching from Therascreen to Cobas as the CDx test for EGFR mutations in clinical practice was observed. However, the detection pattern of the EGFR mutation subtypes between the two CDx tests was slightly different.Key pointsSignificant findings of the studyWe examined the influence of changing the EGFR test in 1228 patients in total. The detection rate of EGFR mutations was similar. However, the detection pattern for EGFR subtype mutations was slightly different between the two tests.What this study addsSwitching CDx tests from target polymerase chain reaction (PCR)‐ to next‐generation sequencing (NGS)‐based methods may lead to obvious changes in clinical practice. When the CDx test is required to change, the investigation of this influence is warranted in future studies.

When should we order a next generation sequencing test in a patient with cancer?

Technical advances in genome sequencing and the implementation of next-generation sequencing (NGS) in clinical oncology have paved the way for individualizing cancer patient therapy based on molecular profiles. When and how to use NGS testing in the clinic is at present an unsolved issue, although new research results provide evidence favoring this approach in some types of advanced cancer. Clinical research is evolving rapidly, from basket and umbrella trials to adaptative design precision oncology clinical studies, and genomic and molecular data often displace the classical clinical validation procedures of biomarkers. In this context, physicians must be aware of the clinical evidence behind these new biomarkers and NGS tests available, in order to use them in the right moment, and with a critical point of view. This review will present the status of currently available targeted drugs that can be effective based on actionable molecular alterations, and the NGS tests that are currently available, offering a practical guide for the application of Clinical Precision Oncology in the real world routine practice.

A combined genomic and immune profile in patients with head and neck squamous cell carcinoma treated with immunotherapy: A pilot study.

e18506 Background: In head and neck squamous cell carcinoma (HNSCC) only a small subset of patients (pts) really benefits from immunotherapy, suggesting the need of validated molecular and immunological predictive biomarkers. The aim of the study was to evaluate both the genomic and immune profile of HNSCC correlating it to early progression to immunotherapy. Methods: This is a pilot study evaluating immune and molecular profile in platinum-refractory HNSCC pts treated with Nivolumab. Blood samples were collected at baseline (T0) and at second cycle of therapy (T1). The immune profile was studied by multiplex assay, evaluating circulating: CD137, CTLA4, PD1, PDL1, PDL2, CD27, TIM3, LAG3, GITR, HVEM, BTLA, CD80, CD28 and IDO. The comprehensive genomic profile was evaluated at baseline on formalin-fixed paraffin-embedded samples of the tumor through Foundation One Cdx test. The results obtained were correlated with early progression (within 3 months from the start of therapy). Results: Ten pts were enrolled in the study. Early progression occurred in 6 pts (60%). Median progression free survival and overall survival were 3 months (1-11) and 6.5 months (1-12), respectively. In all patients ≥ 5 genetic mutations were detected; median number of mutation was 6 (5-14); 8/10 pts had a tumor mutational burden less than 10 Muts/mb. The more frequent genetic alterations involved the cycline-dependent-kynase pathway (9/10), the transcription factor associated gene TP53 (8/10) and the PI3K-Akt-PTEN signaling pathways (5/10). Less frequent alterations involved FGFR family (4/10), NOTCH signaling pathway (3/10) and TERT (4/10). Early progression was slightly associated with the number of mutations detected (p = 0.06). The rising or falling trend of circulating levels of biomarkers were detected in 9/10 pts and included modification in IDO (1/10), LAG3 (3/10), GITR (3/10), BTLA (3/10), CD137 (4/10), CTLA4 (4/10), PD1 (2/10), Tim3 (3/10), CD28 (3/10), HVEM (3/10), CD80 (2/10). No modifications were recorded in PDL1, PDL2 and CD27. Conclusions: Our results, although in a small cohort, highlighted the complexity and heterogeneity of landscape in HNSCC pts. A novel evaluation that combines molecular and immune profile is needed. In the context of a poor prognosis disease the combination of personalized molecular and immune approaches could represent a promising strategy to improve survival.

Olaparib treatment in patients (pts) with platinum-sensitive relapsed (PSR) ovarian cancer (OC) by BRCA mutation (BRCAm) and homologous recombination deficiency (HRD) status: Phase II LIGHT study.

6013 Background: In Study 19 (NCT00753545), olaparib capsules demonstrated improvement in progression-free survival (PFS) vs placebo in the PSR OC maintenance setting, irrespective of BRCAm status (Ledermann et al. Lancet Oncol 2014). LIGHT is the first prospective study to evaluate olaparib tablet treatment in PSR OC pts by BRCAm and HRD status. Methods: This is an open-label, non-randomized study (NCT02983799) that assessed efficacy and safety of olaparib monotherapy (300 mg BID) in pts with PSR, high-grade serous/endometrioid epithelial OC and ≥1 prior line of platinum chemotherapy. Pts were assigned to one of four cohorts: germline (g) BRCAm; somatic (s) BRCAm; HRD+ve (non-BRCAm); HRD–ve; by Myriad BRACAnalysis CDx and myChoice tests. HRD+ve was a score ≥42. Primary endpoint was objective response rate (ORR). Secondary endpoints included: disease control rate (DCR) and investigator-assessed PFS (RECIST v1.1). Primary analysis was to be ~6 months (mo) after the last pt was enrolled. Results: Data cut off was 8/27/19. Of 271 pts treated (median of 31.7 weeks [2.1–96.0]), 270 had measurable disease at baseline and were included in efficacy analyses (Table). The most common treatment-emergent adverse events (AEs) were nausea (66%) and fatigue (62%).Serious AEs and Grade ≥3 AEs were experienced by 25% and 44% of pts, respectively. AEs leading to olaparib dose interruptions, reductions and discontinuations occurred in 33%, 24% and 4% of pts, respectively. Conclusions: Olaparib treatment demonstrated activity across all cohorts. As observed in the maintenance setting, similar efficacy was seen in the gBRCAm and sBRCAm cohorts. For non-BRCAm pts, longer median PFS and higher ORR were observed in the HRD+ve cohort. Olaparib treatment was well tolerated with no new safety signals identified and a safety profile consistent with that seen in the PSR and first-line settings. Clinical trial information: NCT02983799. [Table: see text]

Targeted Molecular Therapy in Palliative Cancer Management

Acknowledging the correlation of response to therapy based on the “targeting the target” concept, FDA demonstrated confidence in precision therapy approaches by approving Foundation One® CDx test in late 2017 as an indicated diagnostic for cancer patients. More than 100 precision therapies involving both solid and liquid malignancy have since been approved by FDA as indicated therapy in a variety of cancer types as related to correlated molecular target. We provide clinical justification of consideration for precision therapy guided by matched molecular target, specifically focusing on PI3K/mTOR/AKT, BRCA, CDK4/6, EGFR and BRAF V600E for advanced disease cancer patients who previously failed optimal NCCN guideline directed standard of care.

Feasibility of next-generation sequencing in clinical practice: results of a pilot study in the Department of Precision Medicine at the University of Campania ‘Luigi Vanvitelli’

BackgroundThe emerging role of next-generation sequencing (NGS) targeted panels is revolutionising our approach to cancer patients, providing information on gene alterations helpful for diagnosis and clinical decision, in a short time and with acceptable costs.Materials and methodsIn this work, we evaluated the clinical application of FoundationOne CDx test, a hybrid capture-based NGS. This test identifies alterations in 324 genes, tumour mutational burden and genomic signatures as microsatellite instability. The decision to obtain the NGS assay for a particular patient was done according to investigator’s choice.ResultsOverall, 122 tumour specimens were analysed, of which 84 (68.85%) succeeded. The success rate was influenced by type of specimen formalin-fixed paraffin embedded (FFPE block vs FFPE slides), by origin of the sample (surgery vs biopsy) and by time of fixation (<5 years vs ≥5 years). The most frequent subgroups of effective reports derived from colorectal cancer (25 samples), non-small-cell lung cancer (16 samples), ovarian cancer (10 samples), biliary tract cancer (9 samples), breast cancer (7 samples), gastric cancer (7 samples). The most frequent alterations found in whole population referred to TP53 (45.9%), KRAS (19.6%) and APC (13.9%). Furthermore, we performed an analysis of patients in whom this comprehensive genomic profiling (CGP) had a relevance for the patient’s disease.ConclusionsOn our opinion, CGP could be proposed in clinical practice in order to select patients that could most benefit from the analysis proposed, like patients with good performance status without any available treatments or with unexpected resistance to a therapy.

Cdx Report Program: Heterogeneity Revealed in Current Reporting Practices for Hemato-Oncology Companion Diagnostic (CDx) Markers in Multiple Countries

Introduction: In the current era of precision medicine, CDx tests have become the basis for optimal patient care and targeted treatment. While technical aspects of a CDx assay's performance are audited regularly by participation in external quality assessment (EQA) programs, the reporting of these CDx markers is rarely scrutinized. The aim of this first of its kind CDx report program is to get a detailed picture of hemato-oncology CDx marker reporting across multiple countries. Therefore, we evaluated multiple laboratory parameters and reviewed actual content of clinical CDx reports. Methods: Thirty-four clinical labs from Germany, Italy, Spain, and the United Kingdom provided information for 7 biomarkers used for treatment decisions in hemato-oncology. An identical program is already running in Australia and the United States and will be initiated shortly in India and China. The CDx markers covered in this CDx report program were: BCR-ABL1 in chronic myeloid leukemia (CML), at diagnosis; BCR-ABL1 in CML, minimal residual disease (MRD); IDH1/2 in acute myeloid leukemia (AML); FLT3-ITD in AML; FLT3-TKD in AML; IGHV in chronic lymphocytic leukemia (CLL); and TP53 in CLL. The information requested from participating laboratories included two anonymized or blank reports: one with a positive/mutant result and one with a negative/wild-type result. The received anonymized reports were reviewed by experts within each country according to pre-agreed criteria. In addition, labs participated in a short online survey evaluating test volumes, turnaround times (TATs), positivity rates, participation in an EQA program, and status regarding accreditation and reimbursement. The results of the questionnaires were forwarded as anonymized and aggregated data to reviewing experts. Results: Overall, we received 184 survey datasets and 179 sets of anonymized reports. The review of the anonymized reports according to pre-defined criteria revealed differences in the way CDx results are represented and interpreted in clinical reports. Since not all markers covered in this program were tested by all participating labs, the number of survey datasets per CDx marker ranged from 16 to 34 (IGHV: 16; TP53: 19; IDH1/2: 24; FLT3-TKD: 28; FLT3-ITD: 30; BCR-ABL1 [MRD]: 33; and BCR-ABL1 [diagnosis]: 34). The 184 survey datasets represented more than 7000 tests per month (Figure). The stated average TAT across all covered markers was 6.9 days, ranging from 5.3 days to 8.6 days in the covered countries. The TATs for the individual markers ranged from 4.4 days to 9.4 days (FLT3-ITD: 4.4 days; FLT3-TKD: 4.6 days; BCR-ABL1 [diagnosis]: 5.3 days; BCR-ABL1 [MRD]: 6.9 days; IDH1/2: 8.4 days; IGHV: 9.3 days; and TP53: 9.4 days). In 103/179 (58%) datasets labs participated in EQA programs, and in 76/179 (42%) datasets labs did not participate in EQA programs. For 84/180 (47%) datasets, labs were ISO15189-accredited; for 12/180 (7%) datasets, labs were College of American Pathologists (CAP)-accredited; and for 84/180 (47%) datasets, labs were not accredited by either ISO15189 or CAP. Conclusion: CDx report program results reveal a broad range globally in CDx reporting practices. The identified differences in laboratory parameters, such as TAT and the actual content of clinical CDx reports, suggest a need for international harmonization of CDx reporting. The anonymized and aggregated data generated provide the basis for other initiatives and may support guideline updates and the international harmonization of CDx reporting. Figure. Estimated number of companion diagnostic tests per month covered in the CDx report program. Figure Disclosures Delic: Diaceutics: Employment, Equity Ownership. Blombery:Janssen: Honoraria; Novartis: Consultancy; Invivoscribe: Honoraria. Calasanz:Janssen: Honoraria; Diaceutics: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria. Colomer:Novartis: Honoraria; Incyte: Honoraria. Evans:Diaceutics: Honoraria; Novartis: Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Mason:Novartis: Honoraria; Jazz Pharmaceuticals: Honoraria; AbbVie: Honoraria. Thiede:Daiichi Sankyo: Honoraria; Diaceutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; AgenDix GmbH: Employment, Equity Ownership. Clark:Diaceutics: Employment, Equity Ownership.

Evaluation of rucaparib and companion diagnostics in the PARP inhibitor landscape for recurrent ovarian cancer therapy.

Rucaparib camsylate (CO-338; 8-fluoro-2-{4-[(methylamino)methyl]phenyl}-1,3,4,5-tetrahydro-6H-azepino[5,4,3-cd]indol-6-one ((1S,4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]hept-1-yl)methanesulfonic acid salt) is a PARP1, 2 and 3 inhibitor. Phase I studies identified a recommended Phase II dose of 600 mg orally twice daily. ARIEL2 Part 1 established a tumor genomic profiling test for homologous recombination loss of heterozygosity quantification using a next-generation sequencing companion diagnostic (CDx). Rucaparib received US FDA Breakthrough Therapy designation for treatment of platinum-sensitive BRCA-mutated advanced ovarian cancer patients who received greater than two lines of platinum-based therapy. Comparable to rucaparib development, other PARP inhibitors, such as olaparib, niraparib, veliparib and talazoparib, are developing CDx tests for targeted therapy. PARP inhibitor clinical trials and CDx assays are discussed in this review, as are potential PARP inhibitor combination therapies and likely resistance mechanisms.

Analytical Performance of Nanostring's Companion Diagnostic (CDx) Lymphoma Subtyping Test (LST) with Diffuse Large B-Cell Lymphoma Core Needle Biopsy Samples

Background: Diffuse large B-cell lymphoma (DLBCL) is an aggressive non-Hodgkin's lymphoma with two distinct molecular cell-of-origin (COO) subtypes known as germinal center B-cell (GCB) or activated B-cell (ABC). DLBCL subtypes have been reported to be prognostic and potentially predictive of treatment benefit, underscoring the need for a precise and accurate CDx test (Roschewski, Nat Rev Clin Oncol, 2014). NanoString's LST was developed to enable identification of the COO subtypes on the nCounter® Dx Analysis System using formalin fixed paraffin embedded (FFPE) tissue specimens based on the previously developed Lymph2Cx gene expression profiling assay (Scott, Blood 2014). The test assigns the COO subtypes based on a Linear Predictor Score (LPS) that is calculated using a weighted sum of the gene expression. The LST is currently being used as a CDx in a phase III global clinical validation trial (clinicaltrials.gov NCT02285062) to identify newly diagnosed DLBCL patients of the ABC type who may preferentially respond to lenalidomide.The LST was previously reported to be a highly precise and accurate method of determining DLBCL subtypes (ABC and GCB) when using FFPE excisional/surgical biopsy tissue specimens as a test input (Wallden, JCO 2015). Although excisional biopsies are recommended for DLBCL diagnosis by current ESMO and NCCN guidelines, recent studies have reported excellent sensitivity and specificity in diagnosing malignant lymphomas by core needle biopsy (CNB) leading to increased usage of this method (Hu Q, Am J Clin Pathol, 2013, de Kerviler, Best Pract Res Cl Ha, 2012). The aim of the current study was to assess the analytical performance of the LST when using CNB samples as a test input.Methods: Recently archived CNB and small biopsy DLBCL samples were procured from Oregon Health & Science University (Portland, OR) (n=23), Hôpital Saint-Louis (Paris, France) (n=19), and Asterand Bioscience (n=12). Both nodal and extra-nodal tissue sites were included. Pathology review was performed on an H&E stained slide from each tissue sample to identify the area of DLBCL. Unstained slide mounted FFPE tissue sections (5 µm) were prepared from each sample for assay processing. The RNA extraction and LST procedures were the same as reported previously for surgical biopsy samples (Wallden, JCO 2015). The number of sections used for each RNA extraction was based on the tumor surface area (TSA) measured on the slide (typically 4-6 slides for TSA<10 mm2; 2-4 slides for TSA≥10 mm2). Multiple extractions were performed and subsequently tested independently for each sample (n=148 total). Additional variables tested in this study were user (n=2), reagent lot (n=2), and instrument (n=2). The reproducibility of the test outputs were evaluated using a linear mixed model (LMM) for LPS variance and concordance for subtype call. Principal component analysis (PCA) was performed on the gene expression profile to determine the major source(s) of variability.Results: Of the 54 total samples tested, 51 provided test results (failures were due to RNA degradation). With the 51 passing samples, an assay pass rate of 97% (144/148) was achieved. The total tissue area input into the test was correlated to RNA yield (R2 =0.54). The LPS was highly reproducible across sample replicates (Figure 1) with a total assay standard deviation of 59 LPS units (<2% of the LPS range). This estimate was similar to surgical biopsy samples (76 LPS units). The subtype concordance was estimated as 96-98% (between users) where 2 of 51 samples with LPS scores close to the thresholds moved from ABC or GCB to unclassified, or vice versa. A PCA of the gene expression data demonstrated that the first principle component is highly correlated to the LPS providing further evidence that the biology underlying the LST is the major driver of algorithm gene expression in CNB.Conclusions: In this study we demonstrated robust performance of the LST using FFPE DLBCL CNB/small biopsy samples. Our data support that sufficient RNA yield and gene expression quality can be achieved from biopsy samples with measured TSA of 2 mm2 or greater. We also show that the analytical performance of the LST is comparable between CNB/small biopsy samples and surgical biopsies and that the principal component of gene expression variation is LPS. The LST sample input requirements for the ongoing phase III trial will be updated to include CNB/small biopsy samples to accommodate clinical practice. [Display omitted] DisclosuresStorhoff:NanoString Technologies, Inc.: Employment, Other: Stock . Wallden:NanoString Technologies, Inc.: Employment, Other: Stock. Braziel:Oregon Health & Sciences University: Employment. Thieblemont:St. Louis Hospital, Paris, France: Employment. Hood:NanoString Technologies, Inc.: Employment, Other: Stock. Ravi:NanoString Technologies, Inc.: Employment, Other: Stock. Dennis:NanoString Technologies, Inc.: Employment, Other: Stock. Dowidar:NanoString Technologies, Inc.: Employment, Other: Stock. Danaher:NanoString Technologies, Inc.: Employment, Other: Stock. Dunlap:Oregon Health & Sciences University: Employment. Briere:St. Louis Hospital, Paris, France: Employment. de Kerviler:St. Louis Hospital, Paris, France: Employment. Ferree:NanoString Technologies, Inc.: Employment, Other: Stock, Patents & Royalties: NanoString Technologies, Inc..

Abstract B128: Comparative assessment of MALDI-TOF platforms for utility in CDx development of a proteomic signature for ficlatuzumab

Abstract BDX004 is a multivariate serum proteomics companion diagnostic (CDx) test currently under development as part of a global, randomized, double-blind Phase 2 clinical study, FOCAL. The test will be used prospectively to select previously untreated, EGFR mut-positive patients with advanced NSCLC to receive one of two treatments: (1) the combination of ficlatuzumab (a humanized mAb against hepatocyte growth factor) and an EGFR tyrosine kinase inhibitor (TKI), or (2) an EGFR TKI with placebo. BDX004 measures the abundance of eight serum protein variants using MALDI-TOF mass spectrometry, the data from which is coupled with pre-processing steps and a k-nearest neighbors (kNN) algorithm to classify subjects into positive or negative groups. To date, clinical oncology assays using mass spectrometry, including those to measure serum proteomic profiles in NSCLC, have exclusively been run on instruments designed for use as research use only. Here, we describe a comparative assessment of three MALDI-TOF platforms (microflex™, autoflex™ and ultraflex™). The microflex LT we used is an RUO version of Bruker Daltonics' FDA cleared MALDI Biotyper CA system. All three instrument platforms were qualified with at least two independent sample sets and achieved &amp;gt;97% classification label concordance (Positive and Negative) with a gold standard data set. In summary, the microflex produced results equivalent to the other, higher performance MALDI-TOF MS instruments. In initial studies, the microflex showed similar resolution (R &amp;gt; 600 at 6632.1 m/z) and sensitivity (S/N ≥ 50 with 500 fmoles BSA in 100 laser shots) performance parameters as the autoflex and ultraflex instruments. The microflex was further evaluated with several additional independent sample sets [n = 4 (8 replicates; 2000 spectra/replicate), n = 67 (3 replicates; 2000 spectra/replicate), and n = 20 (3 replicates; 2000 spectra/replicate)] and achieved 100%, &amp;gt;97%, and 100% classification label concordance against reference data sets. At a quantitative level we also measured the feature value (protein peak) CVs that underlie the classification labels. These were found to be highly comparable across all three platforms at 5.7%, 6.4% and 4.8% for the microflex, autoflex and ultraflex, respectively. We conclude that all three of the platforms tested demonstrated equivalent performance, and the microflex LT is well suited for the development of novel MALDI-TOF CDx tests. Citation Format: Nicholas F. Dupuis, Maximillian Steers, Amanda Weaver, Zhigang Weng, Francis C. Payumo, Michael N. Needle, Gary A. Pestano. Comparative assessment of MALDI-TOF platforms for utility in CDx development of a proteomic signature for ficlatuzumab. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B128.

Abstract 2011: Feasibility of serum proteomic companion diagnostic (CDx) test development on the microflex LT platform

Abstract Clinical oncology assays using mass spectrometry have exclusively been run on instrument platforms designed for use in research laboratories. Here, we report on the use of a bench-top MALDI-TOF mass spectrometer (MS) to run a serum proteomic Laboratory Developed Test (LDT), VeriStrat®. A number of previous studies have shown that this assay measures a serum proteomic fingerprint using MALDI-TOF MS which, following spectral acquisition and data processing, classifies advanced non-small cell lung cancer (NSCLC) patients into treatment categories. An extension of VeriStrat, BDX004, is currently under development as part of a global, randomized, double-blind Phase 2 clinical study, FOCAL. BDX004 will be used prospectively to select previously untreated, EGFR mutation-positive patients with advanced NSCLC to receive one of two treatments: (1) the combination of ficlatuzumab (a humanized monoclonal antibody against hepatocyte growth factor) and an EGFR tyrosine kinase inhibitor (TKI), or (2) an EGFR TKI with placebo. For the development of BDX004 as a companion diagnostic (CDx), we have included the microflex™ LT (LT), a component of Bruker's MALDI Biotyper CA system, which was granted FDA clearance for the identification of Gram negative bacteria. In the present study, we evaluated the LT platform through comparative studies with other MALDI-TOF instruments (autoflex™ III and autoflex SPEED) utilizing samples from patients previously diagnosed with NSCLC. In summary, we found that under common laboratory procedures the LT produces results equivalent to research grade MALDI-TOF MS instrumentation. In initial studies, the microflex LT showed similar resolution (R &amp;gt; 600 at 6632.1 m/z) and sensitivity (S/N ≥ 50 with 500 fmoles BSA in 100 laser shots) performance parameters as the autoflex series research instruments (R &amp;gt; 800 at 6632.1 m/z and S/N ≥ 50 with 500 fmoles BSA in 250 laser shots). The LT was qualified with several independent sample sets [n = 4 (8 replicates; 2000 spectra/replicate); n = 67 (3 replicates; 2000 spectra/replicate) and n = 20 (3 replicates; 2000 spectra/replicate)]. In these studies, the LT achieved 100%, &amp;gt;97%, and 100% concordance with reference data sets previously acquired on the autoflex platforms. As expected, due to the lower laser repetition rate of the LT's nitrogen laser, the acquisition speed was slower on the LT as compared to the autoflex instruments by a factor of two. However, the slower acquisition speed is partially compensated for with increased sensitivity, which is particularly evident in the reference sample mass spectra which have signal-to-noise ratios ∼1.5 greater than those acquired on the autoflex platform. Based on these results, the microflex LT appears to be suitable for measurement of serum proteomic profiles in NSCLC and suggests that the inclusion of the microflex LT in CDx test development has the potential to expand the utility of MALDI-based testing in the clinic. Citation Format: Nicholas Dupuis, Jamie Chang, Maximillian Steers, Zhigang Weng, Jeno Gyuris, Gary Pestano. Feasibility of serum proteomic companion diagnostic (CDx) test development on the microflex LT platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2011. doi:10.1158/1538-7445.AM2015-2011

Economic and technical aspects from biomarker identification to companion diagnostic - Co development

In view of an urgent need for novel therapeutics that have a positive impact on morbidity and mortality of chronic diseases, the field is now moving more quickly towards clinical translation from biomarker identification to the development of an companion diagostic (CDx) test. The implimentation of such CDx test is driven by smart preclinical validation, a better study design and improved surrogate readouts using currently available methodologies and diagnostic techniques. Moreover, upcoming novel biomarkers and diagnostic technologies will soon permit a more accurate and efficient assessment of disease progression and regression. The ability of biomarkers to improve treatment and reduce health-care costs is potentially greater than in any other area of current medical research. For example, the American Society of Clinical Oncology estimates that routinely testing people with colon cancer for mutations in the K-RAS oncogene would save at least US $600 million a year. On the other side, many papers in the course of biomarker discovery projects have been published yet, but only few clinically useful biomarkers have been patented and successful validated for routine clinical practice. The reasons for the limitation to bring biomarkers successful on the market are manifold. Regulatory and scientific hurdles as well as lack in the biomarker validation and lack in the technical validation have been cited as one of the main reasons for the slow growth in this area. Whilst the majority of the published biomarkers will not be translated into diagnostic tests, the growth of biomarker used in clinical diagnostics indicates that the future of the industry will lie in the CDx co-development. The first drug introduced using the CDx co – development – Herceptin (trastuzumab; Roche/Genentech) – has now been on the market for several years. Our study focus on some economics and technical factors that should be considered to successfully develop and market a companion diagnostic test, based on lessons learned from recent case studies. A proposed framework of considerations to be addressed at the various stages of developing effective companion diagnostic test is also presented.

Relative importance of oncologist access and patient pre-selection in limiting the uptake of companion diagnostic tests for solid cancers in the United States.

e22157 Background: The speed of uptake of Companion Diagnostics (CDx) tests in the oncologist community is a product of (1) the number of physicians adopting the test in the months following launch, and (2) the % of patients under those physicians’ care who are being tested. Here, we investigate which one of the two factors was the most significant in terms of limiting the speed of uptake within the initial 3 months after launch of two recently approved CDx tests in the U.S. Methods: A panel of oncologists in the US (N=130) were asked to report on their stated tested levels of EML4-ALK testing for their current stage IIIB and IV NSCLC patients, and BRAF mutation testing for their current stage IV melanoma patients. This data was collected through an online survey between August and November 2012. Results: Of the 130 physicians surveyed, only 5 (4%) did not use EML4-ALK testing for their patients at all, and just under 33% used EML4-ALK testing for all their stage IIIB/IV NSCLC patients. The majority (64%) used EML4-ALK testing for a subset of their stage IIIB/IV NSCLC patients only, indicating they preselected their patients for testing. This compares to as many as 55% of surveyed physicians using BRAF for 100% of their stage IV melanoma patients, with a further 29% using it for a subset of their stage IV melanoma patients and 7% not using the test at all. Conclusions: As the vast majority (96% and 93% respectively) of surveyed physicians used EML4-ALK and BRAF testing for at least a subset of their patients within the first 6 months after launch, a lack of access to the tests did not appear to be a major barrier to uptake. Instead, the % of an individual oncologist’s patients being tested through pre-selection for testing was the primary factor determining the volume of testing. Pre-selection was more common for EML4-ALK testing than for BRAF testing, possibly due to the higher incidence of BRAF mutations than EML4-ALK translocations.