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Therapeutic implications of germline vulnerabilities in DNA repair for precision oncology

  • Author Footnotes
    1 Authors contributed equally.
    Shreya M. Shah
    Footnotes
    1 Authors contributed equally.
    Affiliations
    Cancer Prevention and Control Program, Fox Chase Cancer Center, Philadelphia, PA, United States

    Science Scholars Program, Temple University, Philadelphia, PA, United States
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  • Author Footnotes
    1 Authors contributed equally.
    Elena V. Demidova
    Footnotes
    1 Authors contributed equally.
    Affiliations
    Cancer Prevention and Control Program, Fox Chase Cancer Center, Philadelphia, PA, United States

    Kazan Federal University, Kazan, Russian Federation
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  • Randy W. Lesh
    Affiliations
    Cancer Prevention and Control Program, Fox Chase Cancer Center, Philadelphia, PA, United States

    Geisinger Commonwealth School of Medicine, Scranton, PA, United States
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  • Michael J. Hall
    Affiliations
    Cancer Prevention and Control Program, Fox Chase Cancer Center, Philadelphia, PA, United States

    Department of Clinical Genetics, Fox Chase Cancer Center, Philadelphia, PA, United States
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  • Mary B. Daly
    Affiliations
    Cancer Prevention and Control Program, Fox Chase Cancer Center, Philadelphia, PA, United States

    Department of Clinical Genetics, Fox Chase Cancer Center, Philadelphia, PA, United States
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  • Joshua E. Meyer
    Affiliations
    Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, PA, United States

    Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA, United States
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  • Martin J. Edelman
    Correspondence
    Corresponding authors at: Cancer Prevention and Control Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497, United States (S. Arora); Department of Hematology/Oncology, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497, United States (M.J. Edelman).
    Affiliations
    Department of Hematology/Oncology, Fox Chase Cancer Center, Philadelphia, PA, United States
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  • Sanjeevani Arora
    Correspondence
    Corresponding authors at: Cancer Prevention and Control Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497, United States (S. Arora); Department of Hematology/Oncology, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497, United States (M.J. Edelman).
    Affiliations
    Cancer Prevention and Control Program, Fox Chase Cancer Center, Philadelphia, PA, United States

    Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, PA, United States
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  • Author Footnotes
    1 Authors contributed equally.
Open AccessPublished:January 05, 2022DOI:https://doi.org/10.1016/j.ctrv.2021.102337

      Highlights

      • Growing appreciation that germline variation in DNA repair predicts response to multiple therapeutic strategies.
      • Germline testing has identified clinically actionable variants in multiple DNA repair genes.
      • Increasing evidence that genetic testing can benefit patients who do not meet current guidelines for testing, implications for population testing and benefit for treatment decision.
      • Multiple preclinical and clinical studies are evaluating the therapeutic implications of germline variants in additional DNA repair genes or genes that regulate DNA repair pathways.

      Abstract

      DNA repair vulnerabilities are present in a significant proportion of cancers. Specifically, germline alterations in DNA repair not only increase cancer risk but are associated with treatment response and clinical outcomes. The therapeutic landscape of cancer has rapidly evolved with the FDA approval of therapies that specifically target DNA repair vulnerabilities. The clinical success of synthetic lethality between BRCA deficiency and poly(ADP-ribose) polymerase (PARP) inhibition has been truly revolutionary. Defective mismatch repair has been validated as a predictor of response to immune checkpoint blockade associated with durable responses and long-term benefit in many cancer patients. Advances in next generation sequencing technologies and their decreasing cost have supported increased genetic profiling of tumors coupled with germline testing of cancer risk genes in patients. The clinical adoption of panel testing for germline assessment in high-risk individuals has generated a plethora of genetic data, particularly on DNA repair genes. Here, we highlight the therapeutic relevance of germline aberrations in DNA repair to identify patients eligible for precision treatments such as PARP inhibitors (PARPis), immune checkpoint blockade, chemotherapy, radiation therapy and combined treatment. We also discuss emerging mechanisms that regulate DNA repair.

      Keywords

      Abbreviations:

      BC (breast cancer), BER (base excision repair), CRC (colorectal cancer), dMMR (mismatch repair deficiency), DSB (double-strand breaks), FA (Fanconi Anemia), HLRCC (hereditary leiomyomatosis and renal cell cancer), HRR (homologous recombination repair), ICI (immune checkpoint inhibitor), MMEJ (microhomology-mediated end-joining), MMR (mismatch repair), MSI (microsatellite instability), NER (nucleotide excision repair), NHEJ (non-homologous end joining), OC (ovarian cancer), ORR (objective response rate), OS (overall survival), PARP (poly(ADP-ribose) polymerase), PARPi (PARP inhibitor), PC (pancreatic cancer), pCR (pathological complete response), PFS (progression free survival), PV (pathogenic variant), TMB (tumor mutation burden), VUS (variants of uncertain significance)

      Introduction

      It is estimated that ∼ 5–10% of all cancers are due to pathogenic variants (PV) inherited in the germline.[
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      Pathogenic Variants in Less Familiar Cancer Susceptibility Genes: What Happens After Genetic Testing?.
      ] Germline PVs in DNA repair genes are known to not only increase cancer risk but are also relevant for guiding cancer treatment. DNA repair is crucial for genome stability, with multiple specialized pathways existing in the cell to repair different types of DNA lesions (Fig. 1). These pathways include homologous recombination repair (HRR), non-homologous end-joining (NHEJ), Fanconi anemia (FA), microhomology-mediated end joining (MMEJ), nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), and replication repair.[
      • O’Connor M.J.
      Targeting the DNA Damage Response in Cancer.
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      Figure thumbnail gr1
      Fig. 1A simplified view of DNA damage response and repair: cancer risk and approved therapeutic biomarkers or therapies. DNA damage may be caused by multiple endogenous (metabolites, replication errors) or exogenous (irradiation, UV light, chemotherapy agents) sources.[
      • Roos W.P.
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      DNA damage and the balance between survival and death in cancer biology.
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      Mechanisms of DNA damage, repair, and mutagenesis.
      ] Multiple forms of DNA damage, such as replication errors, single stranded and double stranded breaks can activate the DNA damage response signaling and the checkpoint response. The DNA damage response signaling involves the activation of the sensory kinases, ATM and ATR. The signals from these sensory kinases are amplified by the checkpoint kinases, Chk1 and Chk2, with cells arresting cell cycle in a p53 dependent manner to either repair the damage or to proceed to cell death via apoptosis.[
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      The DNA Damage Response: Making It Safe to Play with Knives.
      ] Different types of DNA damage are repaired by specialized DNA repair pathways, with pathway members listed below that are associated with increased cancer risk and/or currently tested on germline cancer gene panels. These specialized repair pathways are HRR, FA, MMEJ, and NHEJ for DSBs, BER for single strand breakss, NER for bulky DNA adducts, MMR and replication-repair for base–base mismatches.[
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      Non-homologous DNA end joining and alternative pathways to double-strand break repair.
      ,
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      Ionizing Radiation-Induced DNA Damage, Response, and Repair.
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      Base excision repair: A critical player in many games.
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      FANCD2 influences replication fork processes and genome stability in response to clustered DSBs.
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      Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide.
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      DNA mismatch repair and the DNA damage response.
      ]
      Tumors with DNA repair defects are particularly sensitive to DNA damaging agents, such as platinum-based chemotherapeutic drugs and radiation therapy. Table 1 shows DNA repair genes that are associated with therapeutic benefit and cancer risk in preclinical and/or clinical studies. In the early 2000s, it was observed that inhibition of PARP in tumor cells, that carry genetic changes damaging BRCA (BRCA1 or BRCA2) function, led to synthetic lethality.[
      • Helleday T.
      The underlying mechanism for the PARP and BRCA synthetic lethality: Clearing up the misunderstandings.
      ] In 2014, olaparib (Lynparza, AstraZeneca) was the first PARPi that was approved by the Federal Drug Administration (FDA). Olaparib monotherapy was first indicated for advanced ovarian cancer (OC) patients with germline PVs in BRCA who had been treated with three or more prior lines of chemotherapy.[
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      FDA Approval Summary: Olaparib Monotherapy in Patients with Deleterious Germline BRCA-Mutated Advanced Ovarian Cancer Treated with Three or More Lines of Chemotherapy.
      ] Since the first approval, the next generation of PARPis (veliparib, niraparib, rucaparib, and talazoparib) have not only demonstrated increase potency but also expanded to the treatment of tumor types that include PVs in other DNA repair genes.[
      • Shen Y.
      • Aoyagi-Scharber M.
      • Wang B.
      Trapping Poly(ADP-Ribose) Polymerase.
      ] Talazoparib (Talzenna, Pfizer Inc.), which is significantly more potent than olaparib, was approved by the FDA on October 16th, 2018 for HER-2 negative locally advanced or metastatic breast cancer (BC) patients with germline PVs in BRCA.[

      FDA. FDA approves talazoparib for gBRCAm HER2-negative locally advanced or metastatic breast cancer. 2018.

      ,
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      Talazoparib in Patients with Advanced Breast Cancer and a Germline BRCA Mutation.
      ] The POLO trial in patients with metastatic pancreatic cancer and the PROfound trial in patients with metastatic castration-resistant prostate cancer led to the approval of olaparib for patients with germline PVs in BRCA genes and/or other specific HRR genes respectively.[
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      ]
      Table 1DNA repair genes that are associated with therapeutic benefit and cancer risk in preclinical and/or clinical studies.
      PathwayGeneFunction
      BERMUTYHExcises inappropriately paired adenine bases from

      DNA backbone to initiate repair
      • D’Agostino V.G.
      • Minoprio A.
      • Torreri P.
      • Marinoni I.
      • Bossa C.
      • Petrucci T.C.
      • et al.
      Functional analysis of MUTYH mutated proteins associated with familial adenomatous polyposis.
      NTHL1Releases damaged DNA base from DNA through

      cleavage of N-glycosidic bond, leaves an AP site
      • Eide L.
      • Luna L.
      • Gustad E.C.
      • Henderson P.T.
      • Essigmann J.M.
      • Demple B.
      • et al.
      Human Endonuclease III Acts Preferentially on DNA Damage Opposite Guanine Residues in DNA†.
      DNA Damage ResponseARID1AInteracts with ATR and recruited to DSBs
      • Shen J.
      • Peng Y.
      • Wei L.
      • Zhang W.
      • Yang L.
      • Lan L.
      • et al.
      ARID1A Deficiency Impairs the DNA Damage Checkpoint and Sensitizes Cells to PARP Inhibitors.
      ATMAssociated with sensitivity to DNA damaging agents,

      ataxia telangiectasia mutated

      Wood R, Lowery M. Human DNA Repair Genes.

      BLMAssociated with sensitivity to DNA damaging agents,

      Bloom syndrome helicase

      Wood R, Lowery M. Human DNA Repair Genes.

      CHEK1Serine/threonine-protein kinase required for checkpoint-mediated

      cell cycle arrest and activation of DNA repair in response to DNA damage/unreplicated DNA
      • Feijoo C.
      • Hall-Jackson C.
      • Wu R.
      • Jenkins D.
      • Leitch J.
      • Gilbert D.M.
      • et al.
      Activation of mammalian Chk1 during DNA replication arrest.
      CHEK2Serine/threonine-protein kinase required for checkpoint-mediated

      cell cycle arrest, activation of DNA repair and apoptosis in

      response to DNA DSBs
      • Bahassi E.M.
      • Ovesen J.L.
      • Riesenberg A.L.
      • Bernstein W.Z.
      • Hasty P.E.
      • Stambrook P.J.
      The checkpoint kinases Chk1 and Chk2 regulate the functional associations between hBRCA2 and Rad51 in response to DNA damage.
      RECQL4Associated with sensitivity to DNA damaging agents,

      Rothmund-Thompson syndrome

      Wood R, Lowery M. Human DNA Repair Genes.

      WRNWerner syndrome helicase / 3′-exonuclease

      Wood R, Lowery M. Human DNA Repair Genes.

      DNA Replication-RepairPOLEComponent of DNA polymerase epsilon complex for leading DNA strand synthesis
      • Ogi T.
      • Limsirichaikul S.
      • Overmeer R.M.
      • Volker M.
      • Takenaka K.
      • Cloney R.
      • et al.
      Three DNA Polymerases, Recruited by Different Mechanisms, Carry Out NER Repair Synthesis in Human Cells.
      POLD1Component of DNA polymerase delta complexes for lagging DNA strand synthesis and DNA repair
      • Li H.
      • Xie B.
      • Zhou Y.
      • Rahmeh A.
      • Trusa S.
      • Zhang S.
      • et al.
      Functional Roles of p12, the Fourth Subunit of Human DNA Polymerase δ.
      FABRIP1

      (or FANCJ)
      DNA helicase & BRCA1-interacting

      Wood R, Lowery M. Human DNA Repair Genes.

      FANCATolerance & repair of DNA crosslinks & other DNA adducts

      Wood R, Lowery M. Human DNA Repair Genes.

      FANCBTolerance & repair of DNA crosslinks & other DNA adducts

      Wood R, Lowery M. Human DNA Repair Genes.

      PALB2

      (or FANCN)
      Recruit BRCA2 and RAD51 to DNA breaks
      • Xia B.
      • Sheng Q.
      • Nakanishi K.
      • Ohashi A.
      • Wu J.
      • Christ N.
      • et al.
      Control of BRCA2 Cellular and Clinical Functions by a Nuclear Partner, PALB2.
      HRRBAP1Promotes DSB repair by interacting with BARD1
      • Carbone M.
      • Harbour J.W.
      • Brugarolas J.
      • Bononi A.
      • Pagano I.
      • Dey A.
      • et al.
      Biological Mechanisms and Clinical Significance of BAP1 Mutations in Human Cancer.
      ,
      • Kobrinski D.A.
      • Yang H.
      • Kittaneh M.
      BAP1: role in carcinogenesis and clinical implications.
      BARD1E3 ubiquitin-protein ligase
      • Morris J.R.
      • Solomon E.
      BRCA1 : BARD1 induces the formation of conjugated ubiquitin structures, dependent on K6 of ubiquitin, in cells during DNA replication and repair.
      BRCA1

      (or FANCS)
      E3 ubiquitin-protein ligase, mediates formation of Lys-6-linked polyubiquitin chains and plays role in repair by facilitating cellular responses to DNA damage
      • Hiraike H.
      • Wada-Hiraike O.
      • Nakagawa S.
      • Koyama S.
      • Miyamoto Y.
      • Sone K.
      • et al.
      Identification of DBC1 as a transcriptional repressor for BRCA1.
      BRCA2

      (or FANCD1)
      Binds RAD51, promotes assembly of RAD51 onto single-stranded DNA to potentiate HRR
      • Bhatia V.
      • Barroso S.I.
      • García-Rubio M.L.
      • Tumini E.
      • Herrera-Moyano E.
      • Aguilera A.
      BRCA2 prevents R-loop accumulation and associates with TREX-2 mRNA export factor PCID2.
      MRE11A3′ exonuclease, defective in ATLD (ataxia-telangiectasia-like disorder)

      Wood R, Lowery M. Human DNA Repair Genes.

      NBNComponent of MRN complex which plays critical role in cellular response to DNA damage and maintenance of chromosome integrity
      • Trujillo K.M.
      • Yuan S.-S.-F.
      • Lee E.-Y.-H.-P.
      • Sung P.
      Nuclease Activities in a Complex of Human Recombination and DNA Repair Factors Rad50, Mre11, and p95.
      RAD50ATPase in complex with MRE11A, NBS1

      Wood R, Lowery M. Human DNA Repair Genes.

      RAD51C

      (FANCO)
      Binds single-stranded DNA and double-stranded DNA, DNA-dependent ATPase activity
      • Rodrigue A.
      • Coulombe Y.
      • Jacquet K.
      • Gagné J.-P.
      • Roques C.
      • Gobeil S.
      • et al.
      The RAD51 paralogs ensure cellular protection against mitotic defects and aneuploidy.
      RAD51DRad51 homolog

      Wood R, Lowery M. Human DNA Repair Genes.

      RAD54BInvolved in DNA repair and mitotic recombination
      • Miyagawa K.
      A role for RAD54B in homologous recombination in human cells.
      XRCC2

      (or FANCU)
      DNA break and crosslink repair

      Wood R, Lowery M. Human DNA Repair Genes.

      MMRMLH1Heterodimerizes with PMS2 to form MutL alpha
      • Kansikas M.
      • Kariola R.
      • Nyström M.
      Verification of the three-step model in assessing the pathogenicity of mismatch repair gene variants.
      MSH2Forms two different heterodimers: MutS alpha and MutS beta
      • Kansikas M.
      • Kariola R.
      • Nyström M.
      Verification of the three-step model in assessing the pathogenicity of mismatch repair gene variants.
      MSH6Heterodimerizes with MSH2 to form MutS alpha
      • Kansikas M.
      • Kariola R.
      • Nyström M.
      Verification of the three-step model in assessing the pathogenicity of mismatch repair gene variants.
      PMS2Heterodimerizes with MLH1 to form MutL alpha
      • Kansikas M.
      • Kariola R.
      • Nyström M.
      Verification of the three-step model in assessing the pathogenicity of mismatch repair gene variants.
      NERERCC1Structure-specific endonuclease component
      • Friboulet L.
      • Postel-Vinay S.
      • Sourisseau T.
      • Adam J.
      • Stoclin A.
      • Ponsonnailles F.
      • et al.
      ERCC1 function in nuclear excision and interstrand crosslink repair pathways is mediated exclusively by the ERCC1-202 isoform.
      ERCC25′ to 3′ DNA helicase

      Wood R, Lowery M. Human DNA Repair Genes.

      ERCC4

      (or FANCQ)
      Structure-specific endonuclease component, removes interstrand cross-link
      • Svendsen J.M.
      • Smogorzewska A.
      • Sowa M.E.
      • O'Connell B.C.
      • Gygi S.P.
      • Elledge S.J.
      • et al.
      Mammalian BTBD12/SLX4 Assembles A Holliday Junction Resolvase and Is Required for DNA Repair.
      ERCC5Structure-specific DNA endonuclease involved in DNA excision repair, 3′ excision
      • Walsh C.S.
      • Ogawa S.
      • Karahashi H.
      • Scoles D.R.
      • Pavelka J.C.
      • Tran H.
      • et al.
      ERCC5 Is a Novel Biomarker of Ovarian Cancer Prognosis.
      MMEJPOLQDNA polymerase that promotes MMEJ and limits RAD51 accumulation at resected ends
      • Ceccaldi R.
      • Liu J.C.
      • Amunugama R.
      • Hajdu I.
      • Primack B.
      • Petalcorin M.I.R.
      • et al.
      Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair.
      FEN15′ nuclease

      Wood R, Lowery M. Human DNA Repair Genes.

      AP, apurinic/apyrimidinic site; BER, base excision repair; DSB, double-strand breaks; FA, Fanconi anemia; HRR, homologous recombination repair; MMEJ, microhomology-mediated end joining; MMR, mismatch repair; NER, nucleotide excision repair; NHEJ, non-homologous end-joining; pol, polymerase.
      In the last few years, immune checkpoint inhibitors (ICI) have transformed the treatment of solid tumors such as melanoma, lung, head neck, breast, renal, and bladder cancers.[
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      ] This review will summarize the current literature on DNA repair defects in the germline and their relevance to personalized cancer treatment. In this review, we also discuss emerging paradigms of germline DNA repair vulnerabilities that could be potentially exploited as therapeutic targets. This review does not discuss the biology of the loss of the normal allele in cancer or the nature of the PV (e.g., hyper or hypomorphic alleles), rather focuses on the clinical significance of germline aberrations in specific DNA repair genes as a biomarker for treatment strategies as well as clinical benefit of germline genetic testing for treatment decision.

      Germline testing for cancer risk

      In 1895, a family was reported with a spectrum of abdominal cancers in different generations, demonstrating a familial predisposition to cancer.[
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      ] Subsequently, Dr. Michael Stratton linked BRCA2, localized to chromosome 13q12-q13, with increased BC risk.[
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      ] Further groundbreaking work, led by multiple groups over several years, confirmed and elucidated that the BRCA genes are indeed associated with increased risk of BC and OC.[
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      • Albertsen H.M.
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      ,
      • Futreal P.
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      ,
      • Kuchenbaecker K.B.
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      ]
      In the early 1990s, BRCA gene sequence was commercially patented by Myriad Genetic Laboratories, which then developed and marketed the first widely available commercial DNA sequencing test for hereditary BC and OC risk based on sequencing of BRCA1, followed shortly thereafter by the addition of BRCA2 (the BRCAnalysis test). In 2013, a landmark ruling by the Supreme Court that, ‘isolated human genes cannot be patented’, rescinded the patents on BRCA and this changed the landscape of genetic testing for cancer diagnostics. A period of rapid innovation through next-generation sequencing, the introduction of multiple new commercial entities into the genetic testing marketplace, and expansion of genetic testing criteria followed.[
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      • Weinmeyer R.
      Supreme Court to Myriad Genetics: Synthetic DNA is Patentable but Isolated Genes Are Not.
      ] It must also be appreciated that the cloning and widescale marketing of BRCA testing in the 2000s to evaluate cancer risks in women laid the foundation for enormous growth in the field of clinical cancer genetics. Clinical testing has been proven in this time to not only help guide cancer risk assessment for the carrier and their family members but also in its potential to guide patient management and treatment.[
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      Addition of the PARP inhibitor veliparib plus carboplatin or carboplatin alone to standard neoadjuvant chemotherapy in triple-negative breast cancer (BrighTNess): a randomised, phase 3 trial.
      ]
      Like the BRCA genes, multiple other cancer susceptibility genes function in DNA repair pathways (genes in Fig. 1 and Table 1). Today, multigene hereditary cancer panels that include a number of the DNA repair genes listed in Table 1 are used in the clinic to test for germline PVs in cancer susceptibility genes.[
      • Maxwell K.N.
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      • Gracia-Aznarez F.J.
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      Whole Exome Sequencing Suggests Much of Non-BRCA1/BRCA2 Familial Breast Cancer Is Due to Moderate and Low Penetrance Susceptibility Alleles.
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      ,
      • Hilbers F.S.
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      ,
      • Stadler Z.K.
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      Cancer Genomics and Inherited Risk.
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      • Castéra L.
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      ,
      • Couch F.J.
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      Inherited Mutations in 17 Breast Cancer Susceptibility Genes Among a Large Triple-Negative Breast Cancer Cohort Unselected for Family History of Breast Cancer.
      ,
      • Kurian A.W.
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      • Laduca H.
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      ,
      • Tung N.
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      ,
      • Thompson E.R.
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      Exome Sequencing Identifies Rare Deleterious Mutations in DNA Repair Genes FANCC and BLM as Potential Breast Cancer Susceptibility Alleles.
      ] An individual with a family history of cancers may undergo germline testing to quantify their cancer risk. If a PV is found in an individual, their physician(s) can perform cancer screening for early detection, develop preventive management plans, or prevention procedures; or patients may personally maintain their health through behavioral modification and risk mitigation. The results of germline testing may also guide patient treatment.[
      • Pal T.
      • Agnese D.
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      Points to consider: is there evidence to support BRCA1/2 and other inherited breast cancer genetic testingfor all breast cancer patients? A statement of the American College of Medical Geneticsand Genomics (ACMG).
      ] Perhaps equally important, the identification of a PV can motivate family members to undergo testing for cancer risk assessment and risk stratification.[
      • Litton J.K.
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      • Ettl J.
      • Hurvitz S.A.
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      Talazoparib in Patients with Advanced Breast Cancer and a Germline BRCA Mutation.
      ,
      • Pal T.
      • Agnese D.
      • Daly M.
      • Spada A.L.
      • Litton J.
      • Wick M.
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      Points to consider: is there evidence to support BRCA1/2 and other inherited breast cancer genetic testingfor all breast cancer patients? A statement of the American College of Medical Geneticsand Genomics (ACMG).
      ,
      • Robson M.
      • Im S.-A.
      • Senkus E.
      • Xu B.
      • Domchek S.M.
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      • et al.
      Olaparib for Metastatic Breast Cancer in Patients with a Germline BRCA Mutation.
      ]
      Patients are selected for genetic testing based on personal and family cancer history, the assessment of which has been codified in numerous practice guidelines. However, there is increasing evidence to suggest that germline sequencing may also be beneficial for patients who do not meet guidelines for genetic testing. Advanced stage (stage III-IV) cancer patients (n = 1040) were sequenced for 76 cancer predisposition genes and ∼ 17.5% of these patients (182/1040) had clinically actionable germline PVs in cancer risk genes.[
      • Mandelker D.
      • Zhang L.
      • Kemel Y.
      • et al.
      Mutation Detection in Patients With Advanced Cancer by Universal Sequencing of Cancer-Related Genes in Tumor and Normal DNA vs Guideline-Based Germline Testing.
      ] Among the 17.5% patients with germline PVs, over half (∼9.7%, 101/1040) did not meet clinical criteria for testing, and these PVs would not have been detected based on the current guidelines. In pediatric cancer patients (n = 1120), it was found that ∼ 8.5% had a PV or a likely PV.[
      • Zhang J.W.M.
      • Wu G.
      • et al.
      Germline Mutations in Predisposition Genes in Pediatric.
      ] Here, only 40% of the pediatric patients with cancer-predisposing PVs had a family history of cancer. Analysis of whole-exome data from 49,738 participants in the UK Biobank found that ∼ 1% (441/49738) of the cohort harbored PVs in genes associated with familial hypercholesterolemia, hereditary breast and ovarian cancer syndrome, and Lynch syndrome. These individuals in the cohort with the PVs (or likely PVs) were at increased risk for the associated diseases and were not reliably detected by their family history alone.[
      • Patel A.P.
      • Wang M.
      • Fahed A.C.
      • Mason-Suares H.
      • Brockman D.
      • Pelletier R.
      • et al.
      Association of Rare Pathogenic DNA Variants for Familial Hypercholesterolemia, Hereditary Breast and Ovarian Cancer Syndrome, and Lynch Syndrome With Disease Risk in Adults According to Family History.
      ] Germline testing in a prospective cohort of patients with solid tumors found that multigene panel testing identified PVs in 13.3% (397/2,984) of the patients. Here, treatment strategies were modified for 28.2% (42/397) of the patients based on the germline findings.[
      • Samadder N.J.
      • Riegert-Johnson D.
      • Boardman L.
      • Rhodes D.
      • Wick M.
      • Okuno S.
      • et al.
      Comparison of Universal Genetic Testing vs Guideline-Directed Targeted Testing for Patients With Hereditary Cancer Syndrome.
      ] Manickam et. al. found that exome sequencing based screening strategies identify 5 times as many individuals with BRCA GPVs than screening based on family history.[
      • Manickam K.
      • Buchanan A.H.
      • Schwartz M.L.B.
      • et al.
      Exome Sequencing-Based Screening for BRCA1/2 Expected Pathogenic Variants Among Adult Biobank Participants.
      ] Beitsch et. al. found that testing based on current guidelines misses nearly half of the BC patients with a GPV.[
      • Beitsch P.D.
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      • Hughes K.
      • Patel R.
      • Rosen B.
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      Underdiagnosis of Hereditary Breast Cancer: Are Genetic Testing Guidelines a Tool or an Obstacle?.
      ]
      Although the current literature suggests benefit of population-based screening, there are several barriers to genetic testing such as lack of initial referral from the oncologist, poor knowledge of genetics, access to care, health insurance, medical costs, and confidentiality concerns.[
      • Manchanda R.
      • Gaba F.
      Population Based Testing for Primary Prevention: A Systematic Review.
      ,

      National Academies of Sciences E, and Medicine, Division HaM, Policy BoHS, Health RoGaP. Understanding Disparities in Access to Genomic Medicine: Proceedings of a Workshop. Washington (DC): National Academies Press (US); 2018.

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      • Swink A.
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      ] Recent studies are focusing on understanding and addressing these barriers so that more patients can benefit from genetic testing.[

      National Academies of Sciences E, and Medicine, Division HaM, Policy BoHS, Health RoGaP. Understanding Disparities in Access to Genomic Medicine: Proceedings of a Workshop. Washington (DC): National Academies Press (US); 2018.

      ,
      • Swink A.
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      • Hoof P.
      • Matthews A.
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      Barriers to the utilization of genetic testing and genetic counseling in patients with suspected hereditary breast and ovarian cancers.
      ] In conclusion, the increased utilization of next-generation sequencing technology in the clinic, significant reduction in the cost, and broader community acceptance have made it possible to identify clinically actionable germline PVs.[
      • Patel A.P.
      • Wang M.
      • Fahed A.C.
      • Mason-Suares H.
      • Brockman D.
      • Pelletier R.
      • et al.
      Association of Rare Pathogenic DNA Variants for Familial Hypercholesterolemia, Hereditary Breast and Ovarian Cancer Syndrome, and Lynch Syndrome With Disease Risk in Adults According to Family History.
      ,
      • Samadder N.J.
      • Riegert-Johnson D.
      • Boardman L.
      • Rhodes D.
      • Wick M.
      • Okuno S.
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      Comparison of Universal Genetic Testing vs Guideline-Directed Targeted Testing for Patients With Hereditary Cancer Syndrome.
      ,
      • Tan O.
      • Shrestha R.
      • Cunich M.
      • Schofield D.J.
      Application of next-generation sequencing to improve cancer management: A review of the clinical effectiveness and cost-effectiveness.
      ,
      • Grzymski J.J.
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      Population genetic screening efficiently identifies carriers of autosomal dominant diseases.
      ]

      Interpretation of germline variants

      The American College of Medical Genetics and Genomics and the Association for Molecular Pathology have established guidelines for the classification of variants identified through clinical genetic testing further grounding the wide applicability of cancer risk assessment. Variants are classified under five categories of increasing disease severity: benign, likely benign, variants of uncertain significance (VUS), likely pathogenic, and pathogenic.[
      • Esterling L.
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      • Morris B.
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      • Pruss D.
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      Impact of a Cancer Gene Variant Reclassification Program Over a 20-Year Period.
      ] Clinical testing has identified a huge number of rare variants that are currently classified as VUS, and it is not known whether these impact cancer risk.[
      • Esterling L.
      • Wijayatunge R.
      • Brown K.
      • Morris B.
      • Hughes E.
      • Pruss D.
      • et al.
      Impact of a Cancer Gene Variant Reclassification Program Over a 20-Year Period.
      ] For example, while the clinical significance of a germline PV in an MMR gene is straightforward, the interpretation of clinical significance is not straightforward when a patient harbors a germline VUS in an MMR gene. These VUS account for ∼ 20–30% of variants in MMR genes.[
      • Arora S.
      • Huwe P.
      • Sikder R.
      • Shah M.
      • Browne A.
      • Lesh R.
      • Nicolas E.
      • Deshpande S.
      • Hall M.
      • Dunbrack R.
      • Golemis E.
      Functional analysis of rare variants in mismatch repair proteins augments results from computation-based predictive methods.
      ] Thus, a critical aspect in the field of cancer risk and treatment decision is to evaluate the clinical significance of VUS in DNA repair genes.
      The interpretation of VUS is challenging and inclusion of multiple lines of evidence has been suggested, for example, the inclusion of data such as presence in the general population (rare variants with minor allele frequency < 1%), segregation of the variant with the disease, in silico prediction of variant effect as predicted-pathogenic or predicted likely-pathogenic, results from structural modeling, mutation rates, and signatures in the tumor, biochemical assays of the variant.[
      • Arora S.
      • Huwe P.
      • Sikder R.
      • Shah M.
      • Browne A.
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      • Deshpande S.
      • Hall M.
      • Dunbrack R.
      • Golemis E.
      Functional analysis of rare variants in mismatch repair proteins augments results from computation-based predictive methods.
      ] As PVs in MMR and DNA polymerase genes are known to be associated with increased mutation rates, the analysis of tumor sequence data has become increasingly important when a VUS is found in these genes.[
      • Mur P.
      • García-Mulero S.
      • Del Valle J.
      • Magraner-Pardo L.
      • Vidal A.
      • Pineda M.
      • et al.
      Role of POLE and POLD1 in familial cancer.
      ] Intriguingly, primary lymphocytes from familial early-onset cancer patients, that carried predicted-pathogenic germline variants in DNA repair genes, exhibited a constitutional defect in the suppression of double strand breaks (DSBs).[
      • Arora S.
      • Yan H.
      • Cho I.
      • Fan H.-Y.
      • Luo B.
      • Gai X.
      • et al.
      Genetic Variants That Predispose to DNA Double-Strand Breaks in Lymphocytes From a Subset of Patients With Familial Colorectal Carcinomas.
      ,
      • Nicolas E.
      • Arora S.
      • Zhou Y.
      • Serebriiskii I.G.
      • Andrake M.D.
      • Handorf E.D.
      • et al.
      Systematic evaluation of underlying defects in DNA repair as an approach to case-only assessment of familial prostate cancer.
      ] These results suggest functional assays in primary lymphocytes may assist in providing the various lines of evidence required to clinically interpret the relevance of specific germline VUS in DNA repair genes.
      Overall, knowing that a variant is indeed a PV or is benign may aid clinicians in interpreting results from germline testing and guiding patients for their cancer risk as well as personalized treatment.[
      • Arora S.
      • Huwe P.
      • Sikder R.
      • Shah M.
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      • Nicolas E.
      • Deshpande S.
      • Hall M.
      • Dunbrack R.
      • Golemis E.
      Functional analysis of rare variants in mismatch repair proteins augments results from computation-based predictive methods.
      ] Recent studies suggest implementation of multidisciplinary molecular tumor board teams to recommend customized therapeutic solution(s) for cancer patients that have failed existing treatment.[
      • Luchini C.
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      • Scarpa A.
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      • Patel M.
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      • Kurzrock R.
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      ] The multidisciplinary team recommends therapeutic strategies based on a comprehensive integrated review of results from genetic testing (germline and/or somatic, TMB etc.), other laboratory results (imaging, pathology, biomarker etc.), patient’s clinical and family history along with potentially available clinical trials.[
      • Luchini C.
      • Lawlor R.T.
      • Milella M.
      • Scarpa A.
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      ,
      • Patel M.
      • Kato S.M.
      • Kurzrock R.
      Molecular Tumor Boards: Realizing Precision Oncology Therapy.
      ] Kato and colleagues showed that patients who received molecular tumor board recommended treatment had better clinical outcomes.[
      • Kato S.
      • Kim K.H.
      • Lim H.J.
      • Boichard A.
      • Nikanjam M.
      • Weihe E.
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      Real-world data from a molecular tumor board demonstrates improved outcomes with a precision N-of-One strategy.
      ] Currently, there is a need for global standardization of molecular tumor boards to deliver therapeutic recommendations that can facilitate the goals of precision oncology.[
      • Luchini C.
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      ]

      Therapeutic implications for ICIs

      Conventionally, the knowledge of germline PVs in DNA repair genes has informed our understanding of cancer risk. More recently, DNA repair pathways have emerged as playing a major role in the selection of patients for ICI therapy. In 2017, the PD-1 inhibitor pembrolizumab received FDA approval for adult and pediatric patients with metastatic or unresectable tumors with microsatellite instability-high (MSI-H) or MMR deficiency (dMMR) phenotype that has progressed following previous treatments or for whom no other treatment options are available. Five studies contributed data for the FDA application, with a total of 149 patients that had MSI-H or dMMR tumors.[
      • Marcus L.
      • Lemery S.J.
      • Keegan P.
      • Pazdur R.
      FDA Approval Summary: Pembrolizumab for the Treatment of Microsatellite Instability-High Solid Tumors.
      ,
      • Le D.T.
      • Durham J.N.
      • Smith K.N.
      • Wang H.
      • Bartlett B.R.
      • Aulakh L.K.
      • et al.
      Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade.
      ,
      • Le D.T.
      • Uram J.N.
      • Wang H.
      • Bartlett B.R.
      • Kemberling H.
      • Eyring A.D.
      • et al.
      PD-1 Blockade in Tumors with Mismatch-Repair Deficiency.
      ] Of the 149 patients, 90 patients had metastatic CRC, and 59 patients had other cancers. Further, out of the 149 patients, 59 patients responded to treatment (objective response rate (ORR) = 39.6%, 95% CI, 31.7–47.9), with a 7% complete response rate.[
      • Marcus L.
      • Lemery S.J.
      • Keegan P.
      • Pazdur R.
      FDA Approval Summary: Pembrolizumab for the Treatment of Microsatellite Instability-High Solid Tumors.
      ,
      • Le D.T.
      • Durham J.N.
      • Smith K.N.
      • Wang H.
      • Bartlett B.R.
      • Aulakh L.K.
      • et al.
      Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade.
      ,
      • Le D.T.
      • Uram J.N.
      • Wang H.
      • Bartlett B.R.
      • Kemberling H.
      • Eyring A.D.
      • et al.
      PD-1 Blockade in Tumors with Mismatch-Repair Deficiency.
      ]
      The landmark tumor agnostic FDA approval of pembrolizumab was based on the understanding of the biology of dMMR/MSI-H which occurs due to germline or somatic PVs in MMR genes (MLH1, MSH2, MSH6, PMS2). The MMR proteins maintain genome stability by correcting single base nucleotide insertions or deletions.[
      • Westdorp H.
      • Kolders S.
      • Hoogerbrugge N.
      • De Vries I.J.M.
      • Jongmans M.C.J.
      • Schreibelt G.
      Immunotherapy holds the key to cancer treatment and prevention in constitutional mismatch repair deficiency (CMMRD) syndrome.
      ] Germline PVs impairing MMR are associated with the autosomal dominant condition, Lynch Syndrome, which increases the risk of CRC, endometrial cancer, and several other cancers, such as OC, brain, skin, pancreatic, and other gastrointestinal cancers.[
      • Pande M.
      • Wei C.
      • Chen J.
      • Amos C.I.
      • Lynch P.M.
      • Lu K.H.
      • et al.
      Cancer spectrum in DNA mismatch repair gene mutation carriers: results from a hospital based Lynch syndrome registry.
      ,
      • Sinicrope F.A.
      DNA mismatch repair and adjuvant chemotherapy in sporadic colon cancer.
      ] Currently, ∼1–7% of CRCs, and ∼ 13–25% of endometrial cancers are associated with germline PVs in MMR genes.[
      • Nagle C.M.
      • O'Mara T.A.
      • Tan Y.
      • Buchanan D.D.
      • Obermair A.
      • Blomfield P.
      • et al.
      Endometrial cancer risk and survival by tumor MMR status.
      ,
      • Abedalthagafi M.
      Constitutional mismatch repair-deficiency: current problems and emerging therapeutic strategies.
      ,
      • Shirts B.H.
      • Konnick E.Q.
      • Upham S.
      • Walsh R.J.
      • Jacobson A.L.
      • et al.
      Using Somatic Mutations from Tumors to Classify Variants in Mismatch Repair Genes.
      ] Somatic PVs in MMR are also common in tumors; for example, in ∼ 12–15% of localized CRC, ∼3–4% of metastatic CRC, and up to 25% of endometrial cancers.[
      • Sinicrope F.A.
      DNA mismatch repair and adjuvant chemotherapy in sporadic colon cancer.
      ,
      • Pearlman R.
      • Haraldsdottir S.
      • De La Chapelle A.
      • Jonasson J.G.
      • Liyanarachchi S.
      • Frankel W.L.
      • et al.
      Clinical characteristics of patients with colorectal cancer with double somatic mismatch repair mutations compared with Lynch syndrome.
      ,
      • Pearlman R.
      • Frankel W.L.
      • Swanson B.
      • Zhao W.
      • Yilmaz A.
      • Miller K.
      • et al.
      Prevalence and Spectrum of Germline Cancer Susceptibility Gene Mutations Among Patients With Early-Onset Colorectal Cancer.
      ] Epigenetic silencing of the MLH1 promoter due to hypermethylation can also contribute to dMMR tumors.[
      • Lee V.
      • Murphy A.
      • Le D.T.
      • Diaz L.A.
      Mismatch Repair Deficiency and Response to Immune Checkpoint Blockade.
      ,
      • Viale G.
      • Trapani D.
      • Curigliano G.
      Mismatch Repair Deficiency as a Predictive Biomarker for Immunotherapy Efficacy.
      ] Other sources of dMMR include deletions at the 3′ end of EPCAM which causes the inactivation of the nearby MSH2 gene.[
      • Lee V.
      • Murphy A.
      • Le D.T.
      • Diaz L.A.
      Mismatch Repair Deficiency and Response to Immune Checkpoint Blockade.
      ] Although rare, it is also possible to inherit two MMR variants (one from each parent), which can result in either a compound heterozygote or, if the variants are in the same MMR gene, a rare recessive inherited syndrome known as Constitutional MMR Deficiency leading to a wide spectrum of childhood malignancies.[
      • Westdorp H.
      • Kolders S.
      • Hoogerbrugge N.
      • De Vries I.J.M.
      • Jongmans M.C.J.
      • Schreibelt G.
      Immunotherapy holds the key to cancer treatment and prevention in constitutional mismatch repair deficiency (CMMRD) syndrome.
      ] In a case study, two siblings with consitutional dMMR and recurrent glioblastoma were treated with PD-1 inhibitor nivolumab (Opdivo, Bristol-Myers Squibb) which led to a significant clinical benefit.[
      • Bouffet E.
      • Larouche V.
      • Campbell B.B.
      • Merico D.
      • De Borja R.
      • Aronson M.
      • et al.
      Immune Checkpoint Inhibition for Hypermutant Glioblastoma Multiforme Resulting From Germline Biallelic Mismatch Repair Deficiency.
      ] Recently, anti-PD-1 monoclonal antibody, dostarlimab (Jemperli, GlaxoSmithKline) showed clinical benefit in patients with dMMR endometrial cancer.[
      • Oaknin A.
      • Tinker A.V.
      • Gilbert L.
      • Samouëlian V.
      • Mathews C.
      • Brown J.
      • et al.
      Clinical Activity and Safety of the Anti-Programmed Death 1 Monoclonal Antibody Dostarlimab for Patients With Recurrent or Advanced Mismatch Repair-Deficient Endometrial Cancer: A Nonrandomized Phase 1 Clinical Trial.
      ,
      • Oaknin A.
      • Tinker A.V.
      • Gilbert L.
      • Samouëlian V.
      • Mathews C.
      • Brown J.
      • et al.
      Clinical activity and safety of the anti-PD-1 monoclonal antibody dostarlimab for patients with recurrent or advanced dMMR endometrial cancer.
      ] Based on the results of the GARNET trial, dostarlimab was approved by the FDA for patients with recurrent or advanced dMMR endometrial cancer[
      • Oaknin A.
      • Tinker A.V.
      • Gilbert L.
      • Samouëlian V.
      • Mathews C.
      • Brown J.
      • et al.
      Clinical Activity and Safety of the Anti-Programmed Death 1 Monoclonal Antibody Dostarlimab for Patients With Recurrent or Advanced Mismatch Repair-Deficient Endometrial Cancer: A Nonrandomized Phase 1 Clinical Trial.
      ,
      • Oaknin A.
      • Tinker A.V.
      • Gilbert L.
      • Samouëlian V.
      • Mathews C.
      • Brown J.
      • et al.
      Clinical activity and safety of the anti-PD-1 monoclonal antibody dostarlimab for patients with recurrent or advanced dMMR endometrial cancer.
      ].
      It is well-appreciated that dMMR leads to increased TMB and tumor neoantigen production, potentially contributing to the better response to ICIs.[
      • Van Allen E.M.
      • Miao D.
      • Schilling B.
      • Shukla S.A.
      • Blank C.
      • Zimmer L.
      • et al.
      Genomic correlates of response to CTLA-4 blockade in metastatic melanoma.
      ,
      • Rizvi N.A.
      • Hellmann M.D.
      • Snyder A.
      • Kvistborg P.
      • Makarov V.
      • Havel J.J.
      • et al.
      Mutational landscape determines sensitivity to PD-1 blockade in non–small cell lung cancer.
      ,
      • Yarchoan M.
      • Hopkins A.
      • Jaffee E.M.
      Tumor Mutational Burden and Response Rate to PD-1 Inhibition.
      ] Infact, testing for dMMR or MSI-H also can identify patients with TMB-H. In June 2020, accelerated FDA approval for pembrolizumab was indicated for the treatment of TMB-high (or TMB-H, ≥10 mutations/megabase) adult and pediatric unresectable or metastatic tumors that have either progressed on prior therapy or for which no other treatment options are available.[

      FDA approves pembrolizumab for adults and children with TMB-H solid tumors. 2020.

      ] Germline PVs in DNA polymerase genes, POLE (typically in exonuclease domain) and POLD1, increase the risk of CRCs, endometrial cancers, OC, other malignancies, and have been associated with ultra-hypermutation in the tumor (i.e. tumors with > 100 mutations/megabase).[
      • Mur P.
      • García-Mulero S.
      • Del Valle J.
      • Magraner-Pardo L.
      • Vidal A.
      • Pineda M.
      • et al.
      Role of POLE and POLD1 in familial cancer.
      ,
      • Rayner E.
      • Van Gool I.C.
      • Palles C.
      • Kearsey S.E.
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      • Tomlinson I.
      • et al.
      A panoply of errors: polymerase proofreading domain mutations in cancer.
      ,
      • Palles C.
      • Cazier J.-B.
      • Howarth K.M.
      • Domingo E.
      • Jones A.M.
      • Broderick P.
      • et al.
      Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas.
      ,
      • Church D.N.
      • Stelloo E.
      • Nout R.A.
      • Valtcheva N.
      • Depreeuw J.
      • Ter Haar N.
      • et al.
      Prognostic Significance of POLE Proofreading Mutations in Endometrial Cancer.
      ] Latest data suggest that germline PVs in POLE and POLD1 lead to ∼ 0.1–0.4% of familial cancers.[
      • Mur P.
      • García-Mulero S.
      • Del Valle J.
      • Magraner-Pardo L.
      • Vidal A.
      • Pineda M.
      • et al.
      Role of POLE and POLD1 in familial cancer.
      ,
      • Siraj A.K.
      • Bu R.
      • Iqbal K.
      • Parvathareddy S.K.
      • Masoodi T.
      • Siraj N.
      • et al.
      POLE and POLD1 germline exonuclease domain pathogenic variants, a rare event in colorectal cancer from the Middle East.
      ] While several studies have focused on variants in the exonuclease domain, increased TMBs have also been noted with variants outside the exonuclease domain.[
      • Arora S.
      • Xiu J.
      • Sohal D.
      • Lou E.
      • Goldberg R.M.
      • Weinberg B.A.
      • et al.
      The landscape of POLE variants in colorectal and endometrial tumors: Correlation with microsatellite instability (MSI) and tumor mutation burden (TMB).
      ,
      • Campbell B.B.
      • Light N.
      • Fabrizio D.
      • Zatzman M.
      • Fuligni F.
      • De Borja R.
      • et al.
      Comprehensive Analysis of Hypermutation in Human Cancer.
      ] Patients with POLE/POLD1 variants had significantly better clinical outcomes following treatment with ICIs when compared to patients without them.[
      • Silberman R.
      • Steiner D.F.
      • Lo A.A.
      • Gomez A.
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      • Chu G.
      • et al.
      Complete and Prolonged Response to Immune Checkpoint Blockade in POLE-Mutated Colorectal Cancer.
      ,

      Wang F, State Key Laboratory of Oncology in South China CICfCM, Sun Yat-sen University Cancer Center, Guangzhou, China, Zhao Q, State Key Laboratory of Oncology in South China CICfCM, Sun Yat-sen University Cancer Center, Guangzhou, China, Wang Y-N, State Key Laboratory of Oncology in South China CICfCM, Sun Yat-sen University Cancer Center, Guangzhou, China, et al. Evaluation of POLE and POLD1 Mutations as Biomarkers for Immunotherapy Outcomes Across Multiple Cancer Types. JAMA Oncology. 2020;5:1504-6.

      ] No difference in clinical outcomes was observed in patients with POLE/POLD1 variants in the exonuclease domain or outside the exonuclease domain.[

      Wang F, State Key Laboratory of Oncology in South China CICfCM, Sun Yat-sen University Cancer Center, Guangzhou, China, Zhao Q, State Key Laboratory of Oncology in South China CICfCM, Sun Yat-sen University Cancer Center, Guangzhou, China, Wang Y-N, State Key Laboratory of Oncology in South China CICfCM, Sun Yat-sen University Cancer Center, Guangzhou, China, et al. Evaluation of POLE and POLD1 Mutations as Biomarkers for Immunotherapy Outcomes Across Multiple Cancer Types. JAMA Oncology. 2020;5:1504-6.

      ]
      Several trials are underway to determine the efficacy of ICIs in combination with PARPis (Supplemental Table 1).[
      • Wang Z.
      • Zhao J.
      • Wang G.
      • Zhang F.
      • Zhang Z.
      • Zhang F.
      • et al.
      Comutations in DNA Damage Response Pathways Serve as Potential Biomarkers for Immune Checkpoint Blockade.
      ,
      • Li A.
      • Yi M.
      • Qin S.
      • Chu Q.
      • Luo S.
      • Wu K.
      Prospects for combining immune checkpoint blockade with PARP inhibition.
      ] The rationale for this combination arises from the observations that PARP inhibition increases DNA damage, which is particularly pronounced in cells that carry DNA repair defects such as BRCA deficiency. This damage not only leads to increased priming of the immune system in the tumor microenvironment but also increases the tumor intrinsic immunogenicity by modulating the expression of surface markers such as PD-L1 (reviewed in [
      • Stewart R.A.
      • Pilie P.G.
      • Yap T.A.
      Development of PARP and Immune-Checkpoint Inhibitor Combinations.
      ]). In models of small cell lung cancer, it was shown that PARP (and Chk1) inhibition leads to a strong anti-tumor immune response by recruitment of cytotoxic T-lymphocytes via the activation of the STING pathway.[
      • Sen T.
      • Rodriguez B.L.
      • Chen L.
      • Corte C.M.D.
      • Morikawa N.
      • Fujimoto J.
      • et al.
      Targeting DNA Damage Response Promotes Antitumor Immunity through STING-Mediated T-cell Activation in Small Cell Lung Cancer.
      ] Based on these observations, an ongoing non-interventional, prospective clinical study (NCT03495544, Supplemental Table 1) is evaluating the association between germline PVs in hereditary BC genes (such as BRCA1, BRCA2, CHEK2) and PD-L1 expression in breast tumor cells and immune cells. This study will evaluate the relationship between germline PVs, and PD-L1 expression to allow precision selection of ICIs in these patients. Interestingly, a recent preclinical study showed that BRCA deficiency differentially modulates the tumor microenvironment.[
      • Samstein R.M.
      • Krishna C.
      • Ma X.
      • Pei X.
      • Lee K.-W.
      • Makarov V.
      • et al.
      Mutations in BRCA1 and BRCA2 differentially affect the tumor microenvironment and response to checkpoint blockade immunotherapy.
      ] While BRCA1 mutations may drive immune responses that limit benefit from ICI, BRCA2 mutations increased tumor immunogenicity and showed benefit from ICI therapy.[
      • Samstein R.M.
      • Krishna C.
      • Ma X.
      • Pei X.
      • Lee K.-W.
      • Makarov V.
      • et al.
      Mutations in BRCA1 and BRCA2 differentially affect the tumor microenvironment and response to checkpoint blockade immunotherapy.
      ] These observations suggest a mechanistic difference between the BRCA genes and warrant further investigation.[
      • Samstein R.M.
      • Krishna C.
      • Ma X.
      • Pei X.
      • Lee K.-W.
      • Makarov V.
      • et al.
      Mutations in BRCA1 and BRCA2 differentially affect the tumor microenvironment and response to checkpoint blockade immunotherapy.
      ]

      Therapeutic implications for PARPi therapy

      PARPis prevent the repair of single-strand breaks, and in HRR-deficient cells these single strand breaks are converted to DSBs which ultimately lead to unrepaired damage and consequent cell death.[
      • Morales J.C.
      • Li L.
      • Fattah F.J.
      • Dong Y.
      • Bey E.A.
      • Patel M.
      • et al.
      Review of Poly (ADP-ribose) Polymerase (PARP) Mechanisms of Action and Rationale for Targeting in Cancer and Other Diseases.
      ,
      • Kyle S.
      • Thomas H.D.
      • Mitchell J.
      • Curtin N.J.
      Exploiting the Achilles Heel of Cancer: The Therapeutic Potential of poly(ADP-ribose) Polymerase Inhibitors in BRCA2-defective Cancer.
      ,
      • Cannan W.J.
      • Pederson D.S.
      Mechanisms and Consequences of Double-strand DNA Break Formation in Chromatin.
      ] Proof-of-concept studies in 2005 and 2007 provided the rationale for the use of PARPis in cancer therapy.[
      • Kim G.
      • Ison G.
      • Mckee A.E.
      • Zhang H.
      • Tang S.
      • Gwise T.
      • et al.
      FDA Approval Summary: Olaparib Monotherapy in Patients with Deleterious Germline BRCA-Mutated Advanced Ovarian Cancer Treated with Three or More Lines of Chemotherapy.
      ,
      • Farmer H.
      • McCabe N.
      • Lord C.J.
      • Tutt A.N.
      • Johnson D.A.
      • Richardson T.B.
      • et al.
      Targeting the DNA Repair Defect in BRCA Mutant Cells as a Therapeutic Strategy.
      ] In 2009, a phase I trial evaluated the antitumor activity of the PARPi, olaparib, for cancer patients with either wild-type or BRCA (BRCA1 or BRCA2) PVs.[
      • Fong P.C.
      • Boss D.S.
      • Yap T.A.
      • Tutt A.
      • Wu P.
      • Mergui-Roelvink M.
      • et al.
      Inhibition of poly(ADP-ribose) Polymerase in Tumors From BRCA Mutation Carriers.
      ] In the expansion phase of the trial, only BRCA (BRCA1 or BRCA2) PV carriers with OC, BC, and prostate cancer were enrolled and these patients showed clinical benefit from olaparib.[
      • Fong P.C.
      • Boss D.S.
      • Yap T.A.
      • Tutt A.
      • Wu P.
      • Mergui-Roelvink M.
      • et al.
      Inhibition of poly(ADP-ribose) Polymerase in Tumors From BRCA Mutation Carriers.
      ] In 2014, olaparib monotherapy received FDA approval for advanced OC patients with a germline PV in BRCA who have had three or more lines of prior chemotherapy.[
      • Kim G.
      • Ison G.
      • Mckee A.E.
      • Zhang H.
      • Tang S.
      • Gwise T.
      • et al.
      FDA Approval Summary: Olaparib Monotherapy in Patients with Deleterious Germline BRCA-Mutated Advanced Ovarian Cancer Treated with Three or More Lines of Chemotherapy.
      ]
      The efficacy and tolerability of olaparib in advanced OC and BC patients with germline BRCA PVs were assessed in two proofs-of-concept multicenter phase II studies.[
      • Audeh M.W.
      • Carmichael J.
      • Penson R.T.
      • Friedlander M.
      • Powell B.
      • Bell-McGuinn K.M.
      • et al.
      Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and recurrent ovarian cancer: a proof-of-concept trial.
      ,
      • Gelmon K.A.
      • Tischkowitz M.
      • Mackay H.
      • Swenerton K.
      • Robidoux A.
      • Tonkin K.
      • et al.
      Olaparib in patients with recurrent high-grade serous or poorly differentiated ovarian carcinoma or triple-negative breast cancer: a phase 2, multicentre, open-label, non-randomised study.
      ,
      • Wiggans A.J.
      • Cass G.K.
      • Bryant A.
      • Lawrie T.A.
      • Morrison J.
      Poly(ADP-ribose) polymerase (PARP) inhibitors for the treatment of ovarian cancer.
      ,
      • Tutt A.
      • Robson M.
      • Garber J.E.
      • Domchek S.M.
      • Audeh M.W.
      • Weitzel J.N.
      • et al.
      Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and advanced breast cancer: a proof-of-concept trial.
      ] In 2018, the FDA approved olaparib therapy for patients with germline BRCA PVs in HER2-negative metastatic BC patients based on the results from the OlympiAD trial (Supplemental Table 2).[
      • Robson M.
      • Im S.-A.
      • Senkus E.
      • Xu B.
      • Domchek S.M.
      • Masuda N.
      • et al.
      Olaparib for Metastatic Breast Cancer in Patients with a Germline BRCA Mutation.
      ,
      • Tutt A.
      • Robson M.
      • Garber J.E.
      • Domchek S.M.
      • Audeh M.W.
      • Weitzel J.N.
      • et al.
      Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and advanced breast cancer: a proof-of-concept trial.
      ,

      FDA. FDA approves olaparib for germline BRCA-mutated metastatic breast cancer 2018.

      ] While no statistically significant change or improvement in median overall survival (OS) was observed, an OS benefit was observed in patients who had not previously been treated for metastatic BC (hazard ratio: 0.51, 95% CI, 0.29–0.90).[
      • Robson M.E.
      • Tung N.
      • Conte P.
      • Im S.-A.
      • Senkus E.
      • Xu B.
      • et al.
      OlympiAD final overall survival and tolerability results: Olaparib versus chemotherapy treatment of physician’s choice in patients with a germline BRCA mutation and HER2-negative metastatic breast cancer.
      ] Overall, treatment with olaparib significantly increased the ORR and OS of the metastatic BC patients. A pooled analysis was performed to explore the benefit of olaparib in OC patients carrying germline PVs in BRCA who had received multiple lines of prior chemotherapy. Here, all patients had received olaparib monotherapy at relapse, and 91% (273/300) patients were evaluated for response.[
      • Matulonis U.A.
      • Penson R.T.
      • Domchek S.M.
      • Kaufman B.
      • Shapira-Frommer R.
      • Audeh M.W.
      • et al.
      Olaparib monotherapy in patients with advanced relapsed ovarian cancer and a germline BRCA1/2 mutation: a multistudy analysis of response rates and safety.
      ] In the pooled analysis, the ORR was 36% (95% CI, 30–42) and the median duration of response was 7.4 months (95% CI, 5.7–9.1).[
      • Fong P.C.
      • Boss D.S.
      • Yap T.A.
      • Tutt A.
      • Wu P.
      • Mergui-Roelvink M.
      • et al.
      Inhibition of poly(ADP-ribose) Polymerase in Tumors From BRCA Mutation Carriers.
      ] In the subset of patients with measurable disease at baseline (205/300) who had received ≥ 3 lines of prior chemotherapy, the ORR was 31% (95% CI, 25–38) and median duration of response was 7.8 months (95% CI, 5.6–9.5). This analysis demonstrated that olaparib monotherapy was safe and effective not only for patients at baseline but also for the patients who underwent multiple prior chemotherapies (both platinum-sensitive and resistant).[
      • Matulonis U.A.
      • Penson R.T.
      • Domchek S.M.
      • Kaufman B.
      • Shapira-Frommer R.
      • Audeh M.W.
      • et al.
      Olaparib monotherapy in patients with advanced relapsed ovarian cancer and a germline BRCA1/2 mutation: a multistudy analysis of response rates and safety.
      ] In Study 19, a phase II trial of olaparib maintenance monotherapy in relapsed OC patients (n = 265) with BRCA PVs (germline or somatic), olaparib-treated patients had a higher PFS when compared to the patients in the placebo arm (median, 8.4 months vs. 4.8 months; P < 0.00001).[
      • Ledermann J.A.
      • Pujade-Lauraine E.
      Olaparib as maintenance treatment for patients with platinum-sensitive relapsed ovarian cancer. Ther Adv.
      ] A retrospective analysis of these patients found that the benefit of olaparib versus placebo was greater in patients with BRCA PV when compared to patients without BRCA PVs.[
      • Ledermann J.A.
      • Pujade-Lauraine E.
      Olaparib as maintenance treatment for patients with platinum-sensitive relapsed ovarian cancer. Ther Adv.
      ]
      The PROfound trial in metastatic castration-resistant prostate cancer patients with specific HRR gene PVs evaluated their response to olaparib.[
      • Hussain M.
      • Mateo J.
      • Fizazi K.
      • Saad F.
      • Shore N.
      • Sandhu S.
      • et al.
      Survival with Olaparib in Metastatic Castration-Resistant Prostate Cancer.
      ,
      • De Bono J.
      • Mateo J.
      • Fizazi K.
      • Saad F.
      • Shore N.
      • Sandhu S.
      • et al.
      Olaparib for Metastatic Castration-Resistant Prostate Cancer.
      ,
      • Mateo J.
      • Carreira S.
      • Sandhu S.
      • Miranda S.
      • Mossop H.
      • Perez-Lopez R.
      • et al.
      DNA-Repair Defects and Olaparib in Metastatic Prostate Cancer.
      ] ORR was 33% (n = 16/49; 95% Cl, 20% to 48%), and homozygous and/or deleterious somatic or germline aberrations in BRCA, ATM, FANCA, and CHEK2 were identified in 16 out of 49 (33%) patients.[
      • Mateo J.
      • Carreira S.
      • Sandhu S.
      • Miranda S.
      • Mossop H.
      • Perez-Lopez R.
      • et al.
      DNA-Repair Defects and Olaparib in Metastatic Prostate Cancer.
      ] The POLO trial assessed the efficacy of olaparib maintenance therapy in metastatic pancreatic cancer patients with germline BRCA PVs.[
      • Golan T.
      • Hammel P.
      • Reni M.
      • Van Cutsem E.
      • Macarulla T.
      • Hall M.J.
      • et al.
      Maintenance Olaparib for Germline BRCA-Mutated Metastatic Pancreatic Cancer.
      ] This trial showed that the olaparib maintenance therapy can extend PFS in metastatic pancreatic cancer patients.[
      • Golan T.
      • Hammel P.
      • Reni M.
      • Van Cutsem E.
      • Macarulla T.
      • Hall M.J.
      • et al.
      Maintenance Olaparib for Germline BRCA-Mutated Metastatic Pancreatic Cancer.
      ] Recently, two parallel phase II studies concluded that olaparib may be therapeutically efficacious for pretreated advanced pancreatic cancer patients with PVs in other DNA repair genes (Supplemental Table 2).[
      • Javle M.
      • Shacham-Shmueli E.
      • Xiao L.
      • Varadhachary G.
      • Halpern N.
      • Fogelman D.
      • et al.
      Olaparib Monotherapy for Previously Treated Pancreatic Cancer With DNA Damage Repair Genetic Alterations Other Than Germline BRCA Variants.
      ]
      Following the success of olaparib, several next generation PARPis have also received FDA approval including veliparib, niraparib, rucaparib, and talazoparib. Mechanistic studies have increased our understanding of how PARPis exert their anti-cancer activities (Fig. 2), however, this is still not completely understood. Initially, PARPis were thought to exert anti-tumor activity by inhibiting the catalytic activity of PARP1/2.[
      • Shen Y.
      • Aoyagi-Scharber M.
      • Wang B.
      Trapping Poly(ADP-Ribose) Polymerase.
      ,
      • Murai J.
      • S-yN H.
      • Das B.B.
      • Renaud A.
      • Zhang Y.
      • Doroshow J.H.
      • et al.
      Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors.
      ] Subsequently, it was shown that PARPis effectively ‘trap’ the PARP1- and PARP2- DNA complexes in pre-clinical testing (Fig. 2).[
      • Shen Y.
      • Aoyagi-Scharber M.
      • Wang B.
      Trapping Poly(ADP-Ribose) Polymerase.
      ] Olaparib, veliparib, niraparib, rucaparib, and talazoparib all have been shown to have PARP-trapping activity.[
      • Shen Y.
      • Aoyagi-Scharber M.
      • Wang B.
      Trapping Poly(ADP-Ribose) Polymerase.
      ] Mechanistically, these inhibitors function similarly, however it has been shown in biochemical assays that veliparib functions as a catalytic inhibitor with slight trapping activity, while olaparib, niraparib and rucaparib function as PARP trappers, ∼100-fold more efficiently in comparison to veliparib (Fig. 2).[
      • Shen Y.
      • Aoyagi-Scharber M.
      • Wang B.
      Trapping Poly(ADP-Ribose) Polymerase.
      ,
      • Murai J.
      • S-yN H.
      • Das B.B.
      • Renaud A.
      • Zhang Y.
      • Doroshow J.H.
      • et al.
      Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors.
      ] Talazoparib is by the far the most potent PARP trapper with ∼ 100-fold more PARP-trapping efficiency than olaparib, rucaparib, and niraparib (Fig. 2).[
      • Shen Y.
      • Aoyagi-Scharber M.
      • Wang B.
      Trapping Poly(ADP-Ribose) Polymerase.
      ,
      • Murai J.
      • S-yN H.
      • Das B.B.
      • Renaud A.
      • Zhang Y.
      • Doroshow J.H.
      • et al.
      Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors.
      ] Both olaparib and rucaparib (Rubraca, Clovis Oncology) have been approved for treatment-refractory OCs with germline BRCA PVs. Niraparib (Zejula, GlaxoSmithKline) and olaparib have both received FDA-approval for maintenance therapy in patients with recurrent OC.[
      • Mirza M.R.
      • Monk B.J.
      • Herrstedt J.
      • Oza A.M.
      • Mahner S.
      • Redondo A.
      • et al.
      Niraparib Maintenance Therapy in Platinum-Sensitive, Recurrent Ovarian Cancer.
      ,

      FDA approves olaparib tablets for maintenance treatment in ovarian cancer. 2017.

      ] Niraparib was approved for women with recurrent OC in March 2017, for late-line treatment of women with recurrent OC in October 2019, and as the first-line maintenance monotherapy in April 2020 for women with advanced OC regardless of biomarker status (PRIMA trial).[

      FDA. FDA approves niraparib for first-line maintenance of advanced ovarian cancer. 2020.

      ] In the PRIMA trial, newly diagnosed advanced OC patients were matched to either niraparib or the placebo. A statistically significant PFS improvement and higher OS in the niraparib cohort were observed compared to the placebo, regardless of HRR deficiency status.[

      FDA. FDA approves niraparib for first-line maintenance of advanced ovarian cancer. 2020.

      ,
      • González-Martín A.
      • Pothuri B.
      • Vergote I.
      • Depont Christensen R.
      • Graybill W.
      • Mirza M.R.
      • et al.
      Niraparib in Patients with Newly Diagnosed Advanced Ovarian Cancer.
      ]
      Figure thumbnail gr2
      Fig. 2A simplified view of PARP1 function in DNA damage response and PARP1 inhibition mechanisms. When a single-strand break occurs, PARP1 binds to the damaged site and uses NAD + as a substrate for PARylation and auto-PARylation. PARylation leads to the recruitment of DNA repair proteins, and auto-PARylation decreases the affinity of PARP1 to DNA. PARP1 dissociates from DNA and BER proteins repair DNA. PARPis differ significantly in their ability to trap PARP1 and/or inhibition of PARP1 catalytic activity.[
      • Min A.
      • Im S.-A.
      PARP Inhibitors as Therapeutics: Beyond Modulation of PARylation.
      ,
      • Murai J.
      • Huang S.-Y.-N.
      • Renaud A.
      • Zhang Y.
      • Ji J.
      • Takeda S.
      • et al.
      Stereospecific PARP Trapping by BMN 673 and Comparison with Olaparib and Rucaparib.
      ] There are two known mechanisms of PARP1-inhibition, PARP1-trapping (left) and inhibition of PARP1 catalytic activity (right). During PARP1 trapping, the PARPi binds directly to the PARP1 active site, and prevents dissociation of PARP1 from DNA, leading to replication fork stalling and DSBs. Here, the DNA damage might be repaired via HRR, FA, template switching, and other repair proteins.[
      • Murai J.
      • Huang S.-Y.-N.
      • Das B.B.
      • Renaud A.
      • Zhang Y.
      • Doroshow J.H.
      • et al.
      Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors.
      ,
      • Ottaviani D.
      • Lecain M.
      • Sheer D.
      The role of microhomology in genomic structural variation.
      ] In the second mechanism, the binding of NAD + to PARP1 is prevented and thereby PARP1 loses the ability to use its substrate and perform catalysis with downstream replication fork staling and DSBs.[
      • Murai J.
      • Huang S.-Y.-N.
      • Das B.B.
      • Renaud A.
      • Zhang Y.
      • Doroshow J.H.
      • et al.
      Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors.
      ,
      • Dziadkowiec K.N.
      • Gąsiorowska E.
      • Nowak-Markwitz E.
      • Jankowska A.
      PARP inhibitors: review of mechanisms of action and BRCA1/2 mutation targeting.
      ] Here, the DNA damage might be repaired via HRR.[
      • Murai J.
      • Huang S.-Y.-N.
      • Das B.B.
      • Renaud A.
      • Zhang Y.
      • Doroshow J.H.
      • et al.
      Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors.
      ,
      • Ottaviani D.
      • Lecain M.
      • Sheer D.
      The role of microhomology in genomic structural variation.
      ]
      The BrighTNess trial assessed the addition of PARPi, veliparib, plus carboplatin to standard neoadjuvant paclitaxel chemotherapy or carboplatin alone to standard neoadjuvant chemotherapy in stage II-III triple-negative BC patients (n = 634) (Supplemental Table 2).[
      • Loibl S.
      • O'Shaughnessy J.
      • Untch M.
      • Sikov W.M.
      • Rugo H.S.
      • Mckee M.D.
      • et al.
      Addition of the PARP inhibitor veliparib plus carboplatin or carboplatin alone to standard neoadjuvant chemotherapy in triple-negative breast cancer (BrighTNess): a randomised, phase 3 trial.
      ] The pathological complete response (pCR) was higher in patients that received the combination of veliparib, carboplatin, and paclitaxel (53%, n = 68/316) versus the patients that received paclitaxel alone (31%, n = 49/158), but not higher when compared to the group that received carboplatin alone with paclitaxel (58%, n = 92/160).[
      • Loibl S.
      • O'Shaughnessy J.
      • Untch M.
      • Sikov W.M.
      • Rugo H.S.
      • Mckee M.D.
      • et al.
      Addition of the PARP inhibitor veliparib plus carboplatin or carboplatin alone to standard neoadjuvant chemotherapy in triple-negative breast cancer (BrighTNess): a randomised, phase 3 trial.
      ] Here, 51% of the patients (47/92) with a germline BRCA PV achieved a pCR versus 48% of the patients (262/542) without a germline BRCA PV that achieved a pCR.[
      • Loibl S.
      • O'Shaughnessy J.
      • Untch M.
      • Sikov W.M.
      • Rugo H.S.
      • Mckee M.D.
      • et al.
      Addition of the PARP inhibitor veliparib plus carboplatin or carboplatin alone to standard neoadjuvant chemotherapy in triple-negative breast cancer (BrighTNess): a randomised, phase 3 trial.
      ] In the GeparOla trial, early HER-2 negative breast cancer patients with HRR deficiency (germline or somatic BRCA1 or BRCA2 PV and/or HRR deficiency) received either paclitaxel plus olaparib with standard chemotherapy or paclitaxel alone with standard chemotherapy. The addition of olaparib with paclitaxel showed a significantly higher rate of pCR in hormone receptor-positive patients that were < 40 years of age, warranting further investigation in primary BCs.[
      • Fasching P.A.
      • Jackisch C.
      • Rhiem K.
      • Schneeweiss A.
      • Klare P.
      • Hanusch C.
      • et al.
      GeparOLA: A randomized phase II trial to assess the efficacy of paclitaxel and olaparib in comparison to paclitaxel/carboplatin followed by epirubicin/cyclophosphamide as neoadjuvant chemotherapy in patients (pts) with HER2-negative early breast cancer (BC) and homologous recombination deficiency (HRD).
      ] Ongoing trials (NCT02227082, NCT01562210, NCT02229656, Supplemental Table 2) are assessing PARPis as radiosensitizers and assessing the safety and tolerability of the combination treatment.
      Several trials are assessing the activity of PARPis in tumors with mutations in other DNA repair genes.[

      Staff N. PARP Inhibitors Show Promise as Initial Treatment for Ovarian Cancer. NCI; 2019.

      ,
      • Morgan R.D.
      • Clamp A.R.
      • Evans D.G.R.
      • Edmondson R.J.
      • Jayson G.C.
      PARP inhibitors in platinum-sensitive high-grade serous ovarian cancer.
      ,
      • Nakamura K.
      • Aimono E.
      • Tanishima S.
      • Imai M.
      • Nagatsuma A.K.
      • Hayashi H.
      • et al.
      Olaparib Monotherapy for BRIP1-Mutated High-Grade Serous Endometrial Cancer. JCO Precision.
      ,
      • Moore K.M.
      • Markham J.M.
      Management of ovarian cancer associated with BRCA and other genetic mutations.
      ] Yurgelun et al. showed that pancreatic ductal adenocarcinoma patients with germline PVs in other HRR genes had a better OS when compared to patients without them.[
      • Yurgelun M.B.
      • Chittenden A.B.
      • Morales-Oyarvide V.
      • Rubinson D.A.
      • Dunne R.F.
      • Kozak M.M.
      • et al.
      Germline cancer susceptibility gene variants, somatic second hits, and survival outcomes in patients with resected pancreatic cancer.
      ] A phase II trial (NCT04508803, Supplemental Table 2) is assessing the combination of an anti-PD1 monoclonal antibody, HX008, in combination with the PARPi, niraparib in metastatic BC patients with germline PVs in multiple DNA repair genes apart from BRCA. Another phase II, interventional trial (NCT03967938, Supplemental Table 2) is assessing the efficacy of olaparib in advanced cancer patients with germline or somatic PVs in HRR genes. With the availability of germline multigene panel data from cancer patients, several studies are finding that germline PVs in DNA repair genes are overrepresented in cancers where the use of PARPis has not been established.[
      • Hartman T.R.
      • Demidova E.V.
      • Lesh R.W.
      • Hoang L.
      • Richardson M.
      • Forman A.
      • et al.
      Prevalence of pathogenic variants in DNA damage response and repair genes in patients undergoing cancer risk assessment and reporting a personal history of early-onset renal cancer.
      ,
      • Carlo M.I.
      • Mukherjee S.
      • Mandelker D.
      • Vijai J.
      • Kemel Y.
      • Zhang L.
      • et al.
      Prevalence of Germline Mutations in Cancer Susceptibility Genes in Patients With Advanced Renal Cell Carcinoma.
      ,
      • Nassar A.H.
      • Abou Alaiwi S.
      • Aldubayan S.H.
      • Moore N.
      • Mouw K.W.
      • Kwiatkowski D.J.
      • et al.
      Prevalence of pathogenic germline cancer risk variants in high-risk urothelial carcinoma.
      ]
      While PARPi therapy has been revolutionary, de novo or acquired resistance to PARPi has been observed in patients. Several mechanisms have been proposed such as alterations in PARP1 leading to increase in PARP1 catalytic activity or removal of PARP trapping, restoration of HRR via BRCA reversion mutations or demethylation of BRCA promoter as reviewed by Lee and Matulonis.[
      • Lee E.K.
      • Matulonis U.A.
      PARP Inhibitor Resistance Mechanisms and Implications for Post-Progression Combination Therapies.
      ] It is anticipated that the number of patients that are resistant to PARPis will continue to rise and thus it is critical to understand the mechanisms of PARPi resistance for optimizing treatment strategies such as targeted combination therapy.[
      • Lee E.K.
      • Matulonis U.A.
      PARP Inhibitor Resistance Mechanisms and Implications for Post-Progression Combination Therapies.
      ] In summary, several PARPis have been approved as therapeutic and maintenance therapy for multiple cancer types, typically based on the presence of germline or somatic BRCA PVs. Furthermore, PARPi therapy is currently being investigated in patients with alterations in other DNA repair genes and combination with other treatments so that a broader range of patients can achieve clinical benefit.

      Implications for chemotherapy and radiation therapy

      It is now well-appreciated that BRCA deficiency can increase sensitivity to DNA crosslinking agents (such as platinum-based agents) and ionizing radiation.[
      • Mylavarapu S.
      • Das A.
      • Roy M.
      Role of BRCA Mutations in the Modulation of Response to Platinum Therapy.
      ] In 2010, a pilot study assessed a small group of women (n = 12) with BC and a germline BRCA1 PV.[
      • Byrski T.
      • Huzarski T.
      • Dent R.
      • Marczyk E.
      • Jasiowka M.
      • Gronwald J.
      • et al.
      Pathologic complete response to neoadjuvant cisplatin in BRCA1-positive breast cancer patients.
      ] Here, 10 of the 12 (∼83%) women had a pCR to neoadjuvant cisplatin. These findings were validated in a larger study in 2014 in women with early-stage BC and a BRCA1 germline PV. Here, 61% of the women experienced a pCR to the neoadjuvant cisplatin.[
      • Byrski T.
      • Huzarski T.
      • Dent R.
      • Marczyk E.
      • Jasiowka M.
      • Gronwald J.
      • et al.
      Pathologic complete response to neoadjuvant cisplatin in BRCA1-positive breast cancer patients.
      ] In a phase II trial, patients (n = 28) with stage II and stage III triple-negative BC were treated with neoadjuvant cisplatin. Then, after undergoing definitive surgery, these patients received standard adjuvant chemotherapy and radiation therapy. Here, 22% (6/28) of the patients experienced a pCR. Interestingly, of the 6 patients with pCR, only 2 patients had a germline BRCA1 PV. Additionally, 64% of patients had a complete or partial response. These studies showed that while patients with a germline BRCA1 PV have a good response to cisplatin, there is a subset of patients with wild-type BRCA1 and triple negative BC that also respond to cisplatin.[
      • Silver D.P.
      • Richardson A.L.
      • Eklund A.C.
      • Wang Z.C.
      • Szallasi Z.
      • Li Q.
      • et al.
      ] In a phase II non-randomized trial, women with metastatic BC and a germline BRCA1 PV were treated with cisplatin chemotherapy. An ORR of 80% (16/20) was observed, with 45% (9/20) of the patients exhibiting complete clinical response, and 7/20 (35%) patients exhibited a partial response.[
      • Byrski T.
      • Dent R.
      • Blecharz P.
      • et al.
      Results of a phase II open-label, non-randomized trial of cisplatin chemotherapy in patients with BRCA1-positive metastatic breast cancer.
      ] This study demonstrated that cisplatin was highly effective in women with metastatic BC and BRCA1 PVs.
      The INFORM trial prospectively assessed pCR in newly diagnosed HER2-negative BC patients with germline BRCA PVs with neoadjuvant cisplatin versus anthracycline-based therapy (Supplemental Table 3). The study patients were randomly assigned to neoadjuvant single-agent cisplatin or to doxorubicin-cyclophosphamide.[
      • Tung N.
      • Arun B.
      • Hacker M.R.
      • Hofstatter E.
      • Toppmeyer D.L.
      • Isakoff S.J.
      • et al.
      TBCRC 031: Randomized Phase II Study of Neoadjuvant Cisplatin Versus Doxorubicin-Cyclophosphamide in Germline BRCA Carriers With HER2-Negative Breast Cancer (the INFORM trial).
      ] The pCR rates for neoadjuvant cisplatin and doxorubicin-cyclophosphamide were 18% and 26% respectively. This study did not find any differences in pCR and residual cancer burden by either agent in patients with triple negative BC, consistent with those reported by the BrighTNess and Geparsixto trials.[
      • Loibl S.
      • O'Shaughnessy J.
      • Untch M.
      • Sikov W.M.
      • Rugo H.S.
      • Mckee M.D.
      • et al.
      Addition of the PARP inhibitor veliparib plus carboplatin or carboplatin alone to standard neoadjuvant chemotherapy in triple-negative breast cancer (BrighTNess): a randomised, phase 3 trial.
      ,
      • Tung N.
      • Arun B.
      • Hacker M.R.
      • Hofstatter E.
      • Toppmeyer D.L.
      • Isakoff S.J.
      • et al.
      TBCRC 031: Randomized Phase II Study of Neoadjuvant Cisplatin Versus Doxorubicin-Cyclophosphamide in Germline BRCA Carriers With HER2-Negative Breast Cancer (the INFORM trial).
      ,
      • Hahnen E.
      • Lederer B.
      • Hauke J.
      • Loibl S.
      • Kröber S.
      • Schneeweiss A.
      • et al.
      Germline Mutation Status, Pathological Complete Response, and Disease-Free Survival in Triple-Negative Breast Cancer.
      ] The results from the INFORM trial suggest that BRCA PV status might predict sensitivity to any DNA damaging agents and not exclusively to platinum-based agents. Recently, a small trial of neoadjuvant talazoparib without chemotherapy reported a pCR of 53% in 20 patients with operable BC and a germline PV in BRCA, warranting larger investigation including comparison with neoadjuvant chemotherapy.[
      • Schwartz M.D.
      • Lerman C.
      • Brogan B.
      • Peshkin B.N.
      • Hughes Halbert C.
      • Demarco T.
      • et al.
      Impact ofBRCA1/BRCA2Counseling and Testing on Newly Diagnosed Breast Cancer Patients.
      ]
      A 2006 retrospective study compared the outcomes in BC patients with germline BRCA PVs to patients with sporadic BC. All patients in the study had breast conservation surgery and received radiation therapy for their stage I or II BC with no significant differences observed in in-breast tumor recurrence in the carriers versus the non-carriers. This study also did not find any significant difference in recurrence in the carriers versus the non-carriers who underwent bilateral oophorectomy. However, the recurrence rates were twice as high in the carriers who did not undergo oophorectomy. Also, as expected, the study found an increased incidence of contralateral BCs in the carriers versus the non-carriers. While tamoxifen significantly reduced the incidence of contralateral BCs in carriers, the incidence remained higher than sporadic non-carriers.[
      • Pierce L.J.
      • Levin A.M.
      • Rebbeck T.R.
      • Ben-David M.A.
      • Friedman E.
      • Solin L.J.
      • et al.
      Ten-Year Multi-Institutional Results of Breast-Conserving Surgery and Radiotherapy in BRCA1/2-Associated Stage I/II Breast Cancer.
      ] Overall, this study suggests that hormonal intervention and prophylactic bilateral oophorectomy are associated with reduced recurrence or reduced risk of new primary BCs and fewer contralateral BCs in carriers. Despite this, carriers still have a high risk of contralateral BCs, warranting the need for additional strategies in carriers undergoing breast conservation surgery to reduce their risk. A prospective study found that an equal proportion of carriers chose breast conservation surgery and bilateral prophylactic mastectomy for risk reduction, thus warranting assessment of additional risk-reduction strategies for breast conservation surgery.[
      • Schwartz M.D.
      • Lerman C.
      • Brogan B.
      • Peshkin B.N.
      • Hughes Halbert C.
      • Demarco T.
      • et al.
      Impact ofBRCA1/BRCA2Counseling and Testing on Newly Diagnosed Breast Cancer Patients.
      ]
      Beyond BC, women with OC who are carriers of a germline BRCA1 or BRCA2 PV have also shown high response rates to platinum-based therapy. In a study by Gorodnova et al., 34 BRCA1 and 1 BRCA2 germline PV carriers were identified. The total clinical response in the patients with germline PVs in BRCA to platinum-based therapy was determined to be 34% (n = 12/35). In contrast, the clinical response rate of the patients with somatic PVs in BRCA was determined to be 4% (8/190).[
      • Gorodnova T.V.
      • Sokolenko A.P.
      • Ivantsov A.O.
      • Iyevleva A.G.
      • Suspitsin E.N.
      • Aleksakhina S.N.
      • et al.
      High response rates to neoadjuvant platinum-based therapy in ovarian cancer patients carrying germ-line BRCA mutation.
      ] In a retrospective study, men with castration-resistant prostate cancer, with or without germline BRCA2 PVs, were assessed for response to platinum-based therapy. These patients were treated with carboplatin and docetaxel and were categorized based on the germline BRCA2 PV status. This study found a significant association between the germline BRCA2 PV status and the response to platinum-based therapy.[
      • Pomerantz M.M.
      • Spisák S.
      • Jia L.
      • Cronin A.M.
      • Csabai I.
      • Ledet E.
      • et al.
      The association between germline BRCA2 variants and sensitivity to platinum-based chemotherapy among men with metastatic prostate cancer.
      ] Overall, both chemotherapy and radiation therapy act by inducing DNA damage, and vulnerabilities in DNA repair, either germline or somatic, impacts response to these therapies.

      Therapeutic implications of germline variants in other DNA repair genes

      Growing evidence suggests that germline variants in other DNA repair genes may also impact response to cancer therapeutics. Defects in the Fanconi Anemia (FA) pathway lead to impaired ability to perform homology-directed repair and thereby increased sensitivity to DNA cross-linking agents.[
      • Duan W.
      • Gao L.
      • Aguila B.
      • Kalvala A.
      • Otterson G.A.
      • Villalona-Calero M.A.
      Fanconi Anemia Repair Pathway Dysfunction, a Potential Therapeutic Target in Lung Cancer.
      ] Tumors carrying germline defects in the FA are also sensitive to PARPis.[
      • Duan W.
      • Gao L.
      • Aguila B.
      • Kalvala A.
      • Otterson G.A.
      • Villalona-Calero M.A.
      Fanconi Anemia Repair Pathway Dysfunction, a Potential Therapeutic Target in Lung Cancer.
      ] There are 22 FA or FA-like genes including FANCA, B, C, D1 (or BRCA2), D2, E, F, G, I, J (or BRIP1), L, M, N (or PALB2), O (or RAD51C), P (or SLX4), Q (or ERCC4), R, S (or BRCA1), T (or UBE2T), U (or XRCC2), V (or REV7) and W (or RFWD3).[
      • D'Andrea A.D.
      Susceptibility pathways in Fanconi's anemia and breast cancer.
      ,
      • Kottemann M.C.
      • Smogorzewska A.
      Fanconi anemia and the repair of Watson and Crick DNA crosslinks.
      ,
      • Popp I.
      • Punekar M.
      • Telford N.
      • Stivaros S.
      • Chandler K.
      • Minnis M.
      • et al.
      Fanconi anemia with sun-sensitivity caused by a Xeroderma pigmentosum-associated missense mutation in XPF.
      ] The BRCA genes and several associated HRR genes are important members of the FA pathway. FANCD2-FANCI monoubiquitinylation is a critical step in the activation of the FA pathway and can be assessed by nuclear staining of monoubiquitylated FANCD2.[
      • Liang C.C.
      • Li Z.
      • Lopez-Martinez D.
      • Nicholson W.V.
      • Venien-Bryan C.
      • Cohn M.A.
      The FANCD2-FANCI complex is recruited to DNA interstrand crosslinks before monoubiquitination of FANCD2.
      ] In non-small cell lung cancer, it was observed that a subset of tumors was negative for FANCD2 foci staining (22%, n = 23/104) and thus was considered deficient in FA pathway.[
      • Duan W.
      • Gao L.
      • Aguila B.
      • Kalvala A.
      • Otterson G.A.
      • Villalona-Calero M.A.
      Fanconi Anemia Repair Pathway Dysfunction, a Potential Therapeutic Target in Lung Cancer.
      ] These results suggest that at least a subset of lung cancers may be sensitive to DNA damaging therapy and/or PARPi therapy due to their limited ability to perform FA repair.[
      • Duan W.
      • Gao L.
      • Aguila B.
      • Kalvala A.
      • Otterson G.A.
      • Villalona-Calero M.A.
      Fanconi Anemia Repair Pathway Dysfunction, a Potential Therapeutic Target in Lung Cancer.
      ] In a clinical study, 643 tumors (including BC, OC, CRC, endometrial, lung, and several other cancers) were evaluated for FANCD2 nuclear foci and 28.7% (185/643) were found to be negative.[
      • Villalona-Calero M.A.
      • Duan W.
      • Zhao W.
      • Shilo K.
      • Schaaf L.J.
      • Thurmond J.
      • et al.
      Veliparib Alone or in Combination with Mitomycin C in Patients with Solid Tumors With Functional Deficiency in Homologous Recombination Repair.
      ] Here, 61 patients with FANCD2-foci negative tumors received either veliparib alone or in combination with mitomycin C, and 6 patients in the combination arm showed clinical benefit. Subsequent tumor sequencing found germline alterations in the FA pathway. These data suggest that PARPi alone or in combination with a DNA damaging therapy is safe and can lead to clinical benefit in some patients with FA-deficient tumors.[
      • Villalona-Calero M.A.
      • Duan W.
      • Zhao W.
      • Shilo K.
      • Schaaf L.J.
      • Thurmond J.
      • et al.
      Veliparib Alone or in Combination with Mitomycin C in Patients with Solid Tumors With Functional Deficiency in Homologous Recombination Repair.
      ]
      The MMEJ pathway is emerging as a promising therapeutic target; it begins after exposure of microhomology (i.e., short complementary sequence) regions for alignment of DNA ends.[
      • Patterson-Fortin J.
      • D'Andrea A.D.
      Exploiting the Microhomology-Mediated End-Joining Pathway in Cancer Therapy.
      ] Following the alignment, the resulting 5′ flaps are processed, the gaps are filled by a polymerase and then ligated. PARP1 is an essential component of the MMEJ pathway and PARPi can also inhibit the MMEJ pathway.[
      • Patterson-Fortin J.
      • D'Andrea A.D.
      Exploiting the Microhomology-Mediated End-Joining Pathway in Cancer Therapy.
      ] Furthermore, loss of expression of MMEJ proteins such as POLQ and FEN1 in HRR-deficient cells was found to cause synthetic lethality.[
      • Patterson-Fortin J.
      • D'Andrea A.D.
      Exploiting the Microhomology-Mediated End-Joining Pathway in Cancer Therapy.
      ,
      • Ceccaldi R.
      • Liu J.C.
      • Amunugama R.
      • Hajdu I.
      • Primack B.
      • Petalcorin M.I.R.
      • et al.
      Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair.
      ] While MMEJ pathway is emerging as a promising biomarker to guide cancer treatment, only the therapeutic potential of somatic tumor alterations has been assessed so far. Brandalize et. al. identified a germline polymorphism in the promoter region of POLQ that was associated with increased risk of inherited BC, warranting further studies on cancer risk and therapy outcomes.[
      • Brandalize A.P.C.
      • Schüler-Faccini L.
      • Hoffmann J.-S.
      • Caleffi M.
      • Cazaux C.
      • Ashton-Prolla P.
      A DNA repair variant in POLQ (c.-1060A > G) is associated to hereditary breast cancer patients: a case–control study.
      ]
      The core NER genes include ERCC1, 2 (XPD), 3 (XPB), 4 (XPF), 5 (XPG), 6 (CSB), 8 (CSA), XPA, XPC, TFIIH, LIG1, RPA, and DDB1.[
      • Marteijn J.A.
      • Lans H.
      • Vermeulen W.
      • Hoeijmakers J.H.
      Understanding nucleotide excision repair and its roles in cancer and ageing.
      ] The NER pathway excises bulky DNA lesions such as those generated by platinum-based agents.[
      • Bowden N.A.
      Nucleotide excision repair: Why is it not used to predict response to platinum-based chemotherapy?.
      ] Targeting the core NER protein complex, the ERCC1-ERCC4 heterodimer has been shown to improve cisplatin cytotoxicity in several cancer cell line models.[
      • Arora S.
      • Kothandapani A.
      • Tillison K.
      • Kalman-Maltese V.
      • Patrick S.M.
      Downregulation of XPF–ERCC1 enhances cisplatin efficacy in cancer cells☆.
      ] Additionally, it has also been suggested that low nuclear ERCC1 expression may correlate with improved treatment response and confer sensitivity to radiation therapy in head and neck cancer.[
      • Mehra R.
      • Zhu F.
      • Yang D.-H.
      • Cai K.Q.
      • Weaver J.
      • Singh M.K.
      • et al.
      Quantification of Excision Repair Cross-Complementing Group 1 and Survival in p16-Negative Squamous Cell Head and Neck Cancers.
      ] A meta-analysis of 836 non-small cell lung cancer patients found that ERCC1 protein levels could predict better PFS and OS to platinum-based therapy.[
      • Bowden N.A.
      Nucleotide excision repair: Why is it not used to predict response to platinum-based chemotherapy?.
      ,
      • Bepler G.
      • Williams C.
      • Schell M.J.
      • Chen W.
      • Zheng Z.
      • Simon G.
      • et al.
      Randomized International Phase III Trial of ERCC1 and RRM1 Expression-Based Chemotherapy Versus Gemcitabine/Carboplatin in Advanced Non–Small-Cell Lung Cancer.
      ,
      • Chen S.
      • Zhang J.
      • Wang R.
      • Luo X.
      • Chen H.
      The platinum-based treatments for advanced non-small cell lung cancer, is low/negative ERCC1 expression better than high/positive ERCC1 expression? A meta-analysis.
      ] Three regulatory polymorphisms in NER genes were determined to predict better PFS and OS following treatment with cisplatin.[
      • Bowden N.A.
      Nucleotide excision repair: Why is it not used to predict response to platinum-based chemotherapy?.
      ] NER genes were also shown to be differentially expressed in CRC and normal colon tissues, with the expression of ERCC2, ERCC4, and XPC associated with CRC prognosis.[
      • Liu J.
      • Li H.
      • Sun L.
      • Feng X.
      • Wang Z.
      • Yuan Y.
      • et al.
      The Differential Expression of Core Genes in Nucleotide Excision Repair Pathway Indicates Colorectal Carcinogenesis and Prognosis.
      ] Aiello et al. evaluated ERCC1 T19007C and C8092A SNPs in tumor specimens from two independent cohorts of lung cancer patients who had received nivolumab. Here, a better response to nivolumab was observed in patients with ERCC1 C8092A SNP.[
      • Aiello M.M.
      • Solinas C.
      • Santoni M.
      • Battelli N.
      • Restuccia N.
      • Latteri F.
      • et al.
      Excision Repair Cross Complementation Group 1 Single Nucleotide Polymorphisms and Nivolumab in Advanced Non-Small Cell Lung Cancer. Frontiers.
      ] Overall, preclinical studies suggest that defects in several DNA repair genes could potentially predict response to therapy, however it remains to be determined how effective they are for patient stratification.

      Therapeutic implications of germline variants in genes that regulate DNA repair

      Several genes that do not directly mediate DNA repair are emerging as key regulators of DNA repair pathways. Recent work is defining the molecular circuits that govern the relationship between androgen receptor signaling and DNA repair.[
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      Conclusions

      The massive use of next-generation sequencing technology in the clinic has revealed a rich landscape of germline defects in DNA repair genes. The availability of FDA-approved therapies that specifically target DNA repair defects such as PARPis or ICIs that show benefit in patients with DNA repair defects have expanded the clinical options for cancer patients with durable responses and long-term benefits. Given the significance of DNA repair defects for cancer risk and treatment response, it is important in the future to determine if germline vulnerabilities in mediators of DNA repair pathways can be exploited therapeutically. Overall, it is the right time to fully understand the therapeutic extent of germline vulnerabilities in DNA repair and assess their clinical benefit in cancer patients.

      CRediT authorship contribution statement

      S.M.S. and E.V.D. contributed equally and substantially to conceptualization, writing - original draft, writing - review & editing, and visualization. R.W.L., M.J.H., M.B.D. and J.E.M. contributed to writing - original draft, and writing - review & editing. S.A. and M.J.E. contributed equally to conceptualization, writing - original draft and writing - review & editing.

      Declaration of Competing Interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper: [M.J.E. has served on scientific advisory board for Biomarker strategies, as an advisor for Windmil Therapeutics, Sanofi/Regeneron, and Syndax; data safety monitoring boards for Astra Zeneca, Seattle Genetics, Takeda, GSK and has received clinical research funding (to the institution) from Apexigen, BMS, Nektar, Precision Oncology, Windmil and Merck. M.J.H. performs collaborative research (with no funding) with the following: Myriad Genetics, Invitae Corporation, Ambry Genetics, Foundation Medicine, Inc. He also performs collaborative research (with no funding) and is part of a Precision Oncology Alliance funded by Caris Life Sciences (cover travel and meals at meetings). S.A. performs collaborative research (with no funding) with Caris Life Sciences, Foundation Medicine, Inc., Ambry Genetics and Invitae Corporation. J.E.M. performs collaborative research (with no funding) with Foundation Medicine, Inc. and Caris Life Sciences. S.A.’s spouse is employed by Akoya Biosciences and has stocks in Akoya Biosciences, HTG Molecular Diagnostics, Abcam Plc., and Senzo Health. S.A., M.J.H., R.W.L., J.E.M. have patents and/or pending patents related to cancer diagnostics/treatment. M.J.E. has pending patent for radiopharmaceuticals to treat small-cell lung cancer. All other authors declare no competing interests.

      Acknowledgments

      We express gratitude to Dr. Erica Golemis (Fox Chase Cancer Center) for valuable input on the article.

      Funding

      All Fox Chase Cancer Center affiliated authors are in part supported by the NCI Core Grant, P30 CA006927, to the Fox Chase Cancer Center. M.J.E. was supported by the DOD W81XWH-18-1-0196. S.A. was supported by the DOD W81XWH-18-1-0148, and a CEP grant from the Yale Head and Neck Cancer SPORE. R.W.L. was supported by an Alpha Omega Alpha Carolyn L. Kuckein Student Research Fellowship. M.J.H. was supported by funding from the American Cancer Society. M.B.D was supported by the NIH U01 CA164920, R01 CA207365 grants. J.E.M. was supported by the Colorectal Cancer Alliance and Varian Medical Systems.

      Appendix A. Supplementary material

      The following are the Supplementary data to this article:

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