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Non-invasive Cell-free Tumor DNA-based Detection of Breast Cancer-related Copy Number Variations
Abstracts We report on the molecular cytogenetic findings of a 10-yearold male with a history of mild mental retardation (IQ 66), mood disorder, anxiety, disruptive behavior and lazy eye and recent onset of seizures. DNA isolated from peripheral blood was analyzed by chromosome microarray and revealed a mosaic non-integer copy number gain of 29.6 Mb on the short arm of chromosome 5 between bands 5p13.3 and 5p15.3 (copy number state of w2.3) suggesting 30% of cells have a 5p15.3p13.3 duplication. No other relevant copy number variants were detected. The clinical manifestations of patients with partial trisomy of 5p are variable and seem to depend on the position and size of the duplication. More proximal duplications or those involving the entire p-arm usually result in a more severe phenotype in comparison to duplications distal to 5p13. Copy number gains of 5p are uncommon and often the result of an unbalanced translocation. With the exception of a small number of cri-du-chat syndrome patients mosaic for both 5p deletions and 5p duplications, mosaic duplications of this region have not been reported. Additional cytogenetic testing is in progress to further characterize the chromosomal abnormality and to examine the level of mosaicism in other tissues.
Integrative Copy Number and Mutational Analysis Improves Glioma Diagnostics Shakti H. Ramkissoon a,c,f, Wenya L. Bi b,d,e, Steven E. Schumacher a,d,f, Lori A. Ramkissoon c, Sam Haidar c, Adrian M. Dubuc a, Loreal Brown d, Margot Burns d, Jane Cryan a, David A. Reardon d, Eudocia Q. Lee d, Mikael L. Rinne d, Andrew D. Norden d, Lakshmi Nayak d, Sandra Ruland d, Lisa M. Doherty d, Debra C. LaFrankie d, Andrea Russo e, Nils D. Arvold e, Elizabeth B. Claus b, Ossama Al-Mefty b, Mark D. Johnson b, Alexandra Golby b, Ian F. Dunn b, E. Antonio Chiocca b, Sandro Santagata b,f, Rebecca D. Folkerth b,f, Philip Kantoff d, Barrett J. Rollins d, Neal I. Lindeman a, Patrick Y. Wen d, Rameen Beroukhim d, Azra H. Ligon a,f, Brian M. Alexander d, Keith L. Ligon a,f a Department of Pathology, Brigham and Women’s Hospital, Boston, MA; b Department of Neurosurgery, Brigham and Women’s Hospital, Boston, MA; c Center for Molecular Oncologic Pathology, DanaFarber Cancer Institute, Boston, MA; d Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA; e Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA; f Harvard Medical School, Harvard Medical School, Boston, MA
Genomic (copy number, mutational) profiling of formalin-fixed paraffin-embedded (FFPE) samples has potential to improve cancer diagnostics, identifying characteristic aberrations relating to grade and/or histology; however, the use of this information in a CLIA setting is not yet routine. We sought to report our experience with the implementation of comparative hybridization genomic (CGH) and mutation-testing program across a series of > 600 primary brain tumors. For glioblastoma multiforme (GBM), the most common and aggressive primary brain tumor, comparative genomic hybridization (nZ236) and targeted mass spectrometrybased mutation genotyping (nZ94) clinical testing profiles were collected and analyzed. These results were compared to large genomic datasets generated from fresh frozen primary tumors profiled through the TCGA. Across our cohort clinically relevant loci were detected, including: 1p/19q co-deletion, EGFR, EGFRvIII, PDGFRA, CDKN2A and PTEN, as well as recurrent mutations in IDH1, TP53 and PTEN. Patients >40 years old showed a
287 classic copy number profile for GBM, including polysomy 7, EGFR amplification, CDKN2A loss or PTEN deletion.
Detection of Copy Number Variations in Breast Cancer Samples Using Single-nucleotide Polymorphismtargeted Massively Multiplexed PCR Joshua E. Babiarz, Bernhard G. Zimmermann, Tudor Constantin, Ryan Swenerton, Eser Kirkizlar, Nicholas Wayham, Matthew Rabinowitz, Matthew Hill Natera, Inc., San Carlos, CA
Evaluation of copy number variation (CNV) in tumor tissues typically involves SNP microarray or aCGH. These methods have high whole-genome resolution, but require large amounts of input material, have high fixed costs, and falter on FFPE samples. Here, we employ a 28,000-plex SNP-targeted PCR with NGS, targeting chromosomes 1, 2, 13, 18, 21 and X, and regions 4p16, 5p15, 7q11, 15q, 17p, 22q11, and 22q13, to detect CNVs from fresh and FFPE samples. An accuracy of 100% was demonstrated on 96 samples: 25 unaffected and 71 affected (minimum 3 samples per region). Single-molecule sensitivity was established by analyzing single cells. Among five matched tumor and normal cell lines all five tumor samples had multiple amplifications and deletions (median: 13), whereas CNVs were absent in the matched controls. Of 15 fresh frozen and 2 FFPE breast tumors samples, 14 fresh and both FFPEs showed full or partial CNVs in at least one and up to 15 regions (median: 7); all had a CNV of either 1q or 17p, the two most common breast cancer CNVs represented on this panel. Evidence of tumor heterogeneity was also observed. Although not investigated here, the very low input requirements will allow this assay to analyze tumor heterogeneity. A similar assay targeting all chromosome arms would result in a sequencing cost of approximately $5 per sample. Thus, this method offers a powerful, efficient, and scalable approach for investigating large numbers of fresh frozen or FFPE samples.
Non-invasive Cell-free Tumor DNA-based Detection of Breast Cancer-related Copy Number Variations Bernhard G. Zimmermann 1, Eser Kirkizlar 1, Matthew Hill, Tudor Constantin, Styrmir Sigurjonsson, Bin Hoanga, Nikhil Chopra, Matthew Rabinowitz Natera, Inc., San Carlos, CA
Breast cancer screening involves mammography, which has high false positive rates and misses some cancers1-4. Analysis of tumor-derived circulating cell-free DNA (ctDNA) for cancerassociated copy number variations (CNVs) may allow for earlier, safer, and more accurate screening. Here, we employed a singlenucleotide polymorphism (SNP)-based massively multiplex PCR (mmPCR) approach to screen for CNVs in ctDNA isolated from the plasma of breast cancer patients. The mmPCR assay targeted 3,168 SNPs on chromosomes 1, 2, and 22q, which often have CNVs in cancer (e.g., 49% of breast cancer samples have a 22q deletion5). Here, we present case studies of six breast
1
These authors contributed equally.
288 cancer patients: one stage IIa, four stage IIb, and one stage IIIb, with ctDNA fractions of 0.58-4.33%. Each tumor sample had CNVs on one or more of the targeted chromosomes. CNVs were identified in the corresponding plasma samples, including in one stage IIb sample with a ctDNA fraction of 0.58%; detection only required 86 heterozygous SNPs. This demonstrates that focal or whole chromosome arm CNVs, both common in cancer, can be readily detected. To further evaluate sensitivity, we generated 22 artificial mixtures containing a 3Mb 22q CNV, with ctDNA fractions of 0.85-14.7%. The method correctly detected CNVs in 100% of these samples with under $100 sequencing cost per sample. In numerous cancers, such as breast and ovarian, a significant proportion of tumors may only be detectable by CNVs instead of point mutations. Together, this supports that this SNPbased mmPCR approach offers a cost-effective, non-invasive method for detecting these cancers.
References 1. Pace LE, Keating NL. A systematic assessment of benefits and risks to guide breast cancer screening decisions. JAMA 2014; 311(13):1327e1335. 2. Nelson HD, Tyne K, Naik A, Bougatsos C, Chan BK, Humphrey L. Screening for breast cancer: An update for the U.S. Preventive Services Task Force. Ann Intern Med 2009;151(10):727e737. 3. Hubbard RA, Kerlikowske K, Flowers CI, Yankaskas BC, Zhu W, Miglioretti DL; Cumulative probability of false-positive recall or biopsy recommendation after 10 years of screening mammography. A cohort study. Ann Intern Med 2011;155(8): 481e492. 4. Braithwaite D, Zhu W, Hubbard RA, O’Meara ES, Miglioretti DL, Geller B, et al. Screening outcomes in older US women undergoing multiple mammograms in community practice: Does interval, age, or comorbidity score affect tumor characteristics or false positive rates? J Natl Cancer Inst 2013;105(5): 334e341. 5. Forbes SA, Bindal N, Bamford S, Cole C, Kok CY, Beare D, et al. COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res. 2011; 39(suppl 1):D945eD950.
KRAS Amplification in a 73-Year-Old Man with Adenocarcinoma of Lung Hui Chen a, Rajesh R. Singh b, Ronald Abraham b, Meenakshi Mehrotra a, Bal Mukund Mishra a, Sinchita RoyChowdhuri a, Mark Routbort b, Kenneth Aldape a, Russell Broaddus a, Keyur P. Patel a, Rajyalakshmi Luthra b a Department of Pathology, MD Anderson Cancer Center, Houston, TX; b Department of Hematopathology, MD Anderson Cancer Center, Houston, TX
Introduction: Recent discoveries have enabled us to identify common genetic alterations such as EGFR, KRAS, BRAF and ERBB2 mutations and ALK, RET and ROS translocations in adenocarcinoma of lung; however the status of gene copy number variation and allelic imbalances remain under-characterized. In this study, we explored genome-wide copy number analysis on a 37-year-old man with metastatic adenocarcinoma of lung with pan negative results for EGFR, KRAS and BRAF mutations and ALK translocation. Material and Methods: Cytology cell block of lymph node with metastatic adenocarcinoma was used for manual micro-dissection and DNA extraction. Purified DNA was analyzed for EGFR,
Abstracts KRAS and BRAF mutations by next generation sequencing using Ion Torrent PGM and 50 gene hotspot panel (LifeTechnologies, Carlsbad, CA). Genomic DNA was also subjected to molecular inversion probe array by OncoScan FFPE Assay kit (Affymetrix, Santa Clara, CA). Genomic copy number and allelic imbalance calls were performed by OncoScan Console Software and data reviewed by OncoScanÔ Nexus Express Software (BioDiscovery, El Segundo, CA). Results: On next generation sequencing, no mutation was detected in EGFR, KRAS and BRAF; but TP53 and JAK3 mutations identified. However KRAS was preferentially amplified by increased normalized coverage ratio. Further study on copy number variation by MIP array revealed high copy number gain in KRAS (16 copies), which was in good concordance with estimated copy number information from next generation sequencing (10 copies). Discussion: Amplification of KRAS might be an alternative oncogenic mechanism for adenocarcinoma of lung, especially in pan-negative cases for EGFR, KRAS, BRAF mutations and ALK translocation.
Multiple Segmental Chromosomal Aberrations in Low-Risk Neuroblastoma are Associated with Metastatic Relapse Jodi R. Mayfield a, Gordana Raca b, Madina Sukhanova b, Rochelle Bagatell c, Meredith Irwin d, Anthony Little c, Jawhar Rawwas e, Susan L. Cohn a a University of Chicago Department of Pediatrics; b University of Chicago Department of Medicine; c Division of Oncology, The Children’s Hospital of Philadelphia; d Hospital for Sick Children, Department of Pediatrics; e Children’s Hospitals and Clinics of Minnesota
Background: Of children with low-risk neuroblastoma 10-15% of patients will develop recurrent disease. To evaluate for possible biomarkers associated with relapse, we analyzed chromosomal aberrations in low-risk tumors. Methods: Patients with low-risk disease who remained diseasefree for > 3 years and patients who developed relapsed disease in local or metastatic sites were identified at University of Chicago, Children’s Hospital of Philadelphia, Hospital for Sick Children and Children’s Hospitals and Clinics of Minnesota. Formalin fixed, paraffin embedded (FFPE) diagnostic samples were used for whole-genome SNP and copy number analysis using OncoScanÔ FFPE Express 2.0 (Affymetrix, Santa Clara, CA). Numerical and segmental aberrations were determined and associations between aberrations and outcome were evaluated. Results: The analytic cohort consisted of 22 low-risk patients; 9 relapsed locally, 7 relapsed at metastatic sites, and 6 remain in remission. Numerical chromosomal aberrations (NCA) (range 1-19) were identified in 17 patients; 8 with local relapse, 5 with metastatic relapse, and 4 with no relapse. Segmental chromosomal aberrations (SCA) (range 1-12) were detected in 9 patients; 3 with local and 4 with metastatic relapse, and 2 with no relapse. The presence of more than 4 SCA were only detected in tumors from patients who developed metastatic relapse (pZ0.0048). Conclusions: It is feasible to obtain chromosomal copy number data from FFPE neuroblastoma tissue. The presence of > 4 SCA may be predictive of metastatic relapse. Prospective studies to determine the prognostic value of SCA in low-risk neuroblastoma are warranted.
Integrative Copy Number and Mutational Analysis Improves Glioma Diagnostics Shakti H. Ramkissoon a,c,f, Wenya L. Bi b,d,e, Steven E. Schumacher a,d,f, Lori A. Ramkissoon c, Sam Haidar c, Adrian M. Dubuc a, Loreal Brown d, Margot Burns d, Jane Cryan a, David A. Reardon d, Eudocia Q. Lee d, Mikael L. Rinne d, Andrew D. Norden d, Lakshmi Nayak d, Sandra Ruland d, Lisa M. Doherty d, Debra C. LaFrankie d, Andrea Russo e, Nils D. Arvold e, Elizabeth B. Claus b, Ossama Al-Mefty b, Mark D. Johnson b, Alexandra Golby b, Ian F. Dunn b, E. Antonio Chiocca b, Sandro Santagata b,f, Rebecca D. Folkerth b,f, Philip Kantoff d, Barrett J. Rollins d, Neal I. Lindeman a, Patrick Y. Wen d, Rameen Beroukhim d, Azra H. Ligon a,f, Brian M. Alexander d, Keith L. Ligon a,f a Department of Pathology, Brigham and Women’s Hospital, Boston, MA; b Department of Neurosurgery, Brigham and Women’s Hospital, Boston, MA; c Center for Molecular Oncologic Pathology, DanaFarber Cancer Institute, Boston, MA; d Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA; e Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA; f Harvard Medical School, Harvard Medical School, Boston, MA
Genomic (copy number, mutational) profiling of formalin-fixed paraffin-embedded (FFPE) samples has potential to improve cancer diagnostics, identifying characteristic aberrations relating to grade and/or histology; however, the use of this information in a CLIA setting is not yet routine. We sought to report our experience with the implementation of comparative hybridization genomic (CGH) and mutation-testing program across a series of > 600 primary brain tumors. For glioblastoma multiforme (GBM), the most common and aggressive primary brain tumor, comparative genomic hybridization (nZ236) and targeted mass spectrometrybased mutation genotyping (nZ94) clinical testing profiles were collected and analyzed. These results were compared to large genomic datasets generated from fresh frozen primary tumors profiled through the TCGA. Across our cohort clinically relevant loci were detected, including: 1p/19q co-deletion, EGFR, EGFRvIII, PDGFRA, CDKN2A and PTEN, as well as recurrent mutations in IDH1, TP53 and PTEN. Patients >40 years old showed a
287 classic copy number profile for GBM, including polysomy 7, EGFR amplification, CDKN2A loss or PTEN deletion.
Detection of Copy Number Variations in Breast Cancer Samples Using Single-nucleotide Polymorphismtargeted Massively Multiplexed PCR Joshua E. Babiarz, Bernhard G. Zimmermann, Tudor Constantin, Ryan Swenerton, Eser Kirkizlar, Nicholas Wayham, Matthew Rabinowitz, Matthew Hill Natera, Inc., San Carlos, CA
Evaluation of copy number variation (CNV) in tumor tissues typically involves SNP microarray or aCGH. These methods have high whole-genome resolution, but require large amounts of input material, have high fixed costs, and falter on FFPE samples. Here, we employ a 28,000-plex SNP-targeted PCR with NGS, targeting chromosomes 1, 2, 13, 18, 21 and X, and regions 4p16, 5p15, 7q11, 15q, 17p, 22q11, and 22q13, to detect CNVs from fresh and FFPE samples. An accuracy of 100% was demonstrated on 96 samples: 25 unaffected and 71 affected (minimum 3 samples per region). Single-molecule sensitivity was established by analyzing single cells. Among five matched tumor and normal cell lines all five tumor samples had multiple amplifications and deletions (median: 13), whereas CNVs were absent in the matched controls. Of 15 fresh frozen and 2 FFPE breast tumors samples, 14 fresh and both FFPEs showed full or partial CNVs in at least one and up to 15 regions (median: 7); all had a CNV of either 1q or 17p, the two most common breast cancer CNVs represented on this panel. Evidence of tumor heterogeneity was also observed. Although not investigated here, the very low input requirements will allow this assay to analyze tumor heterogeneity. A similar assay targeting all chromosome arms would result in a sequencing cost of approximately $5 per sample. Thus, this method offers a powerful, efficient, and scalable approach for investigating large numbers of fresh frozen or FFPE samples.
Non-invasive Cell-free Tumor DNA-based Detection of Breast Cancer-related Copy Number Variations Bernhard G. Zimmermann 1, Eser Kirkizlar 1, Matthew Hill, Tudor Constantin, Styrmir Sigurjonsson, Bin Hoanga, Nikhil Chopra, Matthew Rabinowitz Natera, Inc., San Carlos, CA
Breast cancer screening involves mammography, which has high false positive rates and misses some cancers1-4. Analysis of tumor-derived circulating cell-free DNA (ctDNA) for cancerassociated copy number variations (CNVs) may allow for earlier, safer, and more accurate screening. Here, we employed a singlenucleotide polymorphism (SNP)-based massively multiplex PCR (mmPCR) approach to screen for CNVs in ctDNA isolated from the plasma of breast cancer patients. The mmPCR assay targeted 3,168 SNPs on chromosomes 1, 2, and 22q, which often have CNVs in cancer (e.g., 49% of breast cancer samples have a 22q deletion5). Here, we present case studies of six breast
1
These authors contributed equally.
288 cancer patients: one stage IIa, four stage IIb, and one stage IIIb, with ctDNA fractions of 0.58-4.33%. Each tumor sample had CNVs on one or more of the targeted chromosomes. CNVs were identified in the corresponding plasma samples, including in one stage IIb sample with a ctDNA fraction of 0.58%; detection only required 86 heterozygous SNPs. This demonstrates that focal or whole chromosome arm CNVs, both common in cancer, can be readily detected. To further evaluate sensitivity, we generated 22 artificial mixtures containing a 3Mb 22q CNV, with ctDNA fractions of 0.85-14.7%. The method correctly detected CNVs in 100% of these samples with under $100 sequencing cost per sample. In numerous cancers, such as breast and ovarian, a significant proportion of tumors may only be detectable by CNVs instead of point mutations. Together, this supports that this SNPbased mmPCR approach offers a cost-effective, non-invasive method for detecting these cancers.
References 1. Pace LE, Keating NL. A systematic assessment of benefits and risks to guide breast cancer screening decisions. JAMA 2014; 311(13):1327e1335. 2. Nelson HD, Tyne K, Naik A, Bougatsos C, Chan BK, Humphrey L. Screening for breast cancer: An update for the U.S. Preventive Services Task Force. Ann Intern Med 2009;151(10):727e737. 3. Hubbard RA, Kerlikowske K, Flowers CI, Yankaskas BC, Zhu W, Miglioretti DL; Cumulative probability of false-positive recall or biopsy recommendation after 10 years of screening mammography. A cohort study. Ann Intern Med 2011;155(8): 481e492. 4. Braithwaite D, Zhu W, Hubbard RA, O’Meara ES, Miglioretti DL, Geller B, et al. Screening outcomes in older US women undergoing multiple mammograms in community practice: Does interval, age, or comorbidity score affect tumor characteristics or false positive rates? J Natl Cancer Inst 2013;105(5): 334e341. 5. Forbes SA, Bindal N, Bamford S, Cole C, Kok CY, Beare D, et al. COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res. 2011; 39(suppl 1):D945eD950.
KRAS Amplification in a 73-Year-Old Man with Adenocarcinoma of Lung Hui Chen a, Rajesh R. Singh b, Ronald Abraham b, Meenakshi Mehrotra a, Bal Mukund Mishra a, Sinchita RoyChowdhuri a, Mark Routbort b, Kenneth Aldape a, Russell Broaddus a, Keyur P. Patel a, Rajyalakshmi Luthra b a Department of Pathology, MD Anderson Cancer Center, Houston, TX; b Department of Hematopathology, MD Anderson Cancer Center, Houston, TX
Introduction: Recent discoveries have enabled us to identify common genetic alterations such as EGFR, KRAS, BRAF and ERBB2 mutations and ALK, RET and ROS translocations in adenocarcinoma of lung; however the status of gene copy number variation and allelic imbalances remain under-characterized. In this study, we explored genome-wide copy number analysis on a 37-year-old man with metastatic adenocarcinoma of lung with pan negative results for EGFR, KRAS and BRAF mutations and ALK translocation. Material and Methods: Cytology cell block of lymph node with metastatic adenocarcinoma was used for manual micro-dissection and DNA extraction. Purified DNA was analyzed for EGFR,
Abstracts KRAS and BRAF mutations by next generation sequencing using Ion Torrent PGM and 50 gene hotspot panel (LifeTechnologies, Carlsbad, CA). Genomic DNA was also subjected to molecular inversion probe array by OncoScan FFPE Assay kit (Affymetrix, Santa Clara, CA). Genomic copy number and allelic imbalance calls were performed by OncoScan Console Software and data reviewed by OncoScanÔ Nexus Express Software (BioDiscovery, El Segundo, CA). Results: On next generation sequencing, no mutation was detected in EGFR, KRAS and BRAF; but TP53 and JAK3 mutations identified. However KRAS was preferentially amplified by increased normalized coverage ratio. Further study on copy number variation by MIP array revealed high copy number gain in KRAS (16 copies), which was in good concordance with estimated copy number information from next generation sequencing (10 copies). Discussion: Amplification of KRAS might be an alternative oncogenic mechanism for adenocarcinoma of lung, especially in pan-negative cases for EGFR, KRAS, BRAF mutations and ALK translocation.
Multiple Segmental Chromosomal Aberrations in Low-Risk Neuroblastoma are Associated with Metastatic Relapse Jodi R. Mayfield a, Gordana Raca b, Madina Sukhanova b, Rochelle Bagatell c, Meredith Irwin d, Anthony Little c, Jawhar Rawwas e, Susan L. Cohn a a University of Chicago Department of Pediatrics; b University of Chicago Department of Medicine; c Division of Oncology, The Children’s Hospital of Philadelphia; d Hospital for Sick Children, Department of Pediatrics; e Children’s Hospitals and Clinics of Minnesota
Background: Of children with low-risk neuroblastoma 10-15% of patients will develop recurrent disease. To evaluate for possible biomarkers associated with relapse, we analyzed chromosomal aberrations in low-risk tumors. Methods: Patients with low-risk disease who remained diseasefree for > 3 years and patients who developed relapsed disease in local or metastatic sites were identified at University of Chicago, Children’s Hospital of Philadelphia, Hospital for Sick Children and Children’s Hospitals and Clinics of Minnesota. Formalin fixed, paraffin embedded (FFPE) diagnostic samples were used for whole-genome SNP and copy number analysis using OncoScanÔ FFPE Express 2.0 (Affymetrix, Santa Clara, CA). Numerical and segmental aberrations were determined and associations between aberrations and outcome were evaluated. Results: The analytic cohort consisted of 22 low-risk patients; 9 relapsed locally, 7 relapsed at metastatic sites, and 6 remain in remission. Numerical chromosomal aberrations (NCA) (range 1-19) were identified in 17 patients; 8 with local relapse, 5 with metastatic relapse, and 4 with no relapse. Segmental chromosomal aberrations (SCA) (range 1-12) were detected in 9 patients; 3 with local and 4 with metastatic relapse, and 2 with no relapse. The presence of more than 4 SCA were only detected in tumors from patients who developed metastatic relapse (pZ0.0048). Conclusions: It is feasible to obtain chromosomal copy number data from FFPE neuroblastoma tissue. The presence of > 4 SCA may be predictive of metastatic relapse. Prospective studies to determine the prognostic value of SCA in low-risk neuroblastoma are warranted.