The Experts below are selected from a list of 1233 Experts worldwide ranked by ideXlab platform
Stephen J. Salipante - One of the best experts on this subject based on the ideXlab platform.
-
Ultrasensitive Detection of Chimerism by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing of Copy Number Deletion Polymorphisms.
Clinical chemistry, 2018Co-Authors: Adam Waalkes, Kelsi Penewit, Stephen J. SalipanteAbstract:BACKGROUND: Genomic chimerism, the co-occurrence of cells from different genetic origins, provides important diagnostic information in diverse clinical contexts, including graft injury detection and longitudinal surveillance of hematopoietic stem cell transplantation patients, but existing assays are limiting. Here we applied single-molecule Molecular Inversion Probes (smMIPs), a high-throughput sequencing technology combining multiplexed target capture with read quantification mediated by unique Molecular identifiers, to detect chimerism based on the presence or absence of polymorphic genomic loci. METHODS: We designed a 159-smMIP panel targeting 40 autosomal regions of frequent homozygous deletion across human populations and 2 sex-linked loci. We developed methods for detecting and quantitating loci absent from 1 cell population but present in another, which could be used to sensitively identify chimeric cell populations. RESULTS: Unrelated individuals and first-degree relatives were highly polymorphic across the loci examined. Using synthetic DNA mixtures, limits of detection of at least 1 in 10000 chimeric cells were demonstrated without prior knowledge of genotypes, and mixtures of up to 4 separate donors could be deconvoluted. Quantitative linearity over 4 orders of magnitude and false-positive rates CONCLUSIONS: smMIPs are scalable to high sensitivity and large numbers of informative markers, enabling ultrasensitive chimerism detection for many clinical purposes.
-
Accurate Pan-Cancer Molecular Diagnosis of Microsatellite Instability by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing.
Clinical chemistry, 2018Co-Authors: Adam Waalkes, Kelsi Penewit, Nahum Smith, Jennifer A. Hempelmann, Eric Q. Konnick, Ronald J. Hause, Colin C. Pritchard, Stephen J. SalipanteAbstract:BACKGROUND: Microsatellite instability (MSI) is an emerging actionable phenotype in oncology that informs tumor response to immune checkpoint pathway immunotherapy. However, there remains a need for MSI diagnostics that are low cost, highly accurate, and generalizable across cancer types. We developed a method for targeted high-throughput sequencing of numerous microsatellite loci with pan-cancer informativity for MSI using single-molecule Molecular Inversion Probes (smMIPs). METHODS: We designed a smMIP panel targeting 111 loci highly informative for MSI across cancers. We developed an analytical framework taking advantage of smMIP-mediated error correction to specifically and sensitively detect instability events without the need for typing matched normal material. RESULTS: Using synthetic DNA mixtures, smMIPs were sensitive to at least 1% MSI-positive cells and were highly consistent across replicates. The fraction of identified unstable microsatellites discriminated tumors exhibiting MSI from those lacking MSI with high accuracy across colorectal (100% diagnostic sensitivity and specificity), prostate (100% diagnostic sensitivity and specificity), and endometrial cancers (95.8% diagnostic sensitivity and 100% specificity). MSI-PCR, the current standard-of-care Molecular diagnostic for MSI, proved equally robust for colorectal tumors but evidenced multiple false-negative results in prostate (81.8% diagnostic sensitivity and 100% specificity) and endometrial (75.0% diagnostic sensitivity and 100% specificity) tumors. CONCLUSIONS: smMIP capture provides an accurate, diagnostically sensitive, and economical means to diagnose MSI across cancer types without reliance on patient-matched normal material. The assay is readily scalable to large numbers of clinical samples, enables automated and quantitative analysis of microsatellite instability, and is readily standardized across clinical laboratories.
-
Ultrasensitive detection of acute myeloid leukemia minimal residual disease using single molecule Molecular Inversion Probes
Haematologica, 2017Co-Authors: Adam Waalkes, Kelsi Penewit, Brent L. Wood, Stephen J. SalipanteAbstract:The identification of minimal residual disease is the primary diagnostic finding which predicts relapse in patients treated for acute myeloid leukemia. Ultrasensitive detection of minimal residual disease would enable better patient risk stratification and could open opportunities for early therapeutic intervention. Herein we apply single molecule Molecular Inversion Probe capture, a technology combining multiplexed targeted sequencing with error correction schemes based on Molecular barcoding, in order to detect mutations identifying minimal residual disease with ultrasensitive and quantitative precision. We designed a single molecule Molecular Inversion Probe capture panel spanning >50 kb and targeting 32 factors relevant to acute myeloid leukemia pathogenesis. We demonstrate linearity and quantitative precision over 100-fold relative abundance of mutant cells (1 in 100 to 1 in 1,500), with estimated error rates approaching 1 in 1,200 base pairs sequenced and maximum theoretical limits of detection exceeding 1 in 60,000 mutant alleles. In 3 of 4 longitudinally collected specimens from patients with acute myeloid leukemia, we find that single molecule Molecular Inversion Probe capture detects somatic mutations identifying minimal residual disease at substantially earlier time points and with greater sensitivity than clinical diagnostic approaches used as current standard of care (flow cytometry and conventional Molecular diagnosis), and identifies persisting neoplastic cells during clinical remission. In 2 patients, single molecule Molecular Inversion Probe capture detected heterogeneous, subclonal acute myeloid leukemia populations carrying distinct mutational signatures. Single molecule Molecular Inversion Probe technology uniquely couples scalable target enrichment with sequence read error correction, providing an integrated, ultrasensitive approach for detecting minimal residual disease identifying mutations.
Adam Waalkes - One of the best experts on this subject based on the ideXlab platform.
-
Development of a precision oncology single molecule Molecular Inversion Probe (smMIP) panel using a community-consensus approach
2018Co-Authors: Erica K. Barnell, Adam Waalkes, Kelsi Penewit, Katie M. Campbell, Zachary L. Skidmore, Colin Pritchard, Todd A. Fehniger, Ravindra Uppaluri, Ramaswamy Govindan, Malachi GriffithAbstract:Clinical targeted sequencing panels are important for identifying actionable variants for cancer patients, however, there are currently no strategies to create impartial and rationally-designed panels to accommodate rapidly growing knowledge within the field. Here we use the Clinical Interpretations of Variants in Cancer database (CIViC) in conjunction with single-molecule Molecular Inversion Probe (smMIP) capture to provide a dynamic and accurate capture panel targeting clinically relevant variants in cancer. In total, 2,027 smMIPs were designed to target 111 eligible CIViC variants. The total genomic region covered by the panel was 61.5 kb. When compared to existing genome or exome sequencing results (n = 27 cancer samples from 5 tumor types), the CIViC smMIP capture panel demonstrated a 95% sensitivity for variant detection (n = 61/64 variants). Variant allele frequency for variants identified on both sequencing platforms were highly concordant (Pearson correlation = 0.885; n = 61 variants). Moreover, for individuals with paired tumor/normal samples (n = 12), 182 clinically relevant variants missed by original sequencing were discovered by the CIViC smMIPs panel. This panel demonstrates the utility of an open-sourced database built on attendant community contributions for each variant with peer-reviewed interpretations. Use of a public repository for variant identification, Probe development, and variant annotation could provide a dynamic and transparent approach to alleviate the analysis bottleneck hindering precision oncology efforts.
-
Accurate Pan-Cancer Molecular Diagnosis of Microsatellite Instability by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing.
Clinical chemistry, 2018Co-Authors: Adam Waalkes, Kelsi Penewit, Nahum Smith, Jennifer A. Hempelmann, Eric Q. Konnick, Ronald J. Hause, Colin C. Pritchard, Stephen J. SalipanteAbstract:BACKGROUND: Microsatellite instability (MSI) is an emerging actionable phenotype in oncology that informs tumor response to immune checkpoint pathway immunotherapy. However, there remains a need for MSI diagnostics that are low cost, highly accurate, and generalizable across cancer types. We developed a method for targeted high-throughput sequencing of numerous microsatellite loci with pan-cancer informativity for MSI using single-molecule Molecular Inversion Probes (smMIPs). METHODS: We designed a smMIP panel targeting 111 loci highly informative for MSI across cancers. We developed an analytical framework taking advantage of smMIP-mediated error correction to specifically and sensitively detect instability events without the need for typing matched normal material. RESULTS: Using synthetic DNA mixtures, smMIPs were sensitive to at least 1% MSI-positive cells and were highly consistent across replicates. The fraction of identified unstable microsatellites discriminated tumors exhibiting MSI from those lacking MSI with high accuracy across colorectal (100% diagnostic sensitivity and specificity), prostate (100% diagnostic sensitivity and specificity), and endometrial cancers (95.8% diagnostic sensitivity and 100% specificity). MSI-PCR, the current standard-of-care Molecular diagnostic for MSI, proved equally robust for colorectal tumors but evidenced multiple false-negative results in prostate (81.8% diagnostic sensitivity and 100% specificity) and endometrial (75.0% diagnostic sensitivity and 100% specificity) tumors. CONCLUSIONS: smMIP capture provides an accurate, diagnostically sensitive, and economical means to diagnose MSI across cancer types without reliance on patient-matched normal material. The assay is readily scalable to large numbers of clinical samples, enables automated and quantitative analysis of microsatellite instability, and is readily standardized across clinical laboratories.
-
Ultrasensitive Detection of Chimerism by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing of Copy Number Deletion Polymorphisms.
Clinical chemistry, 2018Co-Authors: Adam Waalkes, Kelsi Penewit, Stephen J. SalipanteAbstract:BACKGROUND: Genomic chimerism, the co-occurrence of cells from different genetic origins, provides important diagnostic information in diverse clinical contexts, including graft injury detection and longitudinal surveillance of hematopoietic stem cell transplantation patients, but existing assays are limiting. Here we applied single-molecule Molecular Inversion Probes (smMIPs), a high-throughput sequencing technology combining multiplexed target capture with read quantification mediated by unique Molecular identifiers, to detect chimerism based on the presence or absence of polymorphic genomic loci. METHODS: We designed a 159-smMIP panel targeting 40 autosomal regions of frequent homozygous deletion across human populations and 2 sex-linked loci. We developed methods for detecting and quantitating loci absent from 1 cell population but present in another, which could be used to sensitively identify chimeric cell populations. RESULTS: Unrelated individuals and first-degree relatives were highly polymorphic across the loci examined. Using synthetic DNA mixtures, limits of detection of at least 1 in 10000 chimeric cells were demonstrated without prior knowledge of genotypes, and mixtures of up to 4 separate donors could be deconvoluted. Quantitative linearity over 4 orders of magnitude and false-positive rates CONCLUSIONS: smMIPs are scalable to high sensitivity and large numbers of informative markers, enabling ultrasensitive chimerism detection for many clinical purposes.
-
Ultrasensitive detection of acute myeloid leukemia minimal residual disease using single molecule Molecular Inversion Probes
Haematologica, 2017Co-Authors: Adam Waalkes, Kelsi Penewit, Brent L. Wood, Stephen J. SalipanteAbstract:The identification of minimal residual disease is the primary diagnostic finding which predicts relapse in patients treated for acute myeloid leukemia. Ultrasensitive detection of minimal residual disease would enable better patient risk stratification and could open opportunities for early therapeutic intervention. Herein we apply single molecule Molecular Inversion Probe capture, a technology combining multiplexed targeted sequencing with error correction schemes based on Molecular barcoding, in order to detect mutations identifying minimal residual disease with ultrasensitive and quantitative precision. We designed a single molecule Molecular Inversion Probe capture panel spanning >50 kb and targeting 32 factors relevant to acute myeloid leukemia pathogenesis. We demonstrate linearity and quantitative precision over 100-fold relative abundance of mutant cells (1 in 100 to 1 in 1,500), with estimated error rates approaching 1 in 1,200 base pairs sequenced and maximum theoretical limits of detection exceeding 1 in 60,000 mutant alleles. In 3 of 4 longitudinally collected specimens from patients with acute myeloid leukemia, we find that single molecule Molecular Inversion Probe capture detects somatic mutations identifying minimal residual disease at substantially earlier time points and with greater sensitivity than clinical diagnostic approaches used as current standard of care (flow cytometry and conventional Molecular diagnosis), and identifies persisting neoplastic cells during clinical remission. In 2 patients, single molecule Molecular Inversion Probe capture detected heterogeneous, subclonal acute myeloid leukemia populations carrying distinct mutational signatures. Single molecule Molecular Inversion Probe technology uniquely couples scalable target enrichment with sequence read error correction, providing an integrated, ultrasensitive approach for detecting minimal residual disease identifying mutations.
Birgitta Lannering - One of the best experts on this subject based on the ideXlab platform.
-
prognostic effect of whole chromosomal aberration signatures in standard risk non wnt non shh medulloblastoma a retrospective Molecular analysis of the hit siop pnet 4 trial
Lancet Oncology, 2018Co-Authors: Tobias Goschzik, Anja Zur Muehlen, Stefan Rutkowski, Edward C Schwalbe, Debbie Hicks, Amanda Smith, Dominique Figarellabranger, Francois Doz, Birgitta LanneringAbstract:Summary Background Most children with medulloblastoma fall within the standard-risk clinical disease group defined by absence of high-risk features (metastatic disease, large-cell/anaplastic histology, and MYC amplification), which includes 50–60% of patients and has a 5-year event-free survival of 75–85%. Within standard-risk medulloblastoma, patients in the WNT subgroup are established as having a favourable prognosis; however, outcome prediction for the remaining majority of patients is imprecise. We sought to identify novel prognostic biomarkers to enable improved risk-adapted therapies. Methods The HIT-SIOP PNET 4 trial recruited 338 patients aged 4–21 years with medulloblastoma between Jan 1, 2001, and Dec 31, 2006, in 120 treatment institutions in seven European countries to investigate hyperfractionated radiotherapy versus standard radiotherapy. In this retrospective analysis, we assessed the remaining tumour samples from patients in the HIT-SIOP PNET 4 trial (n=136). We assessed the clinical behaviour of the Molecularly defined WNT and SHH subgroups, and identified novel independent prognostic markers and models for standard-risk patients with non-WNT/non-SHH disease. Because of the scarcity and low quality of available genomic material, we used a mass spectrometry-minimal methylation classifier assay (MS-MIMIC) to assess methylation subgroup and a Molecular Inversion Probe array to detect genome-wide copy number aberrations. Prognostic biomarkers and models identified were validated in an independent, demographically matched cohort (n=70) of medulloblastoma patients with non-WNT/non-SHH standard-risk disease treated with conventional therapies (maximal surgical resection followed by adjuvant craniospinal irradiation [all patients] and chemotherapy [65 of 70 patients], at UK Children's Cancer and Leukaemia Group and European Society for Paediatric Oncology (SIOPE) associated treatment centres between 1990 and 2014. These samples were analysed by Illumina 450k DNA methylation microarray. HIT-SIOP PNET 4 is registered with ClinicalTrials.gov, number NCT01351870. Findings We analysed methylation subgroup, genome-wide copy number aberrations, and mutational features in 136 assessable tumour samples from the HIT-SIOP PNET 4 cohort, representing 40% of the 338 patients in the trial cohort. This cohort of 136 samples consisted of 28 (21%) classified as WNT, 17 (13%) as SHH, and 91 (67%) as non-WNT/non-SHH (we considered Group3 and Group4 medulloblastoma together in our analysis because of their similar Molecular and clinical features). Favourable outcomes for WNT tumours were confirmed in patients younger than 16 years, and all relapse events in SHH (four [24%] of 17) occurred in patients with TP53 mutation ( TP53 mut ) or chromosome 17p loss. A novel whole chromosomal aberration signature associated with increased ploidy and multiple non-random whole chromosomal aberrations was identified in 38 (42%) of the 91 samples from patients with non-WNT/non-SHH medulloblastoma in the HIT-SIOP PNET 4 cohort. Biomarkers associated with this whole chromosomal aberration signature (at least two of chromosome 7 gain, chromosome 8 loss, and chromosome 11 loss) predicted favourable prognosis. Patients with non-WNT/non-SHH medulloblastoma could be reclassified by these markers as having favourable-risk or high-risk disease. In patients in the HIT-SIOP PNET4 cohort with non-WNT/non-SHH medulloblastoma, with a median follow-up of 6·7 years (IQR 5·8–8·2), 5-year event-free survival was 100% in the favourable-risk group and 68% (95% CI 57·5–82·7; p=0·00014) in the high-risk group. In the validation cohort, with a median follow-up of 5·6 years (IQR 3·1–8·1), 5-year event-free survival was 94·7% (95% CI 85·2–100) in the favourable-risk group and 58·6% (95% CI 45·1–76·1) in the high-risk group (hazard ratio 9·41, 95% CI 1·25–70·57; p=0·029). Our comprehensive Molecular investigation identified subgroup-specific risk models which allowed 69 (51%) of 134 accessible patients from the standard-risk medulloblastoma HIT-SIOP PNET 4 cohort to be assigned to a favourable-risk group. Interpretation We define a whole chromosomal signature that allows the assignment of non-WNT/non-SHH medulloblastoma patients normally classified as standard-risk into favourable-risk and high-risk categories. In addition to patients younger than 16 years with WNT tumours, patients with non-WNT/non-SHH tumours with our defined whole chromosomal aberration signature and patients with SHH- TP53 wild-type tumours should be considered for therapy de-escalation in future biomarker-driven, risk-adapted clinical trials. The remaining subgroups of patients with high-risk medulloblastoma might benefit from more intensive therapies. Funding Cancer Research UK, Swedish Childhood Cancer Foundation, French Ministry of Health/French National Cancer Institute, and the German Children's Cancer Foundation.
Edward C Schwalbe - One of the best experts on this subject based on the ideXlab platform.
-
prognostic effect of whole chromosomal aberration signatures in standard risk non wnt non shh medulloblastoma a retrospective Molecular analysis of the hit siop pnet 4 trial
Lancet Oncology, 2018Co-Authors: Tobias Goschzik, Anja Zur Muehlen, Stefan Rutkowski, Edward C Schwalbe, Debbie Hicks, Amanda Smith, Dominique Figarellabranger, Francois Doz, Birgitta LanneringAbstract:Summary Background Most children with medulloblastoma fall within the standard-risk clinical disease group defined by absence of high-risk features (metastatic disease, large-cell/anaplastic histology, and MYC amplification), which includes 50–60% of patients and has a 5-year event-free survival of 75–85%. Within standard-risk medulloblastoma, patients in the WNT subgroup are established as having a favourable prognosis; however, outcome prediction for the remaining majority of patients is imprecise. We sought to identify novel prognostic biomarkers to enable improved risk-adapted therapies. Methods The HIT-SIOP PNET 4 trial recruited 338 patients aged 4–21 years with medulloblastoma between Jan 1, 2001, and Dec 31, 2006, in 120 treatment institutions in seven European countries to investigate hyperfractionated radiotherapy versus standard radiotherapy. In this retrospective analysis, we assessed the remaining tumour samples from patients in the HIT-SIOP PNET 4 trial (n=136). We assessed the clinical behaviour of the Molecularly defined WNT and SHH subgroups, and identified novel independent prognostic markers and models for standard-risk patients with non-WNT/non-SHH disease. Because of the scarcity and low quality of available genomic material, we used a mass spectrometry-minimal methylation classifier assay (MS-MIMIC) to assess methylation subgroup and a Molecular Inversion Probe array to detect genome-wide copy number aberrations. Prognostic biomarkers and models identified were validated in an independent, demographically matched cohort (n=70) of medulloblastoma patients with non-WNT/non-SHH standard-risk disease treated with conventional therapies (maximal surgical resection followed by adjuvant craniospinal irradiation [all patients] and chemotherapy [65 of 70 patients], at UK Children's Cancer and Leukaemia Group and European Society for Paediatric Oncology (SIOPE) associated treatment centres between 1990 and 2014. These samples were analysed by Illumina 450k DNA methylation microarray. HIT-SIOP PNET 4 is registered with ClinicalTrials.gov, number NCT01351870. Findings We analysed methylation subgroup, genome-wide copy number aberrations, and mutational features in 136 assessable tumour samples from the HIT-SIOP PNET 4 cohort, representing 40% of the 338 patients in the trial cohort. This cohort of 136 samples consisted of 28 (21%) classified as WNT, 17 (13%) as SHH, and 91 (67%) as non-WNT/non-SHH (we considered Group3 and Group4 medulloblastoma together in our analysis because of their similar Molecular and clinical features). Favourable outcomes for WNT tumours were confirmed in patients younger than 16 years, and all relapse events in SHH (four [24%] of 17) occurred in patients with TP53 mutation ( TP53 mut ) or chromosome 17p loss. A novel whole chromosomal aberration signature associated with increased ploidy and multiple non-random whole chromosomal aberrations was identified in 38 (42%) of the 91 samples from patients with non-WNT/non-SHH medulloblastoma in the HIT-SIOP PNET 4 cohort. Biomarkers associated with this whole chromosomal aberration signature (at least two of chromosome 7 gain, chromosome 8 loss, and chromosome 11 loss) predicted favourable prognosis. Patients with non-WNT/non-SHH medulloblastoma could be reclassified by these markers as having favourable-risk or high-risk disease. In patients in the HIT-SIOP PNET4 cohort with non-WNT/non-SHH medulloblastoma, with a median follow-up of 6·7 years (IQR 5·8–8·2), 5-year event-free survival was 100% in the favourable-risk group and 68% (95% CI 57·5–82·7; p=0·00014) in the high-risk group. In the validation cohort, with a median follow-up of 5·6 years (IQR 3·1–8·1), 5-year event-free survival was 94·7% (95% CI 85·2–100) in the favourable-risk group and 58·6% (95% CI 45·1–76·1) in the high-risk group (hazard ratio 9·41, 95% CI 1·25–70·57; p=0·029). Our comprehensive Molecular investigation identified subgroup-specific risk models which allowed 69 (51%) of 134 accessible patients from the standard-risk medulloblastoma HIT-SIOP PNET 4 cohort to be assigned to a favourable-risk group. Interpretation We define a whole chromosomal signature that allows the assignment of non-WNT/non-SHH medulloblastoma patients normally classified as standard-risk into favourable-risk and high-risk categories. In addition to patients younger than 16 years with WNT tumours, patients with non-WNT/non-SHH tumours with our defined whole chromosomal aberration signature and patients with SHH- TP53 wild-type tumours should be considered for therapy de-escalation in future biomarker-driven, risk-adapted clinical trials. The remaining subgroups of patients with high-risk medulloblastoma might benefit from more intensive therapies. Funding Cancer Research UK, Swedish Childhood Cancer Foundation, French Ministry of Health/French National Cancer Institute, and the German Children's Cancer Foundation.
Kelsi Penewit - One of the best experts on this subject based on the ideXlab platform.
-
Development of a precision oncology single molecule Molecular Inversion Probe (smMIP) panel using a community-consensus approach
2018Co-Authors: Erica K. Barnell, Adam Waalkes, Kelsi Penewit, Katie M. Campbell, Zachary L. Skidmore, Colin Pritchard, Todd A. Fehniger, Ravindra Uppaluri, Ramaswamy Govindan, Malachi GriffithAbstract:Clinical targeted sequencing panels are important for identifying actionable variants for cancer patients, however, there are currently no strategies to create impartial and rationally-designed panels to accommodate rapidly growing knowledge within the field. Here we use the Clinical Interpretations of Variants in Cancer database (CIViC) in conjunction with single-molecule Molecular Inversion Probe (smMIP) capture to provide a dynamic and accurate capture panel targeting clinically relevant variants in cancer. In total, 2,027 smMIPs were designed to target 111 eligible CIViC variants. The total genomic region covered by the panel was 61.5 kb. When compared to existing genome or exome sequencing results (n = 27 cancer samples from 5 tumor types), the CIViC smMIP capture panel demonstrated a 95% sensitivity for variant detection (n = 61/64 variants). Variant allele frequency for variants identified on both sequencing platforms were highly concordant (Pearson correlation = 0.885; n = 61 variants). Moreover, for individuals with paired tumor/normal samples (n = 12), 182 clinically relevant variants missed by original sequencing were discovered by the CIViC smMIPs panel. This panel demonstrates the utility of an open-sourced database built on attendant community contributions for each variant with peer-reviewed interpretations. Use of a public repository for variant identification, Probe development, and variant annotation could provide a dynamic and transparent approach to alleviate the analysis bottleneck hindering precision oncology efforts.
-
Accurate Pan-Cancer Molecular Diagnosis of Microsatellite Instability by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing.
Clinical chemistry, 2018Co-Authors: Adam Waalkes, Kelsi Penewit, Nahum Smith, Jennifer A. Hempelmann, Eric Q. Konnick, Ronald J. Hause, Colin C. Pritchard, Stephen J. SalipanteAbstract:BACKGROUND: Microsatellite instability (MSI) is an emerging actionable phenotype in oncology that informs tumor response to immune checkpoint pathway immunotherapy. However, there remains a need for MSI diagnostics that are low cost, highly accurate, and generalizable across cancer types. We developed a method for targeted high-throughput sequencing of numerous microsatellite loci with pan-cancer informativity for MSI using single-molecule Molecular Inversion Probes (smMIPs). METHODS: We designed a smMIP panel targeting 111 loci highly informative for MSI across cancers. We developed an analytical framework taking advantage of smMIP-mediated error correction to specifically and sensitively detect instability events without the need for typing matched normal material. RESULTS: Using synthetic DNA mixtures, smMIPs were sensitive to at least 1% MSI-positive cells and were highly consistent across replicates. The fraction of identified unstable microsatellites discriminated tumors exhibiting MSI from those lacking MSI with high accuracy across colorectal (100% diagnostic sensitivity and specificity), prostate (100% diagnostic sensitivity and specificity), and endometrial cancers (95.8% diagnostic sensitivity and 100% specificity). MSI-PCR, the current standard-of-care Molecular diagnostic for MSI, proved equally robust for colorectal tumors but evidenced multiple false-negative results in prostate (81.8% diagnostic sensitivity and 100% specificity) and endometrial (75.0% diagnostic sensitivity and 100% specificity) tumors. CONCLUSIONS: smMIP capture provides an accurate, diagnostically sensitive, and economical means to diagnose MSI across cancer types without reliance on patient-matched normal material. The assay is readily scalable to large numbers of clinical samples, enables automated and quantitative analysis of microsatellite instability, and is readily standardized across clinical laboratories.
-
Ultrasensitive Detection of Chimerism by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing of Copy Number Deletion Polymorphisms.
Clinical chemistry, 2018Co-Authors: Adam Waalkes, Kelsi Penewit, Stephen J. SalipanteAbstract:BACKGROUND: Genomic chimerism, the co-occurrence of cells from different genetic origins, provides important diagnostic information in diverse clinical contexts, including graft injury detection and longitudinal surveillance of hematopoietic stem cell transplantation patients, but existing assays are limiting. Here we applied single-molecule Molecular Inversion Probes (smMIPs), a high-throughput sequencing technology combining multiplexed target capture with read quantification mediated by unique Molecular identifiers, to detect chimerism based on the presence or absence of polymorphic genomic loci. METHODS: We designed a 159-smMIP panel targeting 40 autosomal regions of frequent homozygous deletion across human populations and 2 sex-linked loci. We developed methods for detecting and quantitating loci absent from 1 cell population but present in another, which could be used to sensitively identify chimeric cell populations. RESULTS: Unrelated individuals and first-degree relatives were highly polymorphic across the loci examined. Using synthetic DNA mixtures, limits of detection of at least 1 in 10000 chimeric cells were demonstrated without prior knowledge of genotypes, and mixtures of up to 4 separate donors could be deconvoluted. Quantitative linearity over 4 orders of magnitude and false-positive rates CONCLUSIONS: smMIPs are scalable to high sensitivity and large numbers of informative markers, enabling ultrasensitive chimerism detection for many clinical purposes.
-
Ultrasensitive detection of acute myeloid leukemia minimal residual disease using single molecule Molecular Inversion Probes
Haematologica, 2017Co-Authors: Adam Waalkes, Kelsi Penewit, Brent L. Wood, Stephen J. SalipanteAbstract:The identification of minimal residual disease is the primary diagnostic finding which predicts relapse in patients treated for acute myeloid leukemia. Ultrasensitive detection of minimal residual disease would enable better patient risk stratification and could open opportunities for early therapeutic intervention. Herein we apply single molecule Molecular Inversion Probe capture, a technology combining multiplexed targeted sequencing with error correction schemes based on Molecular barcoding, in order to detect mutations identifying minimal residual disease with ultrasensitive and quantitative precision. We designed a single molecule Molecular Inversion Probe capture panel spanning >50 kb and targeting 32 factors relevant to acute myeloid leukemia pathogenesis. We demonstrate linearity and quantitative precision over 100-fold relative abundance of mutant cells (1 in 100 to 1 in 1,500), with estimated error rates approaching 1 in 1,200 base pairs sequenced and maximum theoretical limits of detection exceeding 1 in 60,000 mutant alleles. In 3 of 4 longitudinally collected specimens from patients with acute myeloid leukemia, we find that single molecule Molecular Inversion Probe capture detects somatic mutations identifying minimal residual disease at substantially earlier time points and with greater sensitivity than clinical diagnostic approaches used as current standard of care (flow cytometry and conventional Molecular diagnosis), and identifies persisting neoplastic cells during clinical remission. In 2 patients, single molecule Molecular Inversion Probe capture detected heterogeneous, subclonal acute myeloid leukemia populations carrying distinct mutational signatures. Single molecule Molecular Inversion Probe technology uniquely couples scalable target enrichment with sequence read error correction, providing an integrated, ultrasensitive approach for detecting minimal residual disease identifying mutations.