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Figure 1.  CONSORT Diagram of Patients in the MET1000 Cohort
CONSORT Diagram of Patients in the MET1000 Cohort

NGS indicates next-generation sequencing.

Figure 2.  Clinical Tiering of Molecular Alterations Identified in Metastatic Cancer
Clinical Tiering of Molecular Alterations Identified in Metastatic Cancer

A, Tiering of genomic alterations identified in the MET1000 cohort by clinical relevance. D indicates diagnosis change; G, germline; R, resistance to therapy; and S, somatic. Tier 1 alterations were known to have clinical utility for that individual’s cancer type and included pathogenic germline variants (PGVs) conferring increased cancer risk, changes in cancer diagnosis, and somatic alteration(s) used to estimate clinical benefit from or resistance to a therapy approved by the US Food and Drug Administration (FDA). Tier 2 alterations included somatic events that provided rationale for use of investigational or off-label targeted therapy and alterations postulated from strong preclinical evidence to estimate resistance to an FDA-approved therapy in that indication. Tier 3 included alterations implicated in cancer pathogenesis or molecular events indicative of a known cancer diagnosis but without current therapeutic implications. Genomic alterations in tiers 1 and 2 were considered potentially clinically actionable. B, Percentage of cases in which DNA or RNA sequencing contributed to identifying clinically relevant alterations. C, Classes of clinically relevant alterations identified in the MET1000 cohort. Amp indicates amplification; Del, homozygous deletion; Dx, markers for cancer of unknown primary origin or change of diagnosis; Exp, expression concordant with gene amplification; Fus, gene fusion; Germ, germline; Mut, mutation; and Virus, viral pathogen.

Figure 3.  Patients Receiving Sequencing-Directed Therapy (SDT) in MET1000 Cohort and Exceptional Responses
Patients Receiving Sequencing-Directed Therapy (SDT) in MET1000 Cohort and Exceptional Responses

Bar graphs depict proportion of patients in the MET1000 cohort (n = 1015) who received SDT and ultimately had clinical benefit or exceptional response to treatment.

Figure 4.  Pathogenic Germline Variants (PGVs) Observed in the MET1000 Cohort
Pathogenic Germline Variants (PGVs) Observed in the MET1000 Cohort

A, Among 1015 patients undergoing sequencing, 169 putative PGVs were identified (160 patients [15.8%] of MET1000 cohort). Fifty-five PGVs associated with highly penetrant cancer predisposition syndromes (including PGVs in APC, BAP1, BRCA1, BRCA2, DICER1, FH, HOXB13, MLH1, MSH2, PALB2, PMS2, POT1, and RB1) were identified, which included 14 PGVs known before the patient’s enrollment in the Michigan Oncology Sequencing Program (Mi-ONCOSEQ). Seventy-four PGVs associated with moderately penetrant cancer predisposition syndromes (including PGVs in APC, ATM, BARD1, CHEK2, MITF, MRE11A, MUTYH, RAD50, RAD51C, NF1, and SMARCB1) were identified, which included 2 PGVs known before the patient’s enrollment in Mi-ONCOSEQ. An additional 33 PGVs associated with an autosomal recessive condition conferring increased risk for cancer or lymphoproliferative disorder (including MPL, BLM, CASP8, ERCC1, ERCC2, ERCC3, ERCC4, FANCA, FANCC, FANCG, FANCM, HAX1, NBN, SBDS, and XPC) and 7 PGVs associated with an autosomal recessive condition not known to increase cancer risk (including PARK2, FH, and WRN) were identified. B, Pathogenic germline variants with somatic second-hit events identified by gene. Sixty-nine of the PGVs identified (40.8% of PGVs and 6.8% of MET1000 cohort) harbored a somatic second-hit event in the tumor. Incidental PGVs in highly penetrant cancer predisposition syndromes (ie, BRCA1) were identified in cases where the PGV was not likely related to tumor pathogenesis. C, Pathogenic germline variants with therapeutic targets were identified in 49 patients (4.8% of MET1000 cohort), often in diseases not typically associated with cancer predisposition syndromes in DNA or mismatch repair. Among the 49 PGVs identified with a therapeutic target, 42 (85.7%) harbored a somatic second-hit event in the tumor.

Figure 5.  Cancers of Unknown Primary Origin in MET1000 Cohort
Cancers of Unknown Primary Origin in MET1000 Cohort

A, Among 55 cases of cancer of unknown primary (CUP) origin sequenced, 28 (50.9%) were reclassified to a definitive diagnosis through RNA sequencing tissue of origin predictor. An additional 4 cases in the MET1000 cohort with presumed known diagnoses at study entry were also reclassified. B, Sequencing results were highly informative for patients with CUP, with a total of 34 of 55 CUP cases (61.8%) having at least 1 of the following: (1) a change in cancer diagnosis (28 patients [50.9%]), (2) receipt of sequencing-directed therapy (SDT) (13 patients [23.6%]), or (3) identification of a pathogenic germline variant (PGV) conferring increased cancer risk (8 patients [14.5%]). NET indicates neuroendocrine tumor; NSCLC, non–small cell lung cancer; and SCC, squamous cell carcinoma.

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    2 Comments for this article
    EXPAND ALL
    Status of cancer medicine by cancer genome profiling in Japan
    takuma hayashi, MBBS, DMSci, GMRC, PhD | National Hospital Organization Kyoto Medical Center
    In June 2019, the Cancer Genome Panel Test was approved by the Japanese Ministry of Health, Labor and Welfare as an insurance treatment for cancer patients who do not have standard treatment or have completed standard treatment. At present, at Cancer Genome Medical Core Hospital (11 facilities nationwide), Cancer Genome Medical Center Hospital (34 facilities), Cancer Genome Medical Cooperation Hospital (156 facilities), cancer genome medicine is performed based on the results of the cancer genome panel test.

    From December 2019 to December 2020, in cancer genomic medicine at Japanese national universities, clinical treatments for total of 576 cases with
    cancer (Ncc oncopanel test: 129 cases, F1CDx test: 447 cases) have been investigated.

    From the results of the cancer genome test, a total of 33 cases (Ncc oncopanel test: 8 cases, F1CDx test: 25 cases) were determined to be MSI-H. A total of 25 cases (Ncc oncopanel test: 6 cases, F1CDx test: 19 cases) were determined to be TMB (10 <).

    A total of 18 cases (Ncc oncopanel test: 5 cases, F1CDx test: 13 cases) were determined to be ERBB2 pathogenic variants. A total of 15 cases (Ncc oncopanel test: 3 cases, F1CDx test: 12 cases) were determined to be BRAF pathogenic variants.

    A total of 3 cases (Ncc oncopanel test: 3 cases) were determined to be FGFR 1-4 pathogenic variants. A total of 9 cases (F1CDx test: 9 cases) were determined to be CDK4 / 6 pathogenic variants.

    A total of 9 cases (Ncc oncopanel text: 1 case, F1CDx text: 8 cases) were determined to be EGFR pathogenic variants. A total of 43 cases (Ncc oncopanel test: 3 cases, F1CDx test: 40 cases) were determined to be pathogenic variants of other factors (JAK, KRAS, NF1, etc).

    Of a total of 576 patients, 8 cases were recommended for insured drugs, 30 cases were recommended for non-insured drugs, and 17 were recommended for drugs under the clinical trials.

    When cancer genomic medicine is operated, various issues have surfaced. The results obtained by the cancer genome inspection will be considered by the expert meeting. Therefore, it takes a long time of about 4 to 6 weeks for the results examined at the expert meeting to return to the attending physician. Furthermore, even if a pathogenic variant is found in a gene from the results of a cancer genome test, it is often the case that an appropriate treatment method cannot be obtained. Many problems must be solved in order for the state-of-the-art medical technology called genome analysis to be operated as general cancer medicine.

    Ethical approval and consent to participate.
    This study was reviewed and approved by the Central Ethics Review Board of the National Hospital Organization Headquarters in Japan (Tokyo Japan). The authors attended a 2020 educational lecture on medical ethics supervised by the Japanese government. The completion numbers of the authors are AP0000151756 AP0000151757 AP0000151769 AP000351128.

    Acknowledgments
    We thank all the medical staffs and co-medical staffs for providing and helping medical research at National Hospital Organization Kyoto Medical Center.
    CONFLICT OF INTEREST: None Reported
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    How much to benefit a small#!
    Dilipsinh Solanki, MD | Texas Oncology
    * Qite surprising that 4 of 5 had an actionable genomic abnormalities (which seems very high if these were sporadic cases of cancer).

    * Only 1 out of 7 of these( 1 out of 8 of the total) received targeted therapy.

    * The biggest beneficiaries were patients with CUP that made up just 5% of the patients.

    * Of the CUP chart, o.5% had extended benefit.

    * Clinical benefit of other patients is not compared with treatment they may have received.

    My Q is: what yield in yer s of cost benefit should
    we set or do the authors suggest that is not something we should even bring up.

    In every group discussion , this subject comes yuppie. The most common refrain is a comprehensive panel is more economical than a tailored one. Q is Why? That argument has the feel of BOGO ( how come the price s half just because we buy two instead of one)? t is also not like buying cable subscription where they have a smile switch to connect to more. Here I assume they must do separate testing for mst or many potential targets.
    CONFLICT OF INTEREST: None Reported
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    Original Investigation
    February 25, 2021

    Assessment of Clinical Benefit of Integrative Genomic Profiling in Advanced Solid Tumors

    Author Affiliations
    • 1Department of Internal Medicine, University of Michigan, Ann Arbor
    • 2Michigan Center for Translational Pathology, University of Michigan, Ann Arbor
    • 3Department of Pathology, University of Michigan, Ann Arbor
    • 4Rogel Cancer Center, University of Michigan, Ann Arbor
    • 5Department of Urology, University of Michigan, Ann Arbor
    • 6Howard Hughes Medical Institute, University of Michigan, Ann Arbor
    JAMA Oncol. 2021;7(4):525-533. doi:10.1001/jamaoncol.2020.7987
    Key Points

    Question  What is the clinical utility of genomic profiling for patients with advanced solid tumors?

    Findings  In this cohort study of 1015 patients who underwent integrative genomic profiling, a high rate of pathogenic germline variants and a subset of patients who derive substantial clinical benefit from sequencing information were identified.

    Meaning  These findings support (1) directed germline testing for inherited cancer predisposition in all patients with advanced cancer and (2) use of integrative genomic profiling as a component of standard of care for patients with cancer of unknown origin and other rare malignant neoplasms.

    Abstract

    Importance  Use of next-generation sequencing (NGS) to identify clinically actionable genomic targets has been incorporated into routine clinical practice in the management of advanced solid tumors; however, the clinical utility of this testing remains uncertain.

    Objective  To determine which patients derived the greatest degree of clinical benefit from NGS profiling.

    Design, Setting, and Participants  Patients in this cohort study underwent fresh tumor biopsy and blood sample collection for genomic profiling of paired tumor and normal DNA (whole-exome or targeted-exome capture with analysis of 1700 genes) and tumor transcriptome (RNA) sequencing. Somatic and germline genomic alterations were annotated and classified according to degree of clinical actionability. Results were returned to treating oncologists. Data were collected from May 1, 2011, to February 28, 2018, and analyzed from May 1, 2011, to April 30, 2020.

    Main Outcomes and Measures  Patients’ subsequent therapy and treatment response were extracted from the medical record to determine clinical benefit rate from NGS-directed therapy at 6 months and exceptional responses lasting 12 months or longer.

    Results  During the study period, NGS was attempted on tumors from 1138 patients and was successful in 1015 (89.2%) (MET1000 cohort) (538 men [53.0%]; mean [SD] age, 57.7 [13.3] years). Potentially clinically actionable genomic alterations were discovered in 817 patients (80.5%). Of these, 132 patients (16.2%) received sequencing-directed therapy, and 49 had clinical benefit (37.1%). Exceptional responses were observed in 26 patients (19.7% of treated patients). Pathogenic germline variants (PGVs) were identified in 160 patients (15.8% of cohort), including 49 PGVs (4.8% of cohort) with therapeutic relevance. For 55 patients with carcinoma of unknown primary origin, NGS identified the primary site in 28 (50.9%), and sequencing-directed therapy in 13 patients resulted in clinical benefit in 7 instances (53.8%), including 5 exceptional responses.

    Conclusions and Relevance  The high rate of therapeutically relevant PGVs identified across diverse cancer types supports a recommendation for directed germline testing in all patients with advanced cancer. The high frequency of therapeutically relevant somatic and germline findings in patients with carcinoma of unknown primary origin and other rare cancers supports the use of comprehensive NGS profiling as a component of standard of care for these disease entities.

    Introduction

    Identification of tumor genomic alterations predictive of therapeutic benefit from targeted therapy has improved clinical outcomes across a wide range of malignant neoplasms.1-7 Examples include EGFR-mutant non–small cell lung cancer treated with epidermal growth factor receptor tyrosine kinase inhibitors, ERBB2 (formerly HER2/neu)-amplified breast cancer treated with human epidermal growth factor receptor 2–specific antibodies, and tumors with microsatellite instability treated with immune checkpoint blockade. Because identification of these targets susceptible to drug therapy has improved clinical outcomes, interest in using next-generation sequencing (NGS) testing for advanced solid tumors to identify patients who qualify for enrollment in biomarker-selected clinical trials is increasing. Next-generation sequencing testing can be obtained via commercial or institutional platforms, and these differ significantly in their scope; some are limited to DNA-based testing of a select group of known, targetable cancer-related genes. Others use unbiased, comprehensive DNA- and RNA-based analyses of samples consisting of tumor and normal samples, with additional goals of identifying novel somatic alterations or pathogenic germline variants (PGVs) conferring increased cancer risk.

    Studies of comprehensive NGS testing in patients with advanced cancer8-16 report a wide range of clinically actionable genomic alterations per patient, ranging from 40% to 94%. Furthermore, many studies15-17 note that only 10% to 25% of patients receive therapy informed by sequencing, making it challenging to assess the degree of clinical benefit gained. The only randomized clinical trial to explore the clinical effects of delivering genomically directed therapy to patients undergoing NGS testing in the setting of advanced cancer found no improvement in progression-free survival for patients receiving molecularly matched therapy.18 Other nonrandomized studies11,15,17,19-23 have demonstrated a modest clinical benefit from delivery of molecularly targeted therapy; however, interpretation of these results is often confounded by use of end points in which patients serve as their own control, such as comparison of progression-free survival during targeted therapy with progression-free survival during the patient’s previous line of therapy.

    In this study, we sought to conduct a comprehensive analysis of the clinical outcomes of patients with advanced solid tumors who had NGS performed on metastatic tissue through participation in a prospective clinical cohort study, the Michigan Oncology Sequencing Program (Mi-ONCOSEQ). Robinson et al12 previously summarized the molecular findings from the first 500 patients enrolled, which determined that PGVs were identified at a high rate (12.2%) and that DNA and RNA sequencing were both contributory in identifying clinically actionable targets. Herein, with an expanded cohort of more than 1000 patients linked to clinical outcomes, we determine which patients derived the greatest clinical benefit from sequencing-directed therapy (SDT).

    Methods
    Study Description

    Mi-ONCOSEQ was established in 2011 at the Michigan Center for Translational Pathology24 and is a Clinical Laboratory Improvement Amendments–certified laboratory conducting integrative sequencing of tumor and normal (blood or buccal swab) specimens. Eligibility included advanced or metastatic cancer, being 18 years or older, and the ability to safely undergo fresh tumor sampling by imaging-guided biopsy. All patients provided written informed consent, which included willingness to receive information regarding PGVs if identified. This study was approved by the institutional review board of the University of Michigan and followed the College of American Pathologists reporting guideline.

    Integrative Clinical Sequencing

    Needle biopsy samples or surgically resected tissue samples were flash frozen, and a section was cut for evaluation. Remaining portions of each specimen were retained for nucleic acid extraction. Hematoxylin-eosin–stained frozen sections were reviewed by study pathologists (D.L., S.C.-P., A.M.U., and L.P.K.) to identify areas with highest tumor content. If no tumor was identified, an archival formalin-fixed paraffin-embedded sample was obtained for sequencing if available. Nucleic acid preparation and integrative clinical sequencing were performed using standard protocols.12 A subset of tumor samples was analyzed using a targeted panel of 1700 genes (eTable 1 in Supplement 1). Use of a whole-exome or a targeted panel for each case is described in eTable 2 in Supplement 2. Germline variants were annotated using published literature and public databases (ClinVar and the Human Genome Mutation Database), with pathogenicity determined as per American College of Medical Genetics and Genomics guidelines.25 Only variants previously described as pathogenic or likely pathogenic were disclosed. Sequencing data from all patients can be obtained from the Database of Genotypes and Phenotypes under accession number phs000673.v4.p1.

    Carcinoma of Unknown Primary Tissue of Origin Estimation

    Tissue from a carcinoma of unknown primary (CUP) origin estimation was implemented using a published method,26 but adapted for RNA-sequencing data with modifications. For training, the Cancer Genome Atlas data of primary tumor tissues were artificially contaminated with expression from normal tissues (obtained from the Genotype-Tissue Expression program, the Cancer Genome Atlas, and the Human Protein Atlas). Each estimation was based on an ensemble learning approach aggregating 6 different models (bootstrap aggregation; each model has the same weight). The 6 models were derived from the combination of nu-support vector machine and multinomial lasso classifier machine learning algorithms and 3 ways of contaminating training examples (none, expression from the normal biopsy site, and mean model of expression from all possible biopsy sites).

    Precision Medicine Tumor Board and Results Disclosure

    A monthly multidisciplinary precision medicine tumor board reviewed, interpreted, and discussed sequencing results for patients whose NGS results were of clinical importance (eFigure 1 in Supplement 3 and eTable 2 in Supplement 2). For all patients, summarized results were presented in the form of a report (generated within 4 to 6 weeks of patient enrollment) to treating oncologists, with opportunity to review by the precision medicine tumor board on request (eFigure 1 in Supplement 3). Considerations for targeted therapy included on reports were informed by use of variant annotation databases such as OncoKB, CIViC, My Cancer Genome, and literature review coupled with the National Comprehensive Cancer Network, US Food and Drug Administration guidelines and approvals, and ClinicalTrials.gov. For germline variants, ClinVar, as well as gene-specific databases, was consulted.

    Generation of Genomic Alteration Tiers

    Genomic alterations were classified in a tumor-specific manner and placed in 1 of 3 tiers according to levels of evidence and potential for clinical action. Tier 1 and 2 genomic alterations were considered potentially clinically actionable. The Mi-ONCOSEQ genomic alteration tiers are similar to previously published schemas,27-29 with a few additions: (1) PGVs are categorized separately from somatic variants, distinguishing those that confer increased cancer risk as well as those with direct therapeutic implications; and (2) change in cancer diagnosis as determined by transcriptomic profiling is incorporated.

    Analysis of Clinical Outcomes

    Data were analyzed from May 1, 2011, to April 30, 2020. Medical record reviews were performed by a medical oncologist (E.F.C.). It was determined whether a patient received SDT via review of clinician notes, which often directly referenced the Mi-ONCOSEQ report or precision medicine tumor board discussion. Sequencing-directed therapy could be delivered in clinical trials, off-label, or on-label at the discretion of the treating oncologist. Time receiving treatment and best response to therapy (progressive disease, stable disease, partial response, or complete response) were recorded. For patients treated with SDT during a clinical trial, response evaluation criteria in solid tumors (RECIST) were directly abstracted from the medical record. For patients not treated during a clinical trial, best response to therapy was determined by a medical oncologist (E.F.C.) via review of clinician notes and/or direct measurement of sites of measurable disease on computed tomographic imaging and use of RECIST criteria. Clinical benefit rate was defined as the proportion of patients receiving SDT for 6 months or longer. Exceptional responders received SDT for a duration of 12 months or longer.

    Results
    Cohort Demographics

    From May 1, 2011, to February 28, 2018, 1138 patients underwent biopsy for integrative clinical sequencing. Of these, 1015 had successful NGS testing (MET1000 cohort), whereas 123 did not, owing to the following: (1) patient changed consent, moved, or entered hospice before scheduled biopsy; (2) inability to safely perform planned biopsy owing to tumor location; and (3) inadequate tumor content from biopsy (Figure 1). Mean (SD) patient age at enrollment was 57.7 (13.3) years. A total of 538 patients (53.0%) were men and 477 (47.0%) were women. Types of cancers sequenced and sites of metastatic biopsy are depicted in eFigure 2 in Supplement 3. Eight hundred five patients (79.3%) received systemic therapy before enrollment (eTable 2 in Supplement 2). A mean (SD) of 47 (63) months elapsed from time of the patient’s cancer diagnosis to enrollment in Mi-ONCOSEQ (eTable 2 in Supplement 2).

    Germline and Somatic Molecular Alterations in Patients With Metastatic Solid Tumors

    Among 1015 tumors sequenced, 817 (80.5% of cohort) harbored at least 1 potentially actionable alteration, classified as tier 1 or 2 (Figure 2A). Tumors in 713 patients (70.2%) harbored a somatic molecular alteration that was potentially actionable and could provide rationale for the use of investigational targeted therapy or off-label therapy (designated tier 2 S2). Among tier 1 and 2 molecular alterations, 962 (94.8% of total) were identified by DNA sequencing and 645 (63.5% of total) were identified by RNA sequencing (Figure 2B). Integrative sequencing revealed a variety of alteration classes; DNA sequencing of tumor and normal samples identified mutations, amplifications, homozygous deletions, and germline alterations (Figure 2C). RNA sequencing complemented this analysis and was essential in the identification of 103 clinically actionable molecular events (10.1% of total), particularly with regard to identification of oncogenic gene fusions (Figure 2C). RNA sequencing also provided information on outlier expression concordant with amplification, viral pathogens, and markers for CUP or change of diagnosis. In terms of the contribution of each mode of sequence analyses (mutation, copy number determination, and RNA-seq analysis) to the yield of informative aberrations, in 579 cases (57.0%), informative alterations were detected by all of the 3 modes of integrated sequencing and analysis. In 213 cases (21.0%), clinically relevant aberrations were detected by mutation and copy number analyses, but no reportable aberrations were detected by RNA-seq. In 51 cases (5.0%), only RNA-seq yielded clinically informative events, emphasizing the increased clinical yield of the integrated sequence and analyses approach used here. Clinical tiering information for all molecular events identified in the MET1000 cohort is detailed in eTable 3 in Supplement 4.

    Use of SDT

    Sequencing-directed therapy was initiated in 132 patients (16.2% of 817 patients with clinically actionable alterations) (Figure 3). Median time from study enrollment to initiation of SDT was 3.8 (range, 0.2-44.0) months. Among those receiving SDT, 74 patients were treated during a clinical trial, 43 with off-label therapy, and 15 with on-label therapy (Figure 1; eTable 4 in Supplement 5). Of these, 49 patients (37.1% of those receiving SDT) experienced clinical benefit. The most common cancer types with clinical benefit from SDT were sarcoma (12 of 138 [8.7%]), prostate adenocarcinoma (10 of 154 [6.5%]), and CUP (7 of 55 [12.7%]). Most patients receiving SDT (93 [70.5%]) were treated with targeted small-molecule inhibitors, including CDK4/6 inhibitors in 21 patients, inhibitors of poly–adenosine diphosphate ribose polymerase (PARPis) in 16 patients, and inhibitors of fibroblast growth factor receptor in 11 patients. Additional common SDTs included an immune checkpoint inhibitor (ICI) in 29 patients (22.0%), monoclonal antibodies against human epidermal growth factor receptor 2 in 8 patients (6.1%), and antiestrogen therapy in 2 patients (1.5%).

    Patients With Exceptional Response to SDT

    Twenty-six patients (19.7% of those receiving SDT) received treatment for 12 months or longer (Figure 3, eTable 4 in Supplement 5, and eFigures 3-4 in Supplement 3), ranging from 12.1 to 39.5 months. Genomic alterations in those with an exceptional response included 10 cases with defects in DNA repair, of whom 5 had double-strand DNA repair defects (BRCA1/BRCA2 [OMIM 113705/OMIM 600185]: 4 somatic and 1 PGV) and 5 had defects in DNA mismatch repair (MSH2 [OMIM 609309]: 3 somatic and 2 PGVs). Five cases harbored driver gene fusions, including a novel fusion involving the progesterone receptor (PGR) in a patient who developed chondrosarcoma during pregnancy and had an exceptional response to tamoxifen. Three patients with exceptional responses had tumors with activating hotspot mutations, treated with targeted small-molecule inhibitors. Five cases with amplifications in the CDK4/6 pathway had exceptional response when treated with CDK4/6 inhibitors, and 1 patient with CUP had an exceptional response to dual human epidermal growth factor receptor 2 monoclonal antibody treatment, which was initiated based on ERBB2 amplification. In 2 cases, 1 with breast cancer and 1 with CUP, patients had exceptional responses to programmed cell death protein inhibitor therapy, which was initiated based on high mutation burden by a mechanism other than microsatellite instability (eFigure 3 in Supplement 3). The most common reason for discontinuation of SDT was disease progression (eTable 4 in Supplement 5). Three patients receiving ICI therapy based on high mutation burden by mechanism other than microsatellite instability or alternative mechanism of high mutation burden achieved a complete response and remain without evidence of disease.

    Pathogenic Germline Variants

    A total of 169 PGVs were identified among 160 unique patients (15.8% of cohort), 49 of whom (4.8% of cohort) were designated as having potential therapeutic implications (tier 1, G1*) (Figure 2A and eTable 5 in Supplement 6). Most PGVs (155 of 169 [91.7%]) were unknown before enrollment in Mi-ONCOSEQ and were classified as belonging to 1 of 4 categories shown in Figure 4A (eTable 5 in Supplement 6). One hundred fifteen PGVs were identified (68.0% of total PGVs and 11.3% of cohort) that were indicative of highly or moderately penetrant cancer predisposition syndromes. Sixty-nine of 169 PGVs (40.8%) harbored a somatic second hit event in the tumor (Figure 4B). Pathogenic germline variants associated with therapeutic targets, such as those resulting in defects in double-strand DNA repair (BRCA1, BRCA2, ATM, PALB2, and BRIP1) or DNA mismatch repair (MLH1, MSH2, and PMS2), were identified in 49 patients (29.0% of total PGVs and 4.8% of cohort), 37 of which had not been identified before enrollment. Fourteen PGVs in DNA double-strand repair and 7 PGVs in DNA mismatch repair were identified in cancer types not commonly associated with hereditary breast and ovarian cancer or Lynch syndromes, including CUP and sarcomas (Figure 4C). Seven patients received a PARPi, 3 received an ICI, and 1 received both PARPi and ICI therapy owing to identification of PGVs in DNA repair. Six of these patients achieved clinical benefit from treatment (eFigure 5 in Supplement 3).

    CUP Origin

    Before NGS testing, 55 patients had a diagnosis of CUP. The median number of prior lines of systemic therapy for patients with CUP at Mi-ONCOSEQ enrollment was 0, ranging from 0 to 4 (eTable 6 in Supplement 3). Twenty-eight CUP cases (50.9%) were reclassified to an alternative cancer diagnosis, whereas a definitive diagnosis was not able to be established for the remaining 27 cases. In addition, 4 more cancers that were initially classified as originating from a specific tissue were reclassified using this same method (Figure 5A and eTable 6 in Supplement 3). Sequencing-directed therapy was initiated in 13 instances for patients who initially had CUP (23.6%); 6 patients experienced a clinical benefit, including 1 who had clinical benefit from 2 SDTs administered serially (clinical benefit rate, 53.8%). Five exceptional responses were observed (Figure 5B and eFigure 3 in Supplement 3). Pathogenic germline variants were identified in 8 patients with CUP (14.5%), 4 of which were therapeutically relevant (BRCA1 in 2 patients, BRCA2 in 1 patient, and MSH2 in 1 patient) (eTable 5 in Supplement 6).

    Discussion

    In this study, we found that combined genomic profiling of tumor and normal specimens was a clinically powerful tool. Most importantly, a high incidence of PGVs was observed, approximately 1 in every 6 patients, with many of the PGVs identified having therapeutic relevance and resulting in clinical benefit when targeted therapies were administered. In addition, this finding has significant implications for patients’ family members, who can be offered genetic testing and enhanced screening or risk-reducing interventions if a PGV is identified. Our study also found that patients with CUP derived significant clinical benefit from NGS profiling. At present, CUP has no clear standard treatment paradigm. Among this cohort, 50.9% of CUP tumors were reclassified to a specific cancer diagnosis, allowing some patients to receive standard of care therapy for that disease entity and 13 (23.6%) to receive SDT. Furthermore, a high proportion of these patients harbored PGVs (14.5%).

    Among all patients treated with SDT, 37.1% had clinical benefit from treatment, including 19.7% with exceptional response. None of these therapies would have been recommended per standard of care guidelines, indicating that sequencing information was of significant value. The most common genomic alterations portending exceptional response included defects in DNA double-strand repair and DNA mismatch repair, for which patients received either PARPi or ICI. In many circumstances, DNA repair defects were identified in tumor types not commonly associated with these genomic changes, such as CUP and cholangiocarcinoma. Last, some patients achieved exceptional response to targeted therapy administered on the basis of a driver gene fusion, which would not have been identified without RNA sequencing.

    Assessing the clinical utility of NGS testing in oncology remains challenging for several reasons: (1) the definition of clinically actionable alterations is dynamic as new therapeutics emerge; (2) patients’ tumors often harbor numerous alterations, with uncertainty surrounding which events are most important to target; (3) patients with advanced malignant neoplasms and multiple prior therapies may not qualify for trial enrollment or may tolerate treatment poorly; (4) clinical behavior of diverse cancer types included in studies is highly variable; and (5) availability of biomarker-selected trials may be limited, and insurance may not cover off-label therapies. Indeed, these challenges highlight the need to explore novel clinical trial end points and develop large-scale precision oncology studies with access to a wide range of targeted therapies. Two of the largest clinical trials using this strategy, Molecular Analysis for Therapy Choice (MATCH)30-32 and Targeted Agent and Profiling Utilization Registry (TAPUR),33,34 are ongoing. However, few studies are specifically exploring the effect of identifying incidental PGVs from tumor NGS testing.

    Strengths and Limitations

    Strengths of this study include the use of DNA and RNA sequencing in addition to assessment of tumor and normal specimens, allowing for identification of previously unidentified PGVs, gene fusions, and tissue of origin estimation. Additional strengths are inclusion of large numbers of patients with rare cancers and clinical outcomes analyses for patients treated with SDT. Limitations are that clinical outcome analyses are retrospective and largely descriptive in nature with lack of a comparator or control population to quantify the degree of clinical benefit achieved by testing. However, the diversity of patients, tumor types, and molecular characteristics included within this study make identification of an appropriate control population difficult. Furthermore, the proportion of patients who received SDT is low (13.0%) and in accordance with rates previously reported in studies, which have used more limited sequencing approaches (ie, targeted DNA-based panels),16,21,35 indicating that broader sequencing approaches have not yet translated into improved ability to match patients with targeted therapy. Despite this, we observed that our ability to direct patients toward clinical trials of targeted therapy improved over time (eTable 7 in Supplement 3). Last, our study does not explore other sequencing methods that may be of clinical or scientific value, such as analysis of liquid biopsy specimens in the form of circulating tumor DNA or novel tumor-based sequencing approaches such as methylome profiling.

    Conclusions

    Our data support a recommendation for germline testing of DNA repair genes as standard practice in patients with metastatic solid tumors and comprehensive NGS profiling at diagnosis for patients with CUP. With continued discovery of genomic biomarkers predictive of clinical benefit from anticancer therapies, we anticipate even broader clinical applicability of this technology.

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    Article Information

    Accepted for Publication: December 2, 2020.

    Published Online: February 25, 2021. doi:10.1001/jamaoncol.2020.7987

    Open Access: This is an open access article distributed under the terms of the CC-BY License. © 2021 Cobain EF et al. JAMA Oncology.

    Corresponding Author: Arul M. Chinnaiyan, MD, PhD, Michigan Center for Translational Pathology, University of Michigan, 1500 E Medical Center Dr, 5316 Rogel Cancer Center, Ann Arbor, MI 48109 (arul@umich.edu).

    Author Contributions: Drs Cobain and Wu contributed equally. Drs Robinson, Talpaz, and Chinnaiyan served as co–senior authors. Dr Chinnaiyan had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

    Concept and design: Cobain, Wu, Schott, Jacobs, Rabban, Robinson, Chinnaiyan.

    Acquisition, analysis, or interpretation of data: Cobain, Wu, Vats, Chugh, Worden, Smith, Schuetze, Zalupski, Sahai, Alva, Schott, Caram, Hayes, Stoffel, Jacobs, Kumar-Sinha, Cao, Wang, Lucas, Ning, Bell, Camelo-Piragua, Udager, Cieslik, Lonigro, Kunju, Robinson, Talpaz, Chinnaiyan.

    Drafting of the manuscript: Cobain, Wu, Vats, Zalupski, Stoffel, Ning, Rabban, Bell, Lonigro, Robinson, Chinnaiyan.

    Critical revision of the manuscript for important intellectual content: Cobain, Wu, Chugh, Worden, Smith, Schuetze, Zalupski, Sahai, Alva, Schott, Caram, Hayes, Stoffel, Jacobs, Kumar-Sinha, Cao, Wang, Lucas, Camelo-Piragua, Udager, Cieslik, Kunju, Robinson, Talpaz, Chinnaiyan.

    Statistical analysis: Cieslik, Robinson.

    Obtained funding: Chinnaiyan.

    Administrative, technical, or material support: Cobain, Alva, Caram, Stoffel, Jacobs, Cao, Wang, Lucas, Ning, Rabban, Bell, Camelo-Piragua, Lonigro, Robinson, Chinnaiyan.

    Supervision: Cobain, Hayes, Stoffel, Camelo-Piragua, Kunju, Robinson, Talpaz, Chinnaiyan.

    Other–bioinformatics analysis: Vats.

    Other–enrollment of patients into the clinical study: Schuetze.

    Other–clinical coordinator: Rabban.

    Conflict of Interest Disclosures: Dr Cobain reported receiving personal fees from AstraZeneca, Athenex, Inc, and Ayala Pharmaceuticals outside the submitted work. Dr Chugh reported receiving personal fees from Immune Design and Ipsen Pharma, grants from Epizyme, Inc, AADi, Novartis AG, Medivation, Inc, Advenchen Laboratories, LLC, Plexxikonn, SpringWorks Therapeutics, Mundipharma International, GSK Group, and Qilu Puget Sound Biotherapeutics Corporation, and nonfinancial support from Janssen Global Services, LLC, GSK Group, and SpringWorks Therapeutics outside the submitted work. Dr Worden reported receiving funding for clinical trials from Merck & Co, Eisai Inc, Bristol Myers Squibb, Cue Biopharma, Eli Lilly and Company, and Pfizer, Inc, serving on advisory boards for Merck & Co, Bristol Myers Squibb, Cue Biopharma, and Eli Lilly and Company, personal fees from Bayer AG, and a stipend for a lecture given at the Brazilian Society of Endocrinology and Metabology during the conduct of the study. Dr Smith reported receiving grants from Bristol Myers Squibb, Eli Lilly and Company, Medivation, Inc, Astellas Pharma Inc, Incyte, Novartis AG, Genentech, Inc, and OncoMed-Solutions outside the submitted work. Dr Schuetze reported receiving grants from the National Cancer Institute (NCI) during the conduct of the study and grants from Blueprint Medicines Corporation, Adaptimmune Therapeutics plc, GSK Group, Amgen, Inc, and Karyopharm outside the submitted work. Dr Sahai reported receiving institutional research grant funding from Agios, Inc, Bristol Myers Squibb, Celgene Corporation, Clovis Oncology, Debiopharm, FibroGen Inc, Incyte, Ipsen Pharma, MedImmune, LLC, Merck & Co, NCI, and Rafael Pharmaceuticals, Inc, and consulting for AstraZeneca, Halozyme, Inc, QED Therapeutics, Inc, Incyte, Ipsen Pharma, GSK Group, and Rafael Pharmaceuticals, Inc. Dr Alva reported receiving grants from Pfizer, Inc, Bristol Myers Squibb, Celgene Corporation, AstraZeneca, Prometheus Biosciences, and Astellas Pharma Inc, personal fees from Pfizer, Inc, Bristol Myers Squibb, and AstraZeneca, and nonfinancial support from the American Society of Clinical Oncology outside the submitted work. Dr Hayes reported receiving earnings from Inbiomotion stock options, personal fees for consulting and serving on advisory committees for Cepheid, Freenome Holdings, Inc, Artiman, Lexent Bio, Inc, Agendia, Epic Sciences, Salutogenic Innovations, LLC, and L-Nutra Inc, research support from Merrimack, Eli Lilly and Company, Menarini Silicon Biosystems, Puma Biotechnology, Inc, Pfizer, Inc, and AstraZeneca outside the submitted work, and royalties from licensed technology 08/01/14 licensed to Janssen R&D, LLC (Johnson & Johnson) and transferred to Menarini Silicon Biosystems. Dr Chinnaiyan reported serving on the scientific advisory board of Tempus, which has licensed the Mi-Oncoseq1700 panel and integrative sequencing approach from the University of Michigan; serving on the scientific advisory board of Ascentage; being cofounder and serving on the scientific advisory boards of Oncopia, LynxDx, and Esanik; and receiving support as a Howard Hughes Medical Institute Investigator, A. Alfred Taubman Scholar, and American Cancer Society professor. No other disclosures were reported.

    Funding/Support: This study was supported by the Prostate Cancer Foundation; Clinical Sequencing Exploratory Research Award NIH-1UM1HG006508 from the National Institutes of Health; grant U01-CA214170 from the NCI (Early Detection Research Network); Prostate SPORE grant P50-CA186786 from the NCI; Outstanding Investigator Award R35-CA231996 from the NCI; and grant P30-CA046592 from the NCI (Rogel Cancer Center).

    Role of the Funder/Sponsor: The sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

    Disclaimer: Tempus was not involved in the design of this study, nor did it approve the contents of this study.

    Additional Contributions: We thank the entire Michigan Oncology Sequencing Program (Mi-ONCOSEQ) clinical sequencing team for their contributions to this study and all physicians who referred patients to the Mi-ONCOSEQ program. Jyoti Athanikar, PhD, Michigan Center for Translational Pathology at the University of Michigan, coordinated Mi-ONCOSEQ reports and Stephanie Ellison, PhD, Michigan Center for Translational Pathology at the University of Michigan, assisted with scientific writing and editing. Most importantly, we recognize the generosity and kindness of the patients and their families for participating in this study.

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