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MC13-0060 Analytical validation of the MPACT assay, a targeted next generation sequencing clinical assay for cancer patient treatment selection
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Six Best Poster Abstracts – Oral Presentations MC13-0026 KRAS mutated plasma DNA as predictor of outcome from irinotecan monotherapy in metastatic colorectal cancer K. Spindler 1 , A.L. Appelt 1 , N. Pallisgaard 2 , R.F. Andersen 2 , A. Jakobsen 1 . 1 Oncology, Vejle Hospital, Vejle, Denmark; 2 Biochemistry, Vejle Hospital, Vejle, Denmark Background: A major proportion of colorectal tumors harbour KRAS mutations, a negative predictive marker for outcome of EGFR targeted therapies combined with irinotecan. The role of mutation status for outcome of chemotherapy alone has only been sparsely investigated and with negative results. Purpose/Objective: We investigated the clinical implications of KRAS mutations in patients treated with irinotecan monotherapy when detected in archival tumor tissue and plasma cell free DNA. Materials and Methods: Patients were included in a prospective nonrandomised phase II and biomarker study (Protocol ID S-20090114). Inclusion criteria were; histopathologically verified mCRC, measurable disease according to RECIST, indication for secondline irinotecan monotherapy according to local guidelines, informed consent to therapy and biobank collection and age ≥18. Patients received irinotecan 350 mg/m2 q3w. Response was evaluated according to RECIST v 1.1. Plasma was obtained from a pre-treatment EDTA blood-sample, and KRAS mutations detected in tumor and plasma using an in-house qPCR. Results: The median number of cycles was 4 (range 1–15), response rate (RR) 13%, and disease control rate (DCR) 57%. Median PFS was 4.6 mo (95% CI 3.7–5.8) and OS 9.5 mo (95% CI 8.4–11.8). Ninety-two patients had matching tumor and blood samples and concordance between tumor and plasma KRAS status (pKRAS) was 82% (69/84). Mutation status in archival tumor tissue did not correlate to efficacy, but none of the patients with mutations detectable in plasma responded to therapy (RR=0). The RR in pKRAS wt patients was 19%, (p=0.014). DCR in pKRAS wt patients was 66%, and 37% in the patients with pKRAS mutations (p=0.01). Tumor mutation status was associated with OS but not PFS, whereas pKRAS had a strong influence on both parameters. Median OS was 13.0 mo (95% CI 9.5–15.1) in pKRAS wt patients, and 7.8 mo (4.6–8.4), in patients with pKRAS mutations, HR 2.26 (95% CI 1.31–3.90), p<0.0001. PFS was 4.6 mo (95% CI 3.3–6.4) and 2.7 mo (95% CI 2.1–4.5), respectively, HR 1.69 (95% CI 1.03–2.77), p=0.01. Cox regression analysis confirmed an independent prognostic value of pKRAS status, but not KRAS tumor status. Conclusions: This study indicates that tumor KRAS has minor clinical impact in patients treated with irinotecan monotherapy compared to plasma KRAS which seems to hold important predictive and prognostic information. These data call for reconsideration of the role of KRAS in mCRC and for validation in a randomised trial.
MC13-0049 Two-stage adaptive cutoff design for building and validating a prognostic biomarker signature M. Polley, E. Polley, E. Huang, B. Freidlin, R. Simon. Biometric Research Branch, National Cancer Institute, Bethesda MD, USA Background: The scientific community has committed expansive resources during the last decade to identify useful biomarkers for clinical use. Purpose/Objective: Cancer biomarkers are frequently evaluated using archived specimens. Routine collection of high quality specimens is an expensive and time-consuming process. Therefore, care should be taken to preserve these precious and scarce resources. Here we propose a novel statistical design to allow efficient evaluation of a panel of biomarkers while making economic use of available resources. Materials and Methods: Motivated by the use of futility monitoring for a treatment effect in clinical trials, here we propose a Two-Stage Adaptive Cutoff (TACO) design that affords the possibility to stop performing biomarker assays if an early evaluation of the model performance indicates that the hypothesized biomarker effect will not be confirmed. Our study design includes four components: (a) signature building with a statistically valid test of
the model performance based on data in stage 1; (b) an early futility stopping if the model performance in stage 1 is unsatisfactory; (c) a completely locked-down biomarker signature in stage 1 with a properly cross-validated cutoff to classify patient status; and (d) an independent validation of the locked-down biomarker signature (including the cutoff) in stage 2. Results: We give an example based on a publicly available dataset to illustrate the use of the procedure. Simulation studies are presented to evaluate the operating characteristics of the design. We demonstrate that with the proposed design, substantial savings in specimens is possible under the null hypothesis when the model performance is undesirable. The practical aspects of the proposed design are discussed. Conclusions: In this work, we propose a statistical design useful for developing and validating a biomarkers signature which includes a stopping rule for futility in the event of poor model performance. Novel statistical designs like this are needed to allow conservation of precious tissue specimens for future research.
MC13-0060 Analytical validation of the MPACT assay, a targeted next generation sequencing clinical assay for cancer patient treatment selection C. Lih 1 , D. Sims 1 , E. Polley 2 , Y. Zhao 2 , M. Mehaffey 1 , T. Forbes 1 , R. Harrington 1 , W. Walsh 1 , P. McGregor 1 , R. Simon 2 , B. Conley 3 , S. Kummar 4 , J. Doroshow 4 , P.M. Williams 1 . 1 Molecular Characterization Laboratory, Frederick National Laboratory for Cancer Research, Frederick, USA; 2 Biometric Research Branch, National Cancer Institute, Shady Grove, USA; 3 Cancer Diagnosis Program, National Cancer Institute, Shady Grove, USA; 4 Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, USA Background: Robust and analytically validated assays are essential for development of targeted cancer therapies. MPACT (Molecular Profiling based Assignment of Cancer Therapeutics) is a proposed clinical trial that randomizes patients who failed standard treatment to therapy targeted or not to their own tumor’s molecular abnormalities. Purpose/Objective: Using next generation sequencing, we developed a multi-analyte somatic variant assay to support the treatment selection for MPACT trial. Here we report the results from analytical validation study. Materials and Methods: The MPACT assay interrogates a total of 59,150 bp that represents minimally, 392 variant loci with reported actionable value to guide treatment decisions. In this analytical validation study, we assessed the sensitivity, specificity, and reproducibility for 5 classes of sequence variants, single nucleotide variant (SNV), insertion and deletion (Indel, 3bp or less), large indel (greater than 3bp), SNV at homopolymeric region (HP, greater than 2 identical bases in a row), and Indel at HP. Results: Using DNA samples derived from a hapmap normal cell-line (CEPH, NA12878) with 27 spiked positive control plasmids and from cultured cancer cell-lines or FFPE xenografts derived from cells harboring known variants, we found that the sensitivity is 99.3% in SNVs, 91.1% in SNVs at HP, 100% in Indels, 66.7% in Indels at HP, and 88.9% at large Indels. Reproducibility analysis in a subset of DNA samples revealed that the interand intra-operator concordances are 95.1% and 98.3% respectively, with greater than 0.99 R square values in allele frequency for detected variants. Sanger sequencing of 22 loci in 5 xenograft samples demonstrated 100% accuracy with MPACT assay results. By sequencing three hapmap normal cell-lines (CEPH, Yoruban, Chinese female) multiple times, we showed that the MPACT assay achieved 100% specificity in reportable range for all 5 classes of variants. In addition, we blindly tested 10 unknown tumor specimens that were genotyped previously by a CLIA assay in another lab and found that the MPACT assay reported accurately all 9 previously identified variants at near identical frequency. Finally, we compared the data from MPACT assay and exome capture sequencing using Hiseq for 5 pairs of clinical tumor specimen and matched blood samples. The high concordance confirmed that the MPACT assay results are validated by an independent NGS platform. Conclusions: This validation study demonstrated that the MPACT assay was well-suited for the intended clinical use.
Six Best Poster Abstracts – Oral Presentations MC13-0026 KRAS mutated plasma DNA as predictor of outcome from irinotecan monotherapy in metastatic colorectal cancer K. Spindler 1 , A.L. Appelt 1 , N. Pallisgaard 2 , R.F. Andersen 2 , A. Jakobsen 1 . 1 Oncology, Vejle Hospital, Vejle, Denmark; 2 Biochemistry, Vejle Hospital, Vejle, Denmark Background: A major proportion of colorectal tumors harbour KRAS mutations, a negative predictive marker for outcome of EGFR targeted therapies combined with irinotecan. The role of mutation status for outcome of chemotherapy alone has only been sparsely investigated and with negative results. Purpose/Objective: We investigated the clinical implications of KRAS mutations in patients treated with irinotecan monotherapy when detected in archival tumor tissue and plasma cell free DNA. Materials and Methods: Patients were included in a prospective nonrandomised phase II and biomarker study (Protocol ID S-20090114). Inclusion criteria were; histopathologically verified mCRC, measurable disease according to RECIST, indication for secondline irinotecan monotherapy according to local guidelines, informed consent to therapy and biobank collection and age ≥18. Patients received irinotecan 350 mg/m2 q3w. Response was evaluated according to RECIST v 1.1. Plasma was obtained from a pre-treatment EDTA blood-sample, and KRAS mutations detected in tumor and plasma using an in-house qPCR. Results: The median number of cycles was 4 (range 1–15), response rate (RR) 13%, and disease control rate (DCR) 57%. Median PFS was 4.6 mo (95% CI 3.7–5.8) and OS 9.5 mo (95% CI 8.4–11.8). Ninety-two patients had matching tumor and blood samples and concordance between tumor and plasma KRAS status (pKRAS) was 82% (69/84). Mutation status in archival tumor tissue did not correlate to efficacy, but none of the patients with mutations detectable in plasma responded to therapy (RR=0). The RR in pKRAS wt patients was 19%, (p=0.014). DCR in pKRAS wt patients was 66%, and 37% in the patients with pKRAS mutations (p=0.01). Tumor mutation status was associated with OS but not PFS, whereas pKRAS had a strong influence on both parameters. Median OS was 13.0 mo (95% CI 9.5–15.1) in pKRAS wt patients, and 7.8 mo (4.6–8.4), in patients with pKRAS mutations, HR 2.26 (95% CI 1.31–3.90), p<0.0001. PFS was 4.6 mo (95% CI 3.3–6.4) and 2.7 mo (95% CI 2.1–4.5), respectively, HR 1.69 (95% CI 1.03–2.77), p=0.01. Cox regression analysis confirmed an independent prognostic value of pKRAS status, but not KRAS tumor status. Conclusions: This study indicates that tumor KRAS has minor clinical impact in patients treated with irinotecan monotherapy compared to plasma KRAS which seems to hold important predictive and prognostic information. These data call for reconsideration of the role of KRAS in mCRC and for validation in a randomised trial.
MC13-0049 Two-stage adaptive cutoff design for building and validating a prognostic biomarker signature M. Polley, E. Polley, E. Huang, B. Freidlin, R. Simon. Biometric Research Branch, National Cancer Institute, Bethesda MD, USA Background: The scientific community has committed expansive resources during the last decade to identify useful biomarkers for clinical use. Purpose/Objective: Cancer biomarkers are frequently evaluated using archived specimens. Routine collection of high quality specimens is an expensive and time-consuming process. Therefore, care should be taken to preserve these precious and scarce resources. Here we propose a novel statistical design to allow efficient evaluation of a panel of biomarkers while making economic use of available resources. Materials and Methods: Motivated by the use of futility monitoring for a treatment effect in clinical trials, here we propose a Two-Stage Adaptive Cutoff (TACO) design that affords the possibility to stop performing biomarker assays if an early evaluation of the model performance indicates that the hypothesized biomarker effect will not be confirmed. Our study design includes four components: (a) signature building with a statistically valid test of
the model performance based on data in stage 1; (b) an early futility stopping if the model performance in stage 1 is unsatisfactory; (c) a completely locked-down biomarker signature in stage 1 with a properly cross-validated cutoff to classify patient status; and (d) an independent validation of the locked-down biomarker signature (including the cutoff) in stage 2. Results: We give an example based on a publicly available dataset to illustrate the use of the procedure. Simulation studies are presented to evaluate the operating characteristics of the design. We demonstrate that with the proposed design, substantial savings in specimens is possible under the null hypothesis when the model performance is undesirable. The practical aspects of the proposed design are discussed. Conclusions: In this work, we propose a statistical design useful for developing and validating a biomarkers signature which includes a stopping rule for futility in the event of poor model performance. Novel statistical designs like this are needed to allow conservation of precious tissue specimens for future research.
MC13-0060 Analytical validation of the MPACT assay, a targeted next generation sequencing clinical assay for cancer patient treatment selection C. Lih 1 , D. Sims 1 , E. Polley 2 , Y. Zhao 2 , M. Mehaffey 1 , T. Forbes 1 , R. Harrington 1 , W. Walsh 1 , P. McGregor 1 , R. Simon 2 , B. Conley 3 , S. Kummar 4 , J. Doroshow 4 , P.M. Williams 1 . 1 Molecular Characterization Laboratory, Frederick National Laboratory for Cancer Research, Frederick, USA; 2 Biometric Research Branch, National Cancer Institute, Shady Grove, USA; 3 Cancer Diagnosis Program, National Cancer Institute, Shady Grove, USA; 4 Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, USA Background: Robust and analytically validated assays are essential for development of targeted cancer therapies. MPACT (Molecular Profiling based Assignment of Cancer Therapeutics) is a proposed clinical trial that randomizes patients who failed standard treatment to therapy targeted or not to their own tumor’s molecular abnormalities. Purpose/Objective: Using next generation sequencing, we developed a multi-analyte somatic variant assay to support the treatment selection for MPACT trial. Here we report the results from analytical validation study. Materials and Methods: The MPACT assay interrogates a total of 59,150 bp that represents minimally, 392 variant loci with reported actionable value to guide treatment decisions. In this analytical validation study, we assessed the sensitivity, specificity, and reproducibility for 5 classes of sequence variants, single nucleotide variant (SNV), insertion and deletion (Indel, 3bp or less), large indel (greater than 3bp), SNV at homopolymeric region (HP, greater than 2 identical bases in a row), and Indel at HP. Results: Using DNA samples derived from a hapmap normal cell-line (CEPH, NA12878) with 27 spiked positive control plasmids and from cultured cancer cell-lines or FFPE xenografts derived from cells harboring known variants, we found that the sensitivity is 99.3% in SNVs, 91.1% in SNVs at HP, 100% in Indels, 66.7% in Indels at HP, and 88.9% at large Indels. Reproducibility analysis in a subset of DNA samples revealed that the interand intra-operator concordances are 95.1% and 98.3% respectively, with greater than 0.99 R square values in allele frequency for detected variants. Sanger sequencing of 22 loci in 5 xenograft samples demonstrated 100% accuracy with MPACT assay results. By sequencing three hapmap normal cell-lines (CEPH, Yoruban, Chinese female) multiple times, we showed that the MPACT assay achieved 100% specificity in reportable range for all 5 classes of variants. In addition, we blindly tested 10 unknown tumor specimens that were genotyped previously by a CLIA assay in another lab and found that the MPACT assay reported accurately all 9 previously identified variants at near identical frequency. Finally, we compared the data from MPACT assay and exome capture sequencing using Hiseq for 5 pairs of clinical tumor specimen and matched blood samples. The high concordance confirmed that the MPACT assay results are validated by an independent NGS platform. Conclusions: This validation study demonstrated that the MPACT assay was well-suited for the intended clinical use.