AML cells have low spare reserve capacity in their respiratory chain that renders them susceptible to oxidative metabolic stress.

Mitochondrial respiration is a crucial component of cellular metabolism that can become dysregulated in cancer. Compared with normal hematopoietic cells, acute myeloid leukemia (AML) cells and patient samples have higher mitochondrial mass, without a concomitant increase in respiratory chain complex activity. Hence these cells have a lower spare reserve capacity in the respiratory chain and are more susceptible to oxidative stress. We therefore tested the effects of increasing the electron flux through the respiratory chain as a strategy to induce oxidative stress and cell death preferentially in AML cells. Treatment with the fatty acid palmitate induced oxidative stress and cell death in AML cells, and it suppressed tumor burden in leukemic cell lines and primary patient sample xenografts in the absence of overt toxicity to normal cells and organs. These data highlight a unique metabolic vulnerability in AML, and identify a new therapeutic strategy that targets abnormal oxidative metabolism in this malignancy.

A classification system for clinical relevance of somatic variants identified in molecular profiling of cancer.

PURPOSE: Interpretation systems for clinical laboratory reporting of genetic variants for inherited conditions have been widely published. By contrast, there are no existing systems for interpretation and classification of somatic variants found from molecular testing of cancer. METHODS: We designed an assessment protocol and classification system for somatic variants identified through next-generation sequencing molecular profiling of tumor-derived samples and applied these to a pilot dataset of somatic variants found by next-generation sequencing profiling of 158 tumor samples derived from advanced cancer patients examined at the Princess Margaret Cancer Centre. RESULTS: We present a classification system to interpret the significance of genetic variants in molecular analysis of cancer, including the following key factors: (i) known or predicted pathogenicity of the variant; (ii) primary site and tumor histology in which the variant is found; (iii) recurrence of the variant; and (iv) evidence of clinical actionability. We used these factors to develop a five-category somatic variant classification for simplified reporting of variant interpretations to treating oncologists. CONCLUSION: Our somatic variant classification can be of practical value to other clinical molecular laboratories performing cancer genetic profiling by promoting consistent reporting of somatic variants and permitting harmonization of variant data among laboratories and clinical studies.

Carnitine transporter CT2 (SLC22A16) is over-expressed in acute myeloid leukemia (AML) and target knockdown reduces growth and viability of AML cells.

AML (acute myeloid leukemia) cells have a unique reliance on mitochondrial metabolism and fatty acid oxidation (FAO). Thus, blocking FAO is a potential therapeutic strategy to target these malignant cells. In the current study, we assessed plasma membrane carnitine transporters as novel therapeutic targets for AML. We examined the expression of the known plasma membrane carnitine transporters, OCTN1, OCTN2, and CT2 in AML cell lines and primary AML samples and compared expression to normal hematopoietic cells. Of the three carnitine transporters, CT2 demonstrated the greatest differential expression between AML and normal cells. Using shRNA, we knocked down CT2 and demonstrated that target knockdown impaired the function of the transporter. In addition, knockdown of CT2 reduced the growth and viability of AML cells with high expression of CT2 (OCI-AML2 and HL60), but not low expression. CT2 knockdown reduced basal oxygen consumption without a concomitant increase in glycolysis. Thus, CT2 may be a novel target for a subset of AML.

Select microtubule inhibitors increase lysosome acidity and promote lysosomal disruption in acute myeloid leukemia (AML) cells.

To identify new biological vulnerabilities in acute myeloid leukemia, we screened a library of natural products for compounds cytotoxic to TEX leukemia cells. This screen identified the novel small molecule Deoxysappanone B 7,4' dimethyl ether (Deox B 7,4), which possessed nanomolar anti-leukemic activity. To determine the anti-leukemic mechanism of action of Deox B 7,4, we conducted a genome-wide screen in Saccharomyces cerevisiae and identified enrichment of genes related to mitotic cell cycle as well as vacuolar acidification, therefore pointing to microtubules and vacuolar (V)-ATPase as potential drug targets. Further investigations into the mechanisms of action of Deox B 7,4 and a related analogue revealed that these compounds were reversible microtubule inhibitors that bound near the colchicine site. In addition, Deox B 7,4 and its analogue increased lysosomal V-ATPase activity and lysosome acidity. The effects on microtubules and lysosomes were functionally important for the anti-leukemic effects of these drugs. The lysosomal effects were characteristic of select microtubule inhibitors as only the Deox compounds and nocodazole, but not colchicine, vinca alkaloids or paclitaxel, altered lysosome acidity and induced lysosomal disruption. Thus, our data highlight a new mechanism of action of select microtubule inhibitors on lysosomal function.

A randomized phase II study of cediranib alone versus cediranib in combination with dasatinib in docetaxel resistant, castration resistant prostate cancer patients.

BACKGROUND: Activation of the vascular endothelial growth factor receptor (VEGFR) and the oncogenic Src pathway has been implicated in the development of castration-resistant prostate cancer (CRPC) in preclinical models. Cediranib and dasatinib are multi-kinase inhibitors targeting VEGFR and Src respectively. Phase II studies of cediranib and dasatinib in CRPC have shown single agent activity. METHODS: Docetaxel-pretreated CRPC patients were randomized to arm A: cediranib alone (20 mg/day) versus arm B: cediranib (20 mg/day) plus dasatinib (100 mg/day) given orally on 4-week cycles. Primary endpoint was 12-week progression-free survival (PFS) as per the Prostate Cancer Clinical Trials Working Group (PCWG2). Patient reported outcomes were evaluated using Functional Assessment of Cancer Therapy-Prostate (FACT-P) and Present Pain Intensity (PPI) scales. Correlative studies of bone turnover markers (BTM), including bone alkaline phosphate (BAP) and serum beta-C telopeptide (B-CTx) were serially assayed. Results A total of 22 patients, 11 per arm, were enrolled. Baseline demographics were similar in both arms. Median number of cycles =4 in arm A (range 1-12) and 2 in arm B (range 1-9). Twelve-week PFS was 73 % in arm A versus 18 % in arm B (p = 0.03). Median PFS in months (arm A versus B) was: 5.2 versus 2.6 (95 % CI: 1.9-6.5 versus 1.4-not reached). Most common grade 3 toxicities were hypertension, anemia and thrombocytopenia in arm A and hypertension, diarrhea and fatigue in arm B. One treatment-related death (retroperitoneal hemorrhage) was seen in arm A. FACT-P and PPI scores did not significantly change in either arm. No correlation between BTM and PFS was seen in either arm. CONCLUSIONS: Although limited by small numbers, this randomized study showed that the combination of VEGFR and Src targeted therapy did not result in improved efficacy and may be associated with a worse outcome than VEGFR targeted therapy alone in patients with CRPC. ClinicalTrials.gov number: NCT01260688.

New sources of drugs for hematologic malignancies.

Advancing novel therapeutic agents for the treatment of malignancy into the marketplace is an increasingly costly and lengthy process. As such, new strategies for drug discovery are needed. Drug repurposing represents an opportunity to rapidly advance new therapeutic strategies into clinical trials at a relatively low cost. Known on-patent or off-patent drugs with unrecognized anticancer activity can be rapidly advanced into clinical testing for this new indication by leveraging their known pharmacology, pharmacokinetics, and toxicology. Using this approach, academic groups can participate in the drug discovery field and smaller biotechnology companies can "de-risk" early-stage drug discovery projects. Here, several scientific approaches used to identify drug repurposing opportunities are highlighted, with a focus on hematologic malignancies. In addition, a discussion of the regulatory issues that are unique to drug repurposing and how they impact developing old drugs for new indications is included. Finally, the mechanisms to enhance drug repurposing through increased collaborations between academia, industry, and nonprofit charitable organizations are discussed.

An international consensus on effective, inclusive, and career-spanning short-format training in the life sciences and beyond.

Science, technology, engineering, mathematics, and medicine (STEMM) fields change rapidly and are increasingly interdisciplinary. Commonly, STEMM practitioners use short-format training (SFT) such as workshops and short courses for upskilling and reskilling, but unaddressed challenges limit SFT's effectiveness and inclusiveness. Education researchers, students in SFT courses, and organizations have called for research and strategies that can strengthen SFT in terms of effectiveness, inclusiveness, and accessibility across multiple dimensions. This paper describes the project that resulted in a consensus set of 14 actionable recommendations to systematically strengthen SFT. A diverse international group of 30 experts in education, accessibility, and life sciences came together from 10 countries to develop recommendations that can help strengthen SFT globally. Participants, including representation from some of the largest life science training programs globally, assembled findings in the educational sciences and encompassed the experiences of several of the largest life science SFT programs. The 14 recommendations were derived through a Delphi method, where consensus was achieved in real time as the group completed a series of meetings and tasks designed to elicit specific recommendations. Recommendations cover the breadth of SFT contexts and stakeholder groups and include actions for instructors (e.g., make equity and inclusion an ethical obligation), programs (e.g., centralize infrastructure for assessment and evaluation), as well as organizations and funders (e.g., professionalize training SFT instructors; deploy SFT to counter inequity). Recommendations are aligned with a purpose-built framework-"The Bicycle Principles"-that prioritizes evidenced-based teaching, inclusiveness, and equity, as well as the ability to scale, share, and sustain SFT. We also describe how the Bicycle Principles and recommendations are consistent with educational change theories and can overcome systemic barriers to delivering consistently effective, inclusive, and career-spanning SFT.

The antihelmintic flubendazole inhibits microtubule function through a mechanism distinct from Vinca alkaloids and displays preclinical activity in leukemia and myeloma.

On-patent and off-patent drugs with previously unrecognized anticancer activity could be rapidly repurposed for this new indication given their prior toxicity testing. To identify such compounds, we conducted chemical screens and identified the antihelmintic flubendazole. Flubendazole induced cell death in leukemia and myeloma cell lines and primary patient samples at nanomolar concentrations. Moreover, it delayed tumor growth in leukemia and myeloma xenografts without evidence of toxicity. Mechanistically, flubendazole inhibited tubulin polymerization by binding tubulin at a site distinct from vinblastine. In addition, cells resistant to vinblastine because of overexpression of P-glycoprotein remained fully sensitive to flubendazole, indicating that flubendazole can overcome some forms of vinblastine resistance. Given the different mechanisms of action, we evaluated the combination of flubendazole and vinblastine in vitro and in vivo. Flubendazole synergized with vinblastine to reduce the viability of OCI-AML2 cells. In addition, combinations of flubendazole with vinblastine or vincristine in a leukemia xenograft model delayed tumor growth more than either drug alone. Therefore, flubendazole is a novel microtubule inhibitor that displays preclinical activity in leukemia and myeloma.

The antiparasitic agent ivermectin induces chloride-dependent membrane hyperpolarization and cell death in leukemia cells.

To identify known drugs with previously unrecognized anticancer activity, we compiled and screened a library of such compounds to identify agents cytotoxic to leukemia cells. From these screens, we identified ivermectin, a derivative of avermectin B1 that is licensed for the treatment of the parasitic infections, strongyloidiasis and onchocerciasis, but is also effective against other worm infestations. As a potential antileukemic agent, ivermectin induced cell death at low micromolar concentrations in acute myeloid leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. Ivermectin also delayed tumor growth in 3 independent mouse models of leukemia at concentrations that appear pharmacologically achievable. As an antiparasitic, ivermectin binds and activates chloride ion channels in nematodes, so we tested the effects of ivermectin on chloride flux in leukemia cells. Ivermectin increased intracellular chloride ion concentrations and cell size in leukemia cells. Chloride influx was accompanied by plasma membrane hyperpolarization, but did not change mitochondrial membrane potential. Ivermectin also increased reactive oxygen species generation that was functionally important for ivermectin-induced cell death. Finally, ivermectin synergized with cytarabine and daunorubicin that also increase reactive oxygen species production. Thus, given its known toxicology and pharmacology, ivermectin could be rapidly advanced into clinical trial for leukemia.

Design, synthesis, and in vitro characterization of novel hybrid peptidomimetic inhibitors of STAT3 protein.

Aberrant activation of oncogenic signal transducer and activator of transcription 3 (STAT3) protein signaling pathways has been extensively implicated in human cancers. Given STAT3's prominent dysregulatory role in malignant transformation and tumorigenesis, there has been a significant effort to discover STAT3-specific inhibitors as chemical probes for defining the aberrant STAT3-mediated molecular events that support the malignant phenotype. To identify novel, STAT3-selective inhibitors suitable for interrogating STAT3 signaling in tumor cells, we explored the design of hybrid molecules by conjugating a known STAT3 inhibitory peptidomimetic, ISS610 to the high-affinity STAT3-binding peptide motif derived from the ILR/gp-130. Several hybrid molecules were examined in in vitro biophysical and biochemical studies for inhibitory potency against STAT3. Lead inhibitor 14aa was shown to strongly bind to STAT3 (K(D)=900 nM), disrupt STAT3:phosphopeptide complexes (K(i)=5 µM) and suppress STAT3 activity in in vitro DNA binding activity/electrophoretic mobility shift assay (EMSA). Moreover, lead STAT3 inhibitor 14aa induced a time-dependent inhibition of constitutive STAT3 activation in v-Src transformed mouse fibroblasts (NIH3T3/v-Src), with 80% suppression of constitutively-active STAT3 at 6h following treatment of NIH3T3/v-Src. However, STAT3 activity recovered at 24h after treatment of cells, suggesting potential degradation of the compound. Results further showed a suppression of aberrant STAT3 activity in NIH3T3/v-Src by the treatment with compound 14aa-OH, which is the non-pTyr version of compound 14aa. The effect of compounds 14aa and 14aa-OH are accompanied by a moderate loss of cell viability.

Programmed cell death 4 loss increases tumor cell invasion and is regulated by miR-21 in oral squamous cell carcinoma.

BACKGROUND: The tumor suppressor Programmed Cell Death 4 (PDCD4) has been found to be under-expressed in several cancers and associated with disease progression and metastasis. There are no current studies characterizing PDCD4 expression and its clinical relevance in Oral Squamous Cell Carcinoma (OSCC). Since nodal metastasis is a major prognostic factor in OSCC, we focused on determining whether PDCD4 under-expression was associated with patient nodal status and had functional relevance in OSCC invasion. We also examined PDCD4 regulation by microRNA 21 (miR-21) in OSCC. RESULTS: PDCD4 mRNA expression levels were assessed in 50 OSCCs and 25 normal oral tissues. PDCD4 was under-expressed in 43/50 (86%) OSCCs, with significantly reduced mRNA levels in patients with nodal metastasis (p = 0.0027), and marginally associated with T3-T4 tumor stage (p = 0.054). PDCD4 protein expression was assessed, by immunohistochemistry (IHC), in 28/50 OSCCs and adjacent normal tissues; PDCD4 protein was absent/under-expressed in 25/28 (89%) OSCCs, and marginally associated with nodal metastasis (p = 0.059). A matrigel invasion assay showed that PDCD4 expression suppressed invasion, and siRNA-mediated PDCD4 loss was associated with increased invasive potential of oral carcinoma cells. Furthermore, we showed that miR-21 levels were increased in PDCD4-negative tumors, and that PDCD4 expression may be down-regulated in OSCC by direct binding of miR-21 to the 3'UTR PDCD4 mRNA. CONCLUSIONS: Our data show an association between the loss of PDCD4 expression, tumorigenesis and invasion in OSCC, and also identify a mechanism of PDCD4 down-regulation by microRNA-21 in oral carcinoma. PDCD4 association with nodal metastasis and invasion suggests that PDCD4 may be a clinically relevant biomarker with prognostic value in OSCC.

Assessing the Diagnostic Yield of Targeted Next-Generation Sequencing for Melanoma and Gastrointestinal Tumors.

A common rationale in molecular diagnostic laboratories is that implementation of next-generation sequencing (NGS) enables simultaneous multigene testing, allowing increased information benefit compared with non-NGS assays. However, minimal published data exist to support this justification. The current study compared clinical diagnostic yield of TruSight Tumor 26 Sequencing Panel (TST26) in melanoma, colorectal (CRC), and gastrointestinal stromal (GIST) tumors with non-NGS assays. A total of 1041 formalin-fixed, paraffin-embedded tumors, of melanoma, CRC, and GIST, were profiled. NGS results were compared with non-NGS single-gene or single-variant assays with respect to variant output and diagnostic yield. A total of 79% melanoma and 94% CRC tumors were variant positive by panel testing. TST26 panel improved serine/threonine-protein kinase B-raf (BRAF) variant detection in melanoma compared with single-variant BRAF Val600Glu/Lys (V600E/K) routine tests by 24% and detected variants in genes other than BRAF, NRAS, and KIT, which could impact patient management in 20% additional cases. NGS enhanced diagnostic yield in CRC by 36% when compared with routine single-gene assays. In contrast, no added benefit of NGS-based testing for GIST tumors was observed. TST26 panel either missed or inaccurately called complex insertion/deletion variants in KIT exon 11, which were accurately identified by non-NGS methods. Findings of this study demonstrate the differential impact of cancer site and variant type on diagnostic test information yield from NGS assays.

Latency and interval therapy affect the evolution in metastatic colorectal cancer.

While comparison of primary tumor and metastases has highlighted genomic heterogeneity in colorectal cancer (CRC), previous studies have focused on a single metastatic site or limited genomic testing. Combining data from whole exome and ultra-deep targeted sequencing, we explored possible evolutionary trajectories beyond the status of these mutations, particularly among patient-matched metastatic tumors. Our findings confirm the persistence of known clinically-relevant mutations (e.g., those of RAS family of oncogenes) in CRC primary and metastases, yet reveal that latency and interval systemic therapy affect the course of evolutionary events within metastatic lesions. Specifically, our analysis of patient-matched primary and multiple metastatic lesions, developed over time, showed a similar genetic composition for liver metastatic tumors, which were 21-months apart. This genetic makeup was different from those identified in lung metastases developed before manifestation of the second liver metastasis. These results underscore the role of latency in the evolutionary path of metastatic CRC and may have implications for future treatment options.

Somatic Tumor Variant Filtration Strategies to Optimize Tumor-Only Molecular Profiling Using Targeted Next-Generation Sequencing Panels.

A common approach in clinical diagnostic laboratories to variant assessment from tumor molecular profiling is sequencing of genomic DNA extracted from both tumor (somatic) and normal (germline) tissue, with subsequent variant comparison to identify true somatic variants with potential impact on patient treatment or prognosis. However, challenges exist in paired tumor-normal testing, including increased cost of dual sample testing and identification of germline cancer predisposing variants. Alternatively, somatic variants can be identified by in silico tumor-only variant filtration precluding the need for matched normal testing. The barrier to tumor-only variant filtration is defining a reliable approach, with high sensitivity and specificity to identify somatic variants. In this study, we used retrospective data sets from paired tumor-normal samples tested on small (48 gene) and large (555 gene) targeted next-generation sequencing panels, to model algorithms for tumor-only variants classification. The optimal algorithm required an ordinal filtering approach using information from variant population databases (1000 Genomes Phase 3, ESP6500, ExAC), clinical mutation databases (ClinVar), and information on recurring clinically relevant somatic variants. Overall the tumor-only variant filtration strategy described in this study can define clinically relevant somatic variants from tumor-only analysis with sensitivity of 97% to 99% and specificity of 87% to 94%, and with significant potential utility for clinical laboratories implementing tumor-only molecular profiling.

Improving validation methods for molecular diagnostics: application of Bland-Altman, Deming and simple linear regression analyses in assay comparison and evaluation for next-generation sequencing.

AIMS: A standard approach in test evaluation is to compare results of the assay in validation to results from previously validated methods. For quantitative molecular diagnostic assays, comparison of test values is often performed using simple linear regression and the coefficient of determination (R2), using R2 as the primary metric of assay agreement. However, the use of R2 alone does not adequately quantify constant or proportional errors required for optimal test evaluation. More extensive statistical approaches, such as Bland-Altman and expanded interpretation of linear regression methods, can be used to more thoroughly compare data from quantitative molecular assays. METHODS: We present the application of Bland-Altman and linear regression statistical methods to evaluate quantitative outputs from next-generation sequencing assays (NGS). NGS-derived data sets from assay validation experiments were used to demonstrate the utility of the statistical methods. RESULTS: Both Bland-Altman and linear regression were able to detect the presence and magnitude of constant and proportional error in quantitative values of NGS data. Deming linear regression was used in the context of assay comparison studies, while simple linear regression was used to analyse serial dilution data. Bland-Altman statistical approach was also adapted to quantify assay accuracy, including constant and proportional errors, and precision where theoretical and empirical values were known. CONCLUSIONS: The complementary application of the statistical methods described in this manuscript enables more extensive evaluation of performance characteristics of quantitative molecular assays, prior to implementation in the clinical molecular laboratory.

Minimally Invasive Real-Time Detection of Actionable Mutations in Patients With Metastatic Solid Tumors Using Fine-Needle and Liquid Biopsies.

PURPOSE: Fine-needle biopsy (FNB) and liquid biopsy are minimally invasive methods of tumor sampling that provide feasible means to assess tumor genotypes in real time. However, more data are needed to establish the strength of these methods by benchmarking against the current gold standard methods, core-needle biopsy (CNB) or surgical excision of the tumor. PATIENTS AND METHODS: Eligible patients with advanced solid tumors were prospectively recruited. We performed mutation profiling using matched tumor DNA obtained by CNB, FNB and liquid biopsy, and matrix-assisted laser desorption/ionization time-of-flight custom mass-spectrometry or targeted next-generation DNA sequencing. The actionability of detected mutations was determined using the OncoKB Web tool. Agreement between mutations detected in CNBs, FNBs, and circulating tumor DNA (ctDNA) was examined. RESULTS: Forty-one patients underwent tumor biopsy. Thirty CNBs (73%) and 34 FNBs (83%) had sufficient tumor and DNA for mutation profiling. Median DNA yield from CNB and FNB were 775 ng (interquartile range, 240 to 347 4ng) and 649 ng (interquartile range, 180 to1350 ng), respectively. Of 29 CNB/FNB pairs available for comparison, actionable mutation results were concordant in 28 (96%). Six of nine actionable mutations (67%) that were found by CNB, FNB, or both were detectable in ctDNA. Two additional actionable mutations were found exclusively in ctDNA. CONCLUSION: Optimally processed FNB and liquid biopsy can be used routinely for tumor mutation profiling to identify actionable mutations.

Integration of Technical, Bioinformatic, and Variant Assessment Approaches in the Validation of a Targeted Next-Generation Sequencing Panel for Myeloid Malignancies.

CONTEXT: - Detection of variants in hematologic malignancies is increasingly important because of a growing number of variants impacting diagnosis, prognosis, and treatment response, and as potential therapeutic targets. The use of next-generation sequencing technologies to detect variants in hematologic malignancies in a clinical diagnostic laboratory setting allows for efficient identification of routinely tested markers in multiple genes simultaneously, as well as the identification of novel and rare variants in other clinically relevant genes. OBJECTIVE: - To apply a systematic approach to evaluate and validate a commercially available next-generation sequencing panel (TruSight Myeloid Sequencing Panel, Illumina, San Diego, California) targeting 54 genes. In this manuscript, we focused on the parameters that were used to evaluate assay performance characteristics. DATA SOURCES: - Analytical validation was performed using samples containing known variants that had been identified previously. Cases were selected from different disease types, with variants in a range of genes. Panel performance characteristics were assessed and genomic regions requiring additional analysis or wet-bench approaches identified. CONCLUSIONS: - We validated the performance characteristics of a myeloid next-generation sequencing panel for detection of variants. The TruSight Myeloid Sequencing Panel covers more than 95% of target regions with depth greater than 500×. However, because of unique variant types such as large insertions or deletions or genomic regions of high GC content, variants in CEBPA, FLT3, and CALR required supplementation with non-next-generation sequencing assays or with informatics approaches to address deficiencies in performance. The use of multiple bioinformatics approaches (2 variant callers and informatics scripts) allows for maximizing calling of true positives, while identifying limitations in using either method alone.

Comparison of Next-Generation Sequencing Panels and Platforms for Detection and Verification of Somatic Tumor Variants for Clinical Diagnostics.

Use of next-generation sequencing to detect somatic variants in DNA extracted from formalin-fixed, paraffin-embedded tumor tissues poses a challenge for clinical molecular diagnostic laboratories because of variable DNA quality and quantity, and the potential to detect low allele frequency somatic variants difficult to verify by non-next-generation sequencing methods. We evaluated somatic variant detection performance of the MiSeq and Ion Proton benchtop sequencers using two commercially available panels, the TruSeq Amplicon Cancer Panel and the AmpliSeq Cancer Hotspot Panel Version 2. Both the MiSeq-TruSeq Amplicon Cancer Panel and Ion Proton-AmpliSeq Cancer Hotspot Panel Version 2 were comparable in terms of detection of somatic variants and allele frequency determination using DNA extracted from tumor tissue. Concordance was 100% between the panels for detection of somatic variants in genomic regions tested by both panels, including 27 variants present at low somatic allele frequency (<15%). Use of both the MiSeq and Ion Proton platforms in a combined workflow enabled detection of potentially actionable variants with importance for patient diagnosis, prognosis, or treatment in 49% (305/621) of cases. Overall, a combined workflow using both platforms enabled successful molecular profiling of 96% (621/644) of tumor samples, and provided an approach for verification of somatic variants not amenable to verification by Sanger sequencing (<15% variant allele frequency).

Molecular profiling of advanced solid tumors and patient outcomes with genotype-matched clinical trials: the Princess Margaret IMPACT/COMPACT trial.

BACKGROUND: The clinical utility of molecular profiling of tumor tissue to guide treatment of patients with advanced solid tumors is unknown. Our objectives were to evaluate the frequency of genomic alterations, clinical "actionability" of somatic variants, enrollment in mutation-targeted or other clinical trials, and outcome of molecular profiling for advanced solid tumor patients at the Princess Margaret Cancer Centre (PM). METHODS: Patients with advanced solid tumors aged ≥18 years, good performance status, and archival tumor tissue available were prospectively consented. DNA from archival formalin-fixed paraffin-embedded tumor tissue was tested using a MALDI-TOF MS hotspot panel or a targeted next generation sequencing (NGS) panel. Somatic variants were classified according to clinical actionability and an annotated report included in the electronic medical record. Oncologists were provided with summary tables of their patients' molecular profiling results and available mutation-specific clinical trials. Enrolment in genotype-matched versus genotype-unmatched clinical trials following release of profiling results and response by RECIST v1.1 criteria were evaluated. RESULTS: From March 2012 to July 2014, 1893 patients were enrolled and 1640 tested. After a median follow-up of 18 months, 245 patients (15 %) who were tested were subsequently treated on 277 therapeutic clinical trials, including 84 patients (5 %) on 89 genotype-matched trials. The overall response rate was higher in patients treated on genotype-matched trials (19 %) compared with genotype-unmatched trials (9 %; p < 0.026). In a multi-variable model, trial matching by genotype (p = 0.021) and female gender (p = 0.034) were the only factors associated with increased likelihood of treatment response. CONCLUSIONS: Few advanced solid tumor patients enrolled in a prospective institutional molecular profiling trial were treated subsequently on genotype-matched therapeutic trials. In this non-randomized comparison, genotype-enrichment of early phase clinical trials was associated with an increased objective tumor response rate. TRIAL REGISTRATION: NCT01505400 (date of registration 4 January 2012).