DNA Damage and Repair Biomarkers of Immunotherapy Response

Published OnlineFirst June 19, 2017; DOI: 10.1158/2159-8290.CD-17-0226

REVIEW

Kent W. Mouw 1 ,2 ,3 , Michael S. Goldberg 2 ,4 , Panagiotis A. Konstantinopoulos 2 ,5 ,6 , and Alan D. D’Andrea 1 ,2 ,3 ,6

ABSTRACT DNA-damaging agents are widely used in clinical oncology and exploit defi ciencies in tumor DNA repair. Given the expanding role of immune checkpoint blockade as a therapeutic strategy, the interaction of tumor DNA damage with the immune system has recently come into focus, and it is now clear that the tumor DNA repair landscape has an important role in driving response to immune checkpoint blockade. Here, we summarize the mechanisms by which DNA damage and genomic instability have been found to shape the antitumor immune response and describe clinical efforts to use DNA repair biomarkers to guide use of immune-directed therapies.

Signiﬁ cance: Only a subset of patients respond to immune checkpoint blockade, and reliable predictive biomarkers of response are needed to guide therapy decisions. DNA repair defi ciency is common among tumors, and emerging experimental and clinical evidence suggests that features of genomic instability are associated with response to immune-directed therapies. Cancer Discov; 7(7); 675–93. ©2017 AACR.

DNA REPAIR PATHWAY ALTERATIONS to an environment that is rich in sources of DNA damage ARE CANCER DRIVERS ( 7 ). Further exacerbating the high levels of DNA damage, functional loss of one or more DNA repair pathways DNA is continually exposed to endogenous and exogenous is common in tumors, and recent sequencing-based and sources of damage, and the coordinated activity of multiple functional studies have begun to reveal the broad scope of DNA repair pathways is required to maintain genomic integ- DNA repair pathway defi ciencies across cancer ( 8, 9 ). Due to rity under normal cellular conditions (1, 2). Failure to repair the frequent combination of increased levels of DNA dam- DNA damage in an accurate and timely manner can result in age and decreased DNA repair capacity, most cancer cells a variety of genomic aberrations, including point mutations, accumulate hundreds to thousands of genomic aberrations chromosomal translocations, and gain or loss of chromo- that distinguish them from normal (noncancerous) cells somal segments or entire chromosomes ( 3 ). In some cases, ( 10 ). Although only a minority of these genetic changes may these genomic alterations produce changes in cell physiology be responsible for driving the tumor phenotype, the overall that drive tumor initiation (4–6 ). landscape of DNA alterations provides important informa- In addition to playing a role during the earliest events tion regarding tumor DNA damage exposure and repair in tumorigenesis, loss of DNA repair fi delity has impor- capacity, and it can confer the tumor and microenviron- tant implications for tumor evolution and response to ment with unique properties that have the potential to be therapy. Common tumor features such as high levels of exploited therapeutically. oxidative stress, replicative stress, and loss or suppression of DNA damage–induced cell-cycle checkpoints contribute TUMOR DNA REPAIR ALTERATIONS ARE BIOMARKERS AND THERAPEUTIC TARGETS 1 Department of Radiation Oncology, Brigham & Women’s Hospital/Dana- Farber Cancer Institute, Boston, Massachusetts. 2 Harvard Medical School, Tumor DNA repair defi ciency has been a therapeutic tar- Boston, Massachusetts. 3 Ludwig Center at Harvard, Harvard Medical get in oncology for more than a century, as evidenced by the 4 School, Boston, Massachusetts. Department of Cancer Immunology and widespread use of DNA-damaging chemotherapeutic agents Virology, Dana-Farber Cancer Institute, Boston, Massachusetts. 5 Medi- cal Gynecology Oncology Program, Dana-Farber Cancer Institute, Boston, and ionizing radiation. Several classes of DNA-damaging Massachusetts. 6 Center for DNA Damage and Repair, Dana-Farber Cancer chemotherapy agents—including platinum-based agents, Institute, Boston, Massachusetts. alkylating agents, and DNA intercalators—continue to com- Corresponding Author: Alan D. D’Andrea , Dana-Farber Cancer Institute, prise the backbone of many systemic therapy regimens, and 450 Brookline Avenue, HIM 243, Boston, MA 02215 . Phone: 617-632- radiation (alone or in combination with chemotherapy) is 2080; Fax: 617-632-6069; E-mail: [email protected] used in a variety of curative approaches. Although the mecha- doi: 10.1158/2159-8290.CD-17-0226 nistic underpinnings of increased cancer cell sensitivity to ©2017 American Association for Cancer Research. DNA damage remain to be elucidated in many settings, the

july 2017 CANCER DISCOVERY | 675

REVIEW Mouw et al. therapeutic window for DNA-damaging agents is driven by mutations have also been identified in ovarian tumors, and a deficiency in DNA repair function in cancer cells relative to have important implications for platinum and PARP inhibi- normal cells. tor use in this setting (33). In some cases, the nature of the underlying cancer DNA It is likely that NGS-based studies will continue to uncover repair deficiency has been characterized, and relevant clini- associations between mutation- or expression-based changes cal biomarkers have been developed and are used for both in tumor DNA repair pathway function and response to prognostic and predictive purposes. For example, the asso- conventional and targeted agents. Moreover, the principles ciation between O6-methylguanine DNA methyltransferase that have been used to identify existing DNA repair bio- (MGMT) promoter methylation and response to temozolo- markers will likely remain applicable in the search for novel mide in glioblastoma multiforme (GBM) is one of the best DNA repair biomarkers for emerging therapies, including characterized associations between a specific DNA repair immunotherapy. alteration and response to a DNA-damaging agent (Table 1; refs. 11, 12). Loss of mismatch repair (MMR) function via germline or somatic mutation or gene silencing is a DNA REPAIR DEFECTS DRIVE common event in colorectal and endometrial tumors (13, GENOMIC INSTABILITY AND 14), as well as in a smaller percentage of many other tumor TUMOR IMMUNOGENICITY types. Loss of MMR function confers the microsatellite Although the coordinated activity of DNA repair path- instability (MSI) phenotype that is associated with unique ways swiftly corrects the majority of DNA lesions, delayed clinical features, prognosis, and response to conventional or improper repair can lead to changes in the tumor genome chemotherapy as well as targeted agents (discussed below; that alter the immune balance in the tumor microenvi- refs. 15–17). ronment. The interaction between the tumor and the host Cancer predisposition associated with germline muta- immune system has been appreciated for several decades tions in tumor suppressor genes such as BRCA1 and BRCA2 (34), and therapeutic attempts to activate the host immune has been appreciated for several decades (18–20). BRCA1/2 system to kill tumor cells have shown some clinical efficacy; are central players in the homologous recombination (HR) for example, use of systemic IL2 in metastatic melanoma repair pathway, and loss of BRCA1/2 or other genes in the (35) and renal cell carcinoma (36), and intravesicular Bacillus HR pathway can confer an HR-deficient phenotype in breast, Calmette-Guerin (BCG) in bladder cancer (37). ovarian, prostate, and other tumor types (21–23). HR-defi- However, in the past 5 years, the field of cancer immuno- cient tumors have unique clinical properties, and the recent therapy has been transformed with the clinical implementa- discovery and clinical implementation of poly(ADP ribose) tion of antibodies against inhibitory signaling molecules polymerase (PARP) inhibitors are an example of the poten- on tumor and immune cells (38, 39). The first immune tial to employ DNA repair–directed targeted agents in a syn- checkpoint inhibitor that demonstrated a survival benefit thetic lethal approach to target tumors with a specific DNA was the anti–CTLA-4 antibody ipilimumab in patients with repair pathway deficiency (24–27). Small-molecule inhibitors metastatic melanoma (40, 41). This has been followed by of numerous other DNA repair proteins—many of them evidence of robust clinical activity of agents targeting the pro- kinases involved in DNA damage response pathways (such grammed death-1 (PD-1; refs. 42–44) and PD-1 ligand (PD- as ATM, ATR, CHEK1, CHEK2, WEE1, and DNA-PKcs)—are L1) receptors (45, 46). Monoclonal antibodies that induce now being tested in a variety of DNA repair–deficient and immune checkpoint blockade (ICB) have now been approved DNA repair–proficient tumor settings as single agents and in a variety of advanced and up-front disease settings (47–51), in combinations with conventional DNA-damaging agents and scores of additional trials are under way that are likely to (28–30). further extend the scope of ICB use. Although DNA-damaging chemotherapy and radiation are Despite robust and durable responses to ICB in a subset of used in the treatment of hundreds of thousands of patients tumors, efficacy of ICB varies widely. Even among the tumor each year in the United States, few validated biomarkers are types such as melanoma and non–small cell lung cancer for available to guide the selection of agent and dose. Whereas the which many of the first ICB trials were conducted, only a discovery of the DNA repair–associated biomarkers described subset of patients respond to therapy. As the evidence for ICB above (MGMT, MSI, and BRCA1/2) preceded the widespread use across clinical settings continues to grow, a major unmet availability of next-generation sequencing (NGS), recent need is the identification of reliable biomarkers that predict studies using NGS-based approaches have begun to expand response to ICB. Numerous lines of evidence now suggest the number of apparent associations between specific DNA that DNA repair plays an important role in driving sensitivity repair–deficient states and specific DNA-damaging agents. and response to ICB. For example, ERCC2 is a DNA helicase that plays a central role in the nucleotide excision repair (NER) pathway responsible for repairing adducts created by DNA-damaging agents MUTATIONAL LOAD IS A such as UV light and platinum chemotherapies. Somatic PREDICTOR OF RESPONSE missense mutations in ERCC2 are present in up to 20% of TO IMMUNOTHERAPY primary muscle-invasive bladder cancers (MIBC), and ERCC2- Accumulation of somatic mutations is a hallmark of mutated tumors exhibit improved response to neoadjuvant tumors, but mutational burden varies dramatically both cisplatin-based chemotherapy regimens compared with wild- within and among tumor types (10). The median muta- type ERCC2 tumors (31, 32). In addition, deleterious NER tion burden ranges over several orders of magnitude, from

676 | CANCER DISCOVERY july 2017 www.aacrjournals.org

DNA Repair Biomarkers of Immunotherapy Response REVIEW ( continued ) lomide and ICB are under way in GBM blockade (NCT01876511). cient tumors are MMR-defi planned or under way in HR-defi cient tumors are in HR-defi planned or under way augments response to ICB remains to be determined Clinical ICB response Several clinical trials of temozo- Several Improved response to PD-1 Improved Several clinical trials of ICB Several ciency Whether HR defi PD-1, PD-L1, LAG3, PD-1, PD-L1, LAG3, genes ( CTLA-4 ) enriched in CpG island methylator phenotype (G-CIMP) subtype neoantigens scores: HRD-LOH, HDR-TAI, scores: HRD-LOH, HDR-TAI, HDR-LST neoantigens Genomic biomarkers expression of immune-related ↑ expression MGMT promoter methylation MGMT ↑ mutational burden, predicted MMR mutational signature(s) clinical trials of ICB in Several HR mutational signature T-cell gene signature ↑ cytotoxic transcriptomic HR defi ciency (HRD) HR defi transcriptomic ↑ mutation burden, predicted

+ T T cells, Th1 and CD8 + + ( ↑ CD3 ( ↑ CD8 cells) staining T cells) T Histopathologic features Immune cell infi ltrate ltrate Immune cell infi immune cell infi ltrate ltrate immune cell infi Increased PD-1, PD-L1 ↑ PD-1, PD-L1 staining etc.) BRCA1, BRCA1, sequencing of HR genes ( PALB2, BRCA2, (Lynch syndrome) MLH1, PMS2) assay) PCR Germline/somatic Germline sequencing Clinical diagnostic criteria IHC (MSH2, MSH6, MSI PCR (Bethesda methylation-specifi c methylation-specifi Lynch syndrome, usually clear cell and endometrioid histology) anatomic location) endometrioid subtype) countries) Western 35% with MGMT promoter meth- ∼ 35% with MGMT ylation (varies widely across clinical contexts) castration-resistant tumors castration-resistant gastric, endometrial) Ovarian: 10% (mostly associated with Prostate, breast, glioma: 0%–5% Occasional cases in other tumor types Ovarian: ∼ 50% 20% (varies widely by stage, Colon: ∼ 20% (varies widely by Endometrial: ∼ 20% (highest in in Gastric: 15%–20% (highest rates Glioblastoma multiforme (GBM): Breast: 10%–40% (varies by subtype) Breast: 10%–40% (varies by Prostate: 15%–25% in metastatic Other tumor types (pancreatic, -methylguanine–DNA nation (HR) methyltransferase methyltransferase (MGMT) 6 Homologous recombi- Mismatch repair (MMR) DNA repair pathwayO Common tumor settings Association between deﬁ ciencies in tumor DNA repair and immunotherapy response response ciencies in tumor DNA repair and immunotherapy Table deﬁ 1. Association between

july 2017 CANCER DISCOVERY | 677

REVIEW Mouw et al. responders cient included with MMR-defi planned or tumors in several ongoing ICB trials Clinical ICB response Not reported Case reports of extreme Case reports of extreme POLE-mutant tumors are being or POLD1 or ERCC2 POLE MUTYH mutations ase domain of neoantigens ture other NER gene(s) Genomic biomarkers BER mutational signature Biallelic POLE/POLD1 mutational signatures POLE/POLD1 T-cell gene signature ↑ cytotoxic Somatic point mutation in exonucle- ↑ mutational burden, predicted ERCC2-related mutational signa- Somatic mutation in , +

+ and CD8 + , NK cells, gran- + zyme B) CD8 ( ↑ CD3 T cells) T cell infi ltrate (CD3 ltrate cell infi Histopathologic features ↑ PD-1, PD-L1 staining Clinical diagnostic criteria None in routine use ltrate Immune cell infi None in routine use None described to date Increased mutational burden Not reported None in routine use Prominent immune

MUTYH muta- POLE and mutations also pre- POLD1 POLD1 Germline been associated with tions have a high risk of multiple colorectal adenomas and carcinomas, whereas germline dispose to endometrial cancers. muscle-invasive tumors muscle-invasive are associated with increased risk of colorectal cancer Endometrial: 5%–10% Colon: 2%–5% Rare cases in other tumor types. Bladder: 15%–20% of primary Biallelic germline mutations in POLD1) proofreading repair (NER) (BER) DNA repair pathway (POLE/ Polymerase Common tumor settings Nucleotide excision Nucleotide excision Base excision repair Base excision Association between deﬁ Continued ) response ( ciencies in tumor DNA repair and immunotherapy deﬁ 1. Association between Table

678 | CANCER DISCOVERY july 2017 www.aacrjournals.org

DNA Repair Biomarkers of Immunotherapy Response REVIEW approximately 0.1 mutations/megabase (Mb) of the exome Similar to the principle that one or a small number of in some pediatric tumors to several mutations/Mb in car- genetic alterations in a tumor may be responsible for driving cinogen-induced tumor types such as melanoma, lung, and the tumor phenotype, there is a growing appreciation that bladder. The variation in mutation burden within tumor ICB response may also be driven by host response to a small types is even more dramatic, with the mutation rate com- number of tumor-specific neoantigens. For example, it was monly varying by >1,000× between the most- and least- recently shown that tumors with high levels of clonal neo- mutated samples within a specific tumor type. These vast antigens have improved responses to ICB and that the loss differences in mutation burden reflect significant differences of clonal neoantigens can be associated with ICB resistance in the balance of DNA damage exposure and DNA repair (59, 60). Conversely, increased neoantigen intratumor het- fidelity among tumors. erogeneity characterized by increased number of subclonal The relationship between tumor mutational load and mutations has been associated with poor ICB responses in response to ICB was first described in patients with meta- some cases. static melanoma treated with the CTLA-4–blocking antibod- The mechanisms underlying the role of neoantigen intra- ies ipilimumab or tremelimumab (52). Tumor mutational tumor heterogeneity as a prognostic and predictive bio- load was significantly higher in patients who achieved long- marker have not been fully characterized. DNA-damaging term clinical benefit from anti–CTLA-4 therapy compared agents such as cytotoxic chemotherapy and ionizing radia- with those who had minimal benefit. This association was tion (discussed below) primarily create subclonal mutations, confirmed in a second study demonstrating nonsynonymous and patients who receive more cytotoxic therapy are typically mutation burden was significantly higher among patients those with more aggressive tumors. Thus, in some cases, with disease response and overall survival >1 year (53). subclonal mutations may not drive ICB resistance, but rather The link between tumor mutational burden and response may simply reflect heavily treated, refractory disease. In other to ICB has also been demonstrated for agents targeting cases, subclonal mutations may be a surrogate for the ability the PD-1/PD-L1 axis. In patients with non–small cell lung of the tumor to achieve immune escape, and in yet other con- cancer treated with the anti–PD-1 antibody pembrolizu texts, subclonal mutations may actively dampen the antitu- mab, higher nonsynonymous tumor mutational burden mor immune response by detracting from the host immune was associated with improved response and longer progres- response to clonal neoantigens (61). Conversely, it is even pos- sion-free survival (54). The association between mutational sible that under certain circumstances, subclonal mutations burden and ICB response has now been described in several could promote antitumor immunity through mechanisms cohorts; however, it is becoming increasingly clear that such as epitope spreading (62). high mutational burden alone is not sufficient to drive ICB Finally, evidence to date suggests that the vast major- response. ity of predicted neoantigens result from mutations that are unique to a specific tumor and do not typically involve known oncogenes (55). In addition, it is also worth noting TUMOR MUTATION BURDEN that mutations can also stimulate an immune response CORRELATES WITH PREDICTED through neoantigen-independent mechanisms. For example, NEOANTIGENS AND IMMUNE mutations can alter the immune environment by inducing INFILTRATION changes in gene expression or eliciting an unfolded protein Although there is a correlation between tumor muta- response (63). tional burden and likelihood of response to ICB, there The associations between mutational burden or neo- is no definitive threshold mutational burden that sepa- antigen load and ICB response may be explained by the rates ICB responders from nonresponders. Indeed, there are relationships between each of these factors with intratu- numerous examples of tumors with very few mutations that moral T-cell populations. In melanoma and other solid respond robustly to ICB as well as tumors with many muta- tumors, intratumoral CD8+ T-cell infiltration, both before tions that show no response. Thus, despite the association and during treatment, has been associated with response to between tumor mutational burden and ICB response, a criti- ICB (64). CD4+ T cells are also present within tumors and cal challenge is identifying which mutations drive antitumor contribute to antitumor immune activation by recognizing immune response. MHC class II–bound neoantigens (65–67). Furthermore, Acquired (somatic) mutations in the exome have the total mutation burden and predicted neoantigens are also potential to manifest as changes at the protein level. Mutant associated with T-cell cytolytic activity (as defined by tran- proteins may be processed by the proteasome, and the result- script levels of granzyme A and perforin), further support- ant peptide fragments are bound by MHC class I molecules ing the notion that neoantigens can drive cytotoxic T-cell and presented on the cell surface (55). Several bioinformatics responses (68). approaches have been developed to predict tumor neoanti- In addition to T-cell infiltration and cytolytic activity, gens based on predicted MHC class I binding, T cell–receptor T-cell diversity has been shown to correlate with tumor binding, and patient HLA type (56–58). In many clinical mutation load and response to ICB (69). In one example, studies, including those that first established the relation- potential immunogenic somatic mutations were identified ship between mutational load and ICB response, predicted on the basis of their co-occurrence with CDR3 sequences of neoantigen load is closely associated with overall mutational tumor-infiltrating T cells, highlighting the utility of bioin- burden and thus also associated with clinical response and formatics approaches to connect genomic alterations with survival outcomes (52–54). immune cell infiltrates. Combining tumor immune features

july 2017 CANCER DISCOVERY | 679

REVIEW Mouw et al.

Immune activating

Sources of tumor Point mutations, DC activation genomic instability indels ↑ PD-1/PD-L1 ↑ Type I IFN Altered protein Proteasomal MHC response Exogenous DNA product processing presentation TCR binding damaging agents T-cell activation (chemo, IR) Cytosolic DNA ATP GTP Endogenous damage P sources (replicative/ cGAS binding TBK1 activation oxidative stress) 2’-3’ cGAMP STING P activation Underlying tumor Immune suppressive IRF3 activation DNA repair defect

Copy-number Immune evasion: P changes ↓ Adaptive immune signaling Type I IFN response ↓ Cytotoxic T-cell activity ↓ Cytokine signaling

Figure 1. DNA damage modulates tumor immunity. cGAS, cGAMP synthase; STING, Stimulator of Interferon Genes; TCR, T-cell receptor.

with genomic characteristics such as mutational load may neoantigen levels than HR-proficient tumors (74). Like- yield a stronger prognostic indicator than mutational load wise, wild-type BRCA1/2 ovarian tumors with another alone (70, 71). genomic event predicted to result in HR loss (such as mutations in RAD51, ATM, or ATR; PTEN deletion; or BRCA1 promoter hypermethylation) had higher predicted TUMOR DNA REPAIR DEFICIENCIES neoantigen levels than tumors predicted to be HR profi- AFFECT RESPONSE TO IMMUNOTHERAPY cient. BRCA1/2-mutated ovarian tumors also had higher Genomic instability is a hallmark of tumors, and muta- levels of CD3+ and CD8+ tumor-infiltrating lymphocytes tions can arise due to increased DNA damage exposure and/ (TIL) as well as higher immunohistochemical levels of or decreased DNA repair capacity. Several reports have now PD-1 and PD-L1 compared with HR-proficient tumors (75, linked a specific DNA damage exposure or a specific DNA 76). Despite this compelling preclinical data, early data repair pathway deficiency with ICB response. For example, from a clinical trial of avelumab (an anti–PD-L1 agent) tumors with a mutational landscape dominated by C>A have not shown improved response among BRCA1/2- transversions, a pattern linked to tobacco exposure (72), were mutated ovarian tumors, although the numbers are small more likely to benefit from ICB, and this genomic smoking and the results are preliminary (77). signature was more predictive of ICB response than patient- Patients with biallelic germline mutations in the base reported smoking history (54). Moreover, in the same study, excision repair (BER) gene MUTYH are at increased risk of several of the patients who achieved durable benefit from developing colorectal cancer. Histologically, MUTYH-associ- ICB had tumors with somatic alterations in genes involved ated tumors show dense lymphocytic infiltration (78), and in DNA replication or repair (such as POLE, POLD1, and tumor sequencing reveals a distinct pattern of C>A transver- MSH2), suggesting that loss of normal DNA repair fidelity sions (79, 80). These findings provide another example of may have contributed to increased mutational burden and a link between a specific DNA repair deficiency and tumor ICB response in these tumors (Fig. 1). immune properties, and raise the possibility that ICB may Additional studies have also shown a similar corre- be a useful treatment strategy in MUTYH-associated colo- lation among alterations in specific DNA repair genes, rectal cancer. mutational burden, and ICB response. In a cohort of 38 Tumors with somatic point mutations in the exonucle- patients with metastatic melanoma treated with pembroli- ase (“proofreading”) domain of polymerase epsilon (POLE) or zumab or nivolumab, 6 of 21 ICB responders harbored a polymerase delta (POLD1), the two polymerases responsible predicted deleterious mutation in BRCA2 versus only 1 of for the majority of nuclear DNA replication, have some of the 17 nonresponders (73). Supporting this, BRCA2-mutated highest mutational burdens identified to date (13, 81). These melanomas had significantly higher mutational burdens “ultramutated” tumors are predicted to express many neoan- than BRCA2 wild-type tumors. Among ovarian tumors in tigens, and analysis of endometrial tumors with POLE exonu- The Cancer Genome Atlas (TCGA) dataset, tumors with clease domain mutations has, indeed, shown high levels of TIL BRCA1/2 alterations had significantly higher predicted infiltration and PD-1/PD-L1 expression (Table 1; refs. 82, 83).

680 | CANCER DISCOVERY july 2017 www.aacrjournals.org

DNA Repair Biomarkers of Immunotherapy Response REVIEW

Perhaps driven by genomic instability and increased immu- trials for MSI tumors (Fig. 2). For example, a trial of avelumab nogenicity, improved survival has been reported for POLE- (anti–PD-L1) in MSI-high (MSI-H) or POLE-mutated meta- mutated tumors in the non-ICB setting (13, 84), and recent static endometrial cancer will be opening soon at our institu- case reports also describe dramatic responses to ICB (85, 86). tion (NCT02912572). The most robust current evidence for the association Given its important prognostic implications, many institu- between DNA repair deficiency and ICB activity is in tumors tions have implemented routine MSI testing (using IHC and/ with loss of MMR function (87). Initial evidence for an or PCR-based assays) for all patients with newly diagnosed interaction among MMR deficiency, the immune micro- colorectal and endometrial tumors. In addition, increased use environment, and clinical outcomes came from immuno- of targeted sequencing panels—which can be used to estimate histochemical and genomic studies that showed colorectal mutational load, infer MSI status, and predict clinical benefit tumors with an activated immune microenvironment had to PD-1 blockade—are now becoming standard at many can- improved prognosis and frequently harbored defects in the cer centers (93, 94). MMR pathway (88, 89). Among these MMR-deficient tumors, the activated immune environment was counterbalanced DNA REPAIR FACTORS BEYOND by upregulation of immune checkpoints including PD-1, MUTATIONAL LOAD CAN ALSO AFFECT PD-L1, and CTLA-4 (90). Thus, these tumors appear to avoid ANTITUMOR IMMUNITY host immune-mediated elimination through activation of immune checkpoints, raising the possibility that checkpoint The STING Pathway Is Activated by blockade may represent an effective treatment strategy for DNA-Damaging Agents MMR-deficient tumors. An emerging body of data supports a role for non–neoanti- The first clinical evidence for activity of ICB in MMR- gen-based mechanisms of tumor cell recognition and target- deficient tumors came from a study conducted primarily in ing by the host immune system. The DNA damage response patients with colorectal cancer (91). Interestingly, the earli- (DDR) is directly linked to innate immunity, as cells are adept est trials of ICB did not reveal significant activity in colorec- at sensing damaged and foreign DNA (95). The Stimulator of tal cancer (42, 43). However, a patient who did respond was Interferon Genes (STING) pathway was originally character- later noted to have a tumor with MSI, a marker of MMR ized as a mechanism by which cells sense DNA viruses, but deficiency (92). Based on this finding and the known asso- is also activated in the setting of microbial infections and ciation among MMR deficiency, high somatic point muta- certain autoimmune inflammatory conditions. Now, several tional burden, and prominent T-cell infiltrate, a small phase lines of evidence suggest that the STING pathway also plays II trial was initiated to test the activity of pembrolizumab in a role in tumor detection (96). three cohorts of patients with treatment-refractory disease: The STING pathway is activated when cGAMP synthase (i) mismatch repair deficient (MMRd) colorectal cancer, (ii) (cGAS) interacts with cytosolic DNA and catalyzes the syn- MMR-proficient colorectal cancer, and (iii) MMRd non– thesis of cGAMP, a cyclic dinucleotide that acts as a second colorectal cancer tumors (91). Both the MMRd colorectal messenger to activate STING (Fig. 1; ref. 97). Upon activa- cancer and MMRd non–colorectal cancer cohorts included tion, STING undergoes a conformation change that results patients with inherited germline MMR deficiency (Lynch in its shuttling from the endoplasmic reticulum to perinu- syndrome) as well as patients with sporadic MMRd tumors. clear endosomes, where it activates and is phosphorylated by Among patients with MMRd tumors, the immune-related TBK1. TBK1 also phosphorylates interferon regulatory factor objective response rate was 40% and 71% for patients with 3 (IRF3), which translocates to the nucleus to drive transcrip- colorectal cancer and non–colorectal cancer MMRd tumors, tion of type I IFN genes, including IFNβ (98). respectively, versus 0% in patients with MMR-proficient The host STING pathway appears to be the primary innate colorectal cancer. Within the MMRd colorectal cancer immune sensing pathway for detection of tumors, and STING cohort, patients with Lynch syndrome had lower response pathway activation within antigen-presenting cells (APC) in rates than patients with sporadic MMRd colorectal cancer the tumor microenvironment drives T-cell priming against (3/11 vs. 6/6), raising the possibility that higher background tumor-associated antigens. This is supported by data demon- mutational activity in patients with a germline DNA repair strating that animals deficient inSTING or IRF3 have a defect deficiency such as Lynch syndrome may shape the immune in T-cell priming and fail to reject immunogenic tumors (99). system, resulting in a more immune tolerant microenviron- Although the mechanism has not yet been fully elucidated, ment, reduced immunosuppressive signaling, and decreased current evidence supports a model in which dendritic cells sensitivity to ICB. (DC) engulf dying tumor cells, sense free tumor DNA, and Both the somatic mutational burden and the number of subsequently upregulate type I IFN signaling pathways to predicted neoantigens were higher in MMRd tumors com- activate T cells (100, 101). pared with MMR-proficient tumors, with an average of 1,782 Alterations in the DNA damage response, mediated by versus 73 mutations and 578 versus 21 predicted neoanti- either exposure to cytotoxic agents or loss of normal DNA gens, respectively. Similarly, the density of CD8+ lymphoid repair capacity, may contribute to STING pathway–mediated cells and the fraction of PD-L1–positive cells was higher antitumor immunity. A recent study compared immune in MMRd tumors compared with MMR-proficient tumors. activity in DNA damage response-deficient (DDRD) breast Together, the results of this trial provide compelling evi- tumors with non-DDRD tumors. DDRD tumors were dence that MMR deficiency is a predictive biomarker for ICB defined by a 44-gene expression signature associated with response, and have led to a number of planned and ongoing loss of the S phase–specific DNA damage response and

july 2017 CANCER DISCOVERY | 681

REVIEW Mouw et al.

Tumor type Clinical setting Agents Phase Clinical trial no.

GBM up-front RT + TMZ + pembrolizumab I-II NCT02530502

GBM MGMT methylated RT + TMZ + nivolumab vs. placebo rII NCT02667587

HNSCC intermediate/high risk RT + cisplatin +/– nivolumab I,III NCT02764593

NSCLC metastatic/unresectable pembrolizumab + cytotoxic/targeted agents I-II NCT02039674

NSCLC resectable Stage IIIA cisplatin + docetaxel + durvalumab II NCT02572843

Breast advanced BRCA-mutated pembrolizumab II NCT03025035

Breast metastatic TNBC pembrolizumab + capecitabine or paclitaxel II NCT02734290

Esophageal resectable neoadjuvant durvalumab + chemoRT I-II NCT02735239

ipilumimab + nivolumab vs. 5-FU + oxaliplatin Gastric advanced/metastatic III NCT02872116 vs. 5-FU + oxaliplatin + nivolumab

Pancreatic unresectable nonmetastatic SBRT + durvalumab vs. tremelimumab vs. both Ib NCT02868632

Colorectal metastatic MSI-H pembrolizumab vs. FOLFOX/FOLFIRI III NCT02563002

Endometrial/ unresectable/metastatic with nivolumab + ipilimumab II NCT02982486 sarcoma somatic MMR deficiency

persistent/recurrent Endometrial pembrolizumab II NCT02899793 hypermutated or ultramutated

Stage 3-4 previously untreated Ovarian platinum-based chemotherapy +/– avelumab III NCT02718417 epithelial histology

platinum resistant/refractory PEGylated liposomal Ovarian III NCT02580058 epithelial histology doxorubicin +/– avelumab

muscle-invasive/ Bladder durvalumab + RT Ib-II NCT02891161 locally advanced

Prostate mCRPC with DNA repair defects nivolumab II NCT03040791

Figure 2. Representative clinical trials of ICB agents in combination with DNA-damaging agents or in DNA repair-deficient settings. 5-FU, 5-fluorouracil; HNSCC, head and neck squamous cell carcinoma; mCRPC, metastatic castration-resistant prostate cancer; NSCLC, non–small cell lung cancer; RT, radiation therapy; SBRT, stereotactic body radiation therapy; TMZ, temozolomide; TNBC, triple-negative breast cancer. improved response to DNA-damaging chemotherapy (102). also been observed following exposure to other clinically rel- In the study, DDRD tumors had increased IFN-related evant agents, including etoposide, camptothecin, mitomycin gene expression compared with non-DDRD tumors as well C, and adriamycin (106). Interestingly, STING activation also as increased levels of CD4+ and CD8+ T cells in the tumor occurs following Cre-mediated DNA cutting (107), raising and stroma (103). Subsequent cell-based assays demon- the possibility that in vivo gene-editing techniques that gener- strated that expression of the chemokines CXCL10 and ate targeted DNA lesions, such as Cre/loxP, CRISPR/Cas9, CCL5, which play a key role in CD4+ and CD8+ T-cell and TALEN systems, may be accompanied by activation of chemotaxis, increased following siRNA-mediated depletion the innate immune system and an immune response against of individual DNA repair genes (such as BRCA1, BRCA2, the edited cell. and FANCD2). Increased IRF3 and TBK1 phosphoryla- The STING pathway relies on activation by cytosolic tions were observed in BRCA1/2-deficient compared with DNA, and increased levels of cytosolic DNA are present in BRCA1/2-corrected cell lysates, and conditioned media BRCA1/2- or ATM-deficient cell lines compared with their from BRCA1/2-mutant or BRCA1/2-depleted cells led to wild-type counterparts (103, 104). DNA-damaging agents increased migration of peripheral lymphocytes. Similar such as cisplatin and etoposide can also increase cytosolic STING-mediated upregulation of IFN signaling has been DNA levels in the absence of known DNA repair defects observed in cells from Ataxia-Telangiectasia (AT) patients (108). However, the mechanism by which this free DNA and ATM−/− mice (104). arises is currently not understood. It is possible that reestab- In addition to being upregulated in the setting of DNA lishing DNA replication at stalled or damaged forks during S repair deficiency, the STING pathway is activated following phase and/or gap synthesis mediated by DNA damage toler- exposure to DNA-damaging agents. Chemotherapy-induced ance pathways during G1 phase may liberate free DNA, and genotoxic stress drove a type I IFN response across a panel of there is evidence that canonical DNA repair proteins such as breast cancer cell lines, and STING pathway silencing abro- MRE11 play a role in cytosolic DNA processing and cGAS gated this response (105). Damage-induced IFN response has activation (109).

682 | CANCER DISCOVERY july 2017 www.aacrjournals.org

DNA Repair Biomarkers of Immunotherapy Response REVIEW

Not all activities of the STING pathway exert an antitu- outcomes was demonstrated in two melanoma cohorts treated mor effect. The STING pathway plays an important role with anti–CTLA-4 therapy (114, 115). Moreover, combining in promoting inflammation-induced tumorigenesis through SCNA status with tumor mutational burden provided better augmentation of inflammatory cytokine signaling, and predictive power than either factor alone. STING−/− mice are resistant to inflammation-induced skin In these analyses, the effect of SCNA burden on survival cancers (108). STING pathway activation also drives PD-L1 appeared to be more prominent in patients receiving anti– expression on cancer cells and infiltrating lymphocytes fol- CTLA-4 therapy than in separate cohorts of patients not treated lowing DNA damage, thereby dampening immune-mediated with ICB. However, given that aneuploidy is associated with tumor killing despite increased numbers of CD4+ and CD8+ worse prognosis in many tumor settings (independent of treat- T cells (110, 111). This observation provides a potential ment), similar analyses in randomized trials of ICB will help to biological rationale for combining a STING-inducing DNA- clarify the extent to which the lack of ICB response contributes damaging agent (such as cisplatin) with anti–PD-1/PD-L1 to worse outcomes in the high-SCNA setting. In addition, fur- therapy (discussed below). ther defining the role of factors such as the tumor–stroma ratio Together, these findings are consistent with a model in which and aneuploidy characteristics (i.e., extent of chromosomal a shift in the balance of DNA damage and repair driven by gain/loss) will be required to more fully understand the link either exposure to exogenous DNA-damaging agents or loss between aneuploidy and the antitumor immune response. of a DNA repair pathway can stimulate a STING-mediated Taken together, these data reveal a relationship between innate immune response. Given the potent antitumor immune another manifestation of tumor genomic instability—ane- response driven by STING, direct STING activation represents uploidy—and response to immune checkpoint blockade. an attractive therapeutic strategy, and several cyclic dinucleotide When combined with previous studies that show a posi- mimetics that activate STING have demonstrated activity in pre- tive correlation between point mutational burden and ICB clinical studies (101, 112). Given that STING signaling activates response, tumors that would be predicted to have the best type I IFN signaling, tumors that lack baseline type I IFN signal- response to ICB are those with high mutational burden but ing may be ideal targets for treatment with a STING agonist, and few SCNAs. POLE-mutated tumors are an extreme example STING agonists have been shown to promote IFN signaling and of this genomic context, as they have extremely high point extend survival in two AML mouse models (113). mutational burdens but are typically near-diploid, and several impressive responses of POLE-mutated tumors to ICB have Tumor Aneuploidy Is Associated with been reported (discussed above). Immune Evasion In addition to driving increased point mutational burden, genomic instability can also result in gain or loss of chro- INTERACTIONS BETWEEN THE mosomal segments or entire chromosomes. Aneuploidy is IMMUNE SYSTEM AND DNA DAMAGE/ a common feature of tumors, and recent studies investigat- REPAIR CAN MEDIATE RESISTANCE ing the role of somatic copy-number alterations (SCNA) on As clinical implementation of ICB therapy continues tumor properties have revealed an apparent link between to expand and collective experience grows, examples of SCNAs and immune suppression (Fig. 1). Analyzing more acquired resistance to ICB have emerged. Numerous mech- than 5,000 TCGA tumors across 12 tumor types, Davoli anisms have been described and are reviewed in detail and colleagues noted a correlation between high levels of elsewhere (38, 116). Examples include upregulation of SCNAs (gain or loss) and reduced expression of cytotoxic inhibitory signaling through indole amine 2,3-dioxygenase immune cell markers (114). Although there was a positive (IDO), PD-L1, or regulatory T cells (Treg; refs. 110, 117); correlation between SCNAs and tumor mutational burden stabilization of PD-L1 through disruption of the 3′-UTR in most tumor types, and although tumor mutational bur- (118); and upregulation of alternative immune checkpoints den has been associated with immune activation in several such as TIM3 (119). tumor contexts (as discussed above), increased SCNAs cor- In addition to acquired resistance to ICB, some tumors related with reduced immune activation in all tumor types display intrinsic resistance. Clinically, this has been evident except brain tumors. The association between SCNAs and in the earliest trials of ICB agents, as only a subset of reduced immune activation was driven primarily by arm- patients respond to primary ICB. Several recent studies have level and whole-chromosome gain or loss rather than by attempted to identify genomic features that mediate innate gain or loss of focal chromosomal segments, suggesting that resistance to ICB. In patients with melanoma, innate resist- global gene dosage effects rather than expression changes ance to PD-1 blockade has been associated with a tran- in single genes may be responsible for altering the immune scriptional signature characterized by upregulation of genes landscape. involved in mesenchymal transition and extracellular matrix Analysis of clinical cohorts receiving ICB agents have revealed remodeling (73). WNT/β-catenin pathway activity has also similar trends. In patients with metastatic melanoma treated been associated with the absence of a T-cell gene expression with anti–CTLA-4 therapy followed by anti–PD-1 therapy, the signature and lack of response to anti–CTLA-4/anti–PD-L1 burden of copy-number loss was higher in pretreatment biop- therapy in melanoma (120), and CTNNB1 mutations (which sies from patients who did not respond to either anti–CTLA-4 can drive WNT/β-catenin pathway activity) were associated or anti–PD-1 therapy than in anti–CTLA-4 responders (115). with lower neoantigen load among copy number–low/endo- Similarly, an association between increased SCNAs and worse metrioid endometrial tumors (121). Loss of PTEN has also

july 2017 CANCER DISCOVERY | 683

REVIEW Mouw et al. been associated with decreased T-cell infiltration and worse ICB resistance involving gene mutation(s) will continue to outcomes for patients with melanoma treated with anti– emerge as ICB experience grows. PD-1 therapy (122). The role of the immune microenvironment in mediat- COMBINING DNA DAMAGE AND ing resistance to DNA-damaging agents has recently come REPAIR–BASED THERAPIES WITH into focus. Fibroblasts are a major cellular component of IMMUNOTHERAPY the tumor microenvironment, and a recent study using mouse models of high-grade epithelial ovarian cancer (HG- DNA-Damaging Chemotherapy EOC) revealed that fibroblasts mediate cisplatin resistance Traditionally, most conventional chemotherapies, includ- through release of thiols such as glutathione and cysteine, ing direct DNA-damaging agents, have been considered and that this protective effect is countered by CD8+ T to be immunosuppressive, and lymphopenia remains one cell–mediated IFNγ signaling (123). In a clinical cohort of the most common dose-limiting toxicities of cytotoxic of patients with HG-EOC, stromal fibroblasts were nega- chemotherapy. However, an increasing body of empiric and tively associated with chemotherapy response and patient experimental evidence now suggests that some chemothera- survival, whereas CD8+ T-cell levels were associated with pies, delivered at standard doses, can promote immunogenic improved treatment response and overall survival. Thus, tumor cell death and shape the tumor microenvironment to an additional mechanism through which ICB may promote promote antitumor immunity. tumor cell killing is by overcoming protective effects medi- The first mechanism by which chemotherapy has been ated by stromal cells. shown to activate the host immune system is through induction of immunogenic cell death pathways (125). Unlike apoptosis, which is typically considered to be nonimmuno- RELIABLE DNA REPAIR BIOMARKERS genic, chemotherapy can result in cell killing accompanied ARE NEEDED TO IDENTIFY PAST AND by release of tumor cell antigens. Chemotherapy-induced cel- PRESENT DNA REPAIR DEFICIENCY lular stress promotes surface expression and secretion of dan- IN TUMORS ger-associated molecular patterns (DAMP), which increase Whereas resistance to DNA repair–based therapies is the cell’s immunogenicity (100). In addition to cytosolic often driven by restoration of DNA repair pathway func- DNA, a potent DAMP, a variety of cellular proteins have also tion (such as restoration of HR in tumors with acquired been found to act as DAMPs, including High Mobility Group PARP inhibitor resistance), reversal of an underlying tumor Box 1 (HMGB1), calreticulin, hyaluronan, and heat-shock DNA repair defect has not been described as a mechanism proteins (125). Released DAMPs bind receptors on cancer and of resistance to ICB. Although a DNA repair deficiency stromal cells and elicit a host immune response that resem- may contribute to ICB sensitivity through generation of bles the response to pathogens. DAMP activation promotes tumor-specific neoantigens, correcting an underlying DNA secretion of type I IFN and other chemokines, which are repair defect would not reverse the hundreds or thousands required by DCs to activate tumor-specific CD8+ T cells (126). of somatic alterations that had accumulated across the life- The importance of DAMP-mediated immune activation has time of a cancer cell. been demonstrated in studies linking polymorphisms in This principle highlights one of the fundamental limita- DAMP receptors such as toll-like receptor 4 (TLR4) and the tions of using features such as tumor mutational burden or purinergic receptor P2FX7 with decreased response to DNA- mutational signatures to define the DNA repair status of a damaging agents and worse prognosis in several tumor types tumor: Mutational burdens (and mutational signatures) rep- (127, 128). resent a “historical” record of DNA damage and repair events Numerous chemotherapy classes have been shown to in a cell, but do not reflect the current DNA repair functional induce immunogenic cell death (129). For example, status. Therefore, developing and validating assays that pro- increased numbers of TILs were observed following neoad- vide information regarding real-time DNA repair function juvant paclitaxel in a cohort of patients with breast cancer, (such as expression-based or functional tests) remains an and the extent of TIL response correlated with clinical important challenge. response (130). Anthracyclines activate expression of TLR3 Although a clear example has not yet been described, it is and type I IFN secretion, resulting in immunogenic tumor plausible that a specific somatic mutation responsible for cell death (131), and a type I IFN gene signature predicted generating a neoantigen that elicits a strong host immune response to anthracycline therapy in several cohorts of response could be reversed or silenced as a mechanism to patients with breast cancer (132). Recently, anthracycline- decrease tumor immunogenicity (if the mutation is not a can- induced antitumor immunity was shown to require for- cer driver). Along these lines, deletion of large chromosomal myl peptide receptor 1 (FPR1) to mediate the interaction segments has been described as a mechanism to eliminate between dying cancer cells and host T cells (133). Elegant clonal neoantigens (60). Perhaps, the clearest examples of work in mice has shown that autophagy is required for coding mutations leading to clinical ICB resistance are trun- immune cell recruitment following chemotherapy and that cating mutations in JAK1 or JAK2 that result in abrogation of suppression of autophagy inhibits release of the inflam- IFNγ-mediated signaling and a truncating mutation in β-2- masome inducer ATP from dying tumor cells (134). DNA- microglobulin (B2M) that results in loss of cancer cell MHC damaging chemotherapy can also increase expression of class I expression (124). It is likely that novel mechanisms of MHC class I and cancer-testis antigens as well as decrease

684 | CANCER DISCOVERY july 2017 www.aacrjournals.org

DNA Repair Biomarkers of Immunotherapy Response REVIEW expression of inhibitory mediators such as PD-L1 from the using a phase dosing schedule (ipilimumab delivered with cancer cell surface (135, 136). cycles 3–6 of carboplatin/paclitaxel) resulted in improved A second mechanism by which DNA-damaging chemo- immune-related progression-free survival (PFS), whereas therapies can elicit antitumor immunity is through effects on the addition of ipilimumab using a concurrent schedule the tumor microenvironment, including immune-regulatory (delivered with cycles 1–4 of carboplatin/paclitaxel) was not cell activity and the tumor vasculature. Numerous feed- significantly better than carboplatin/paclitaxel alone (150, back mechanisms exist to curtail the host immune response, 151). and chemotherapies have been shown to downregulate these Numerous combination chemotherapy–immunotherapy inhibitory signals in several settings. For instance, drugs such trials are now under way and are testing a variety of agents as gemcitabine, cyclophosphamide, paclitaxel, fludarabine, in different dosing and timing regimens (Fig. 2). Given the and 5-fluorouracil have each been shown to suppress Treg or complex interaction between DNA-damaging agents and myeloid-derived suppressor cell (MDSC) function in experi- immune function, it is likely that the optimal timing of mental models (137–140). Immune activation can also be DNA-damaging chemotherapy with immune checkpoint achieved through upregulation of DC function, and chemo- blockade (or other immune-directed agents) will vary across therapy agents such as cyclophosphamide have been shown disease settings. For example, ICB prior to chemotherapy to increase the number and activity of DCs (141, 142). In may improve response by reducing the impact of chem- many cases, these effects appear to be dependent on the dose otherapy-induced PD-L1 upregulation in some settings, and timing of chemotherapy administration, and additional whereas in other instances, DNA damage–induced tumor studies are needed to determine if they are active at clinically immunogenicity may be required to drive subsequent ICB relevant doses and schedules. response. Given the interplay between DNA-damaging agents and the tumor immune response, it is perhaps not surprising that Ionizing Radiation several lines of evidence now suggest cytotoxic chemotherapy The interaction between the immune system and ionizing can sensitize tumors to ICB. For example, pretreatment with radiation has been appreciated for many decades. Thera- oxaliplatin and cyclophosphamide was sufficient to induce peutic radiation creates numerous types of DNA damage, sensitivity to host T-cell immunity in a lung adenocarcinoma including double-strand breaks (DSB; ref. 152). Historically, mouse model (143). Similarly, decitabine enhanced lympho- radiation was associated with immunosuppression due to cyte function and synergized with CTLA-4 blockade in ovarian the exquisite sensitivity of lymphocytes to DNA damage– cancer (144). Furthermore, the combination of gemcitabine induced apoptosis (153). However, technical improvements and CTLA-4 blockade induced robust antitumor immune in radiation delivery now allow high doses of radiation to responses in two nonimmunogenic lung cancer models be delivered to tumors while avoiding excessive bone mar- (145), and CTLA-4 blockade reduced tumor cell repopulation row exposure, and recently focus has turned to the potential between cisplatin cycles in a murine mesothelioma model therapeutic interactions between radiation and the immune (146). Reciprocally, knockdown of IDO, a negative immune system. regulator, sensitizes cells to gemcitabine and methoxyamine, The immune system can affect radiation-associated tumor a novel inhibitor of BER (147). control both locally (within the radiation field) and distantly Clinical attempts to combine conventional chemotherapy (at unirradiated tumor sites). Radiation has been shown with immune-modulating agents date back more than 20 to have numerous immunostimulatory effects on the local years. Several “biochemotherapy” approaches involving con- tumor environment (154), including a dose-dependent current delivery of cytototoxic chemotherapy with immune- upregulation of MHC class I (155, 156) and co-stimulatory stimulating agents such as IL2 and/or IFNα have been molecules such as CD86 and CD70 on DCs (157). Radiation investigated, primarily in metastatic melanoma. Although activates chemokine release that stimulates DCs (158), pro- some combinations appeared to be active and several trials motes cross-presentation of tumor antigens (159), attracts showed an improvement in some disease-related endpoints, TILs (160), and enhances TIL extravasation via upregulation the lack of overall survival benefit and concerns regarding of cell adhesion molecules (161). Additionally, radiotherapy toxicity limited widespread clinical incorporation of these stimulates release of DAMPs such as HMGB1 (162) and approaches (148, 149). increases FAS expression to promote caspase-induced apop- The first clinical trial comparing ICB plus chemotherapy tosis (163, 164). with chemotherapy alone was reported in 2011 and included Case reports of systemic responses following focal radia- patients with metastatic melanoma randomized to dacar- tion—the so-called “abscopal effect”—date back more than bazine with or without ipilimumab (41). The addition of ipili- half a century (165, 166). However, despite intense interest, mumab led to an improvement in median overall survival of the molecular underpinnings of the abscopal effect remain approximately 2 months; however, it did not appear that the incompletely understood. Some of the earliest experimental combination provided a benefit compared with ipilimumab evidence for the role of the immune system in mediating a alone and may have contributed to an increase in observed radiation-induced abscopal effect came from experiments hepatic toxicity. performed in mice bearing bilateral syngeneic tumors. Treat- Similar combination trials of chemotherapy and CTLA-4 ment with FLT3-ligand increased the DC population and blockade have been reported in lung cancer. In two studies, promoted regression of an unirradiated tumor following the addition of ipilimumab to carboplatin and paclitaxel radiation of the contralateral tumor (167). One of the first

july 2017 CANCER DISCOVERY | 685

REVIEW Mouw et al. attempts to harness the immune system to increase the effi- DNA Repair–Targeted Therapies cacy of radiation in a clinical setting involved treatment with Finally, there is emerging evidence that combining ICB granulocyte-macrophage colony-stimulating factor (GM- with DNA repair targeted agents such as PARP inhibi- CSF) and local radiation to a metastatic site of disease, and tors may be a useful therapeutic strategy. Administration abscopal responses were observed in approximately 25% of of BMN 673, a PARP1 inhibitor, increased intratumoral patients (168). CD8+ T cells and drove production of IFNγ and TNFα in An abscopal effect has also been observed in patients syngeneic BRCA1-deficient murine ovarian tumors (181), receiving localized radiation and immune checkpoint block- and adding CTLA-4 blockade to PARP inhibition fur- ade. For example, a patient with metastatic melanoma receiv- ther increased IFNγ production and T-cell activation and ing anti–CTLA-4 therapy had marked regression of multiple extended survival compared with PARP inhibition alone metastatic lesions following hypofractionated radiation to a (182). However, PARP inhibition may also induce immu- symptomatic paraspinal mass (169). Postradiation antibody nosuppressive effects such as upregulation of PD-L1 (183), titers against the cancer-testis antigen NY-ESO-1 increased providing further rationale for combining ICB with PARP 30-fold relative to preradiation levels, and changes in inhibition. immune cell profiles were consistent with radiation-induced Further work is needed to uncover the mechanisms by T-cell activation. Numerous additional examples of shrink- which PARP inhibitors or other DNA repair–directed agents age of nonirradiated lesions in patients receiving combined (such as ATM, ATR, or DNA-PKcs inhibitors) modulate the radiation and anti–CTLA-4 therapy have now been reported tumor immune environment and affect sensitivity to ICB. (170, 171). However, it is reasonable to hypothesize that genomic insta- Several studies have now begun to unravel the mechanisms bility induced by disruption of normal DNA repair path- by which ICB increases radiation sensitivity and improves way function could result in increased tumor mutational radiotherapy-mediated tumor control. Radiation increases burden, neoantigens, and/or STING pathway activation, PD-L1 expression on tumor cells, and this inhibitory effect all of which could contribute to heightened ICB sensitivity. can be overcome with PD-L1 blockade (172, 173). An elegant Several clinical trials are now under way to test the safety study showed that radiation, anti–CTLA-4, and anti–PD-1/ and efficacy of combinations of DNA repair–targeted agents PD-L1 agents function in nonredundant ways to activate the with ICB agents in both DNA repair–deficient and DNA immune system (174). Data from patients with melanoma repair–proficient settings (Table 2; ref. 184). As clinical and preclinical melanoma models suggest that although experience with both DNA repair–targeted agents and ICB radiation diversifies the T-cell repertoire and anti–CTLA-4 agents grows, and as the multifaceted cellular interactions therapy inhibits Tregs, anti–PD-L1 therapy is necessary to between DNA repair and the immune system come into overcome resistance driven by increased PD-L1 expression on focus, it is likely that combination approaches will continue tumor cells. to emerge. Despite the plethora of mechanisms by which radiotherapy appears to stimulate the immune system, conventionally fractionated radiotherapy alone does not typically produce an abscopal effect, suggesting that the immunostimulatory CONCLUSIONS AND ONGOING EFFORTS effects of radiotherapy that have been extensively charac- Genomic instability is a hallmark of tumors and under- terized in preclinical systems are insufficient to overcome lies many fundamental cancer cell properties. Recently, the immunosuppressive microenvironment in most human it has become clear that DNA damage and repair have a tumors. In addition to upregulation of PD-L1 on tumor cells, major impact on the interaction between the tumor and the radiation can activate Tregs in some settings (175, 176) and immune system, and furthermore that the DNA damage and dampen the immune response via TGFβ-mediated signaling repair landscape has important therapeutic implications in (177). Interestingly, in a mouse model of lung cancer, PD-1 this context. blockade synergized with radiation in the up-front setting Immune-directed therapies are rapidly reshaping the but had no therapeutic benefit (and instead induced T-cell landscape of clinical oncology. Immune checkpoint inhibi- inhibitory markers) in tumors that had relapsed after radia- tors are approved in a variety of settings, and numerous tion (178). additional approvals are inevitable in the coming years. Numerous clinical trials are now under way that combine Although ICB induces durable responses in many patients, radiation with ICB in both the up-front and metastatic set- response rates vary significantly both within and across tings (179). The optimal radiation dose, timing, and frac- tumor types. Therefore, a major challenge remains to tionation for maximizing the therapeutic interaction with identify predictive biomarkers that can guide the use of ICB are currently not known, but it is likely that a one-size- ICB agents as monotherapy or as part of a combined treat- fits-all approach will not apply. Given that cellular effects of ment strategy involving DNA-damaging agents or targeted radiation-induced DNA damage are strongly dose- and cell therapies. context–dependent, it is reasonable to assume that the down- Several lines of evidence suggest that DNA repair rep- stream immunologic effects of DNA damage vary depending resents an important biomarker of ICB response. Tumors on the tumor DNA repair landscape, which in turn var- with MMR deficiency have high response rates to ICB, ies widely across histology, molecular subtype, and clinical and the FDA very recently approved use of the anti–PD-1 context (180). agent pembrolizumab in patients with refractory MSI-H or

686 | CANCER DISCOVERY july 2017 www.aacrjournals.org

DNA Repair Biomarkers of Immunotherapy Response REVIEW

Table 2. Trials combining agents targeting DNA repair and ICB

Patients/estimated Study identiﬁ er Agents Class of drugs Design accrual Primary endpoint NCT02657889 Niraparib plus PARP inhibitor plus Phase I/II Recurrent triple nega- RP2D (Phase I) pembrolizumab anti–PD-1 antibody tive breast cancer (TNBC) or ovarian cancer (OC) N = 114 ORR (Phase II) NCT02660034 BGB-290 plus PARP inhibitor plus Phase IA/IB Advanced solid tumors RP2D (Phase I) BGB-A317 anti–PD-1 antibody N = 124 ORR (Phase II) NCT02849496 Veliparib plus PARP inhibitor plus Phase II, Open Label, Unresectable stage PFS atezolizumab anti–PD-L1 antibody Randomized III or IV BRCA1/2- mutated TNBC N = 90 NCT02734004 Olaparib plus PARP inhibitor plus Phase I/II Advanced/recurrent Safety and disease durvalumab anti–PD-L1 antibody solid tumors control rate N = 133 NCT02484404 Olaparib plus PARP inhibitor plus Phase I/II Advanced/recurrent RP2D (Phase I) durvalumab plus anti–PD-L1 antibody solid tumors cediranib plus VEGFR1,2,3 inhibitor N = 338 ORR or PFS (Phase II) NCT02571725 Olaparib plus PARP inhibitor plus Phase I/II Recurrent BRCA1/2 - RP2D (Phase I) tremelimumab anti–CTLA-4 mutated OC antibody N = 50 ORR (Phase II) NCT02953457 Olaparib plus PARP inhibitor plus Phase I/II Recurrent BRCA1/2 - MTD (Phase I) durvalumab plus anti–PD-L1 antibody mutated OC tremelimumab plus anti–CTLA-4 antibody N = 39 PFS (Phase II) NCT02264678 AZD6738 plus ATR inhibitor plus anti– Phase I Advanced head and RP2D durvalumab PD-L1 antibody neck squamous cell carcinoma or non– small cell lung cancer N = 114 (all arms, including other combinations)

Abbreviations: R2PD, recommended phase II dose; ORR, overall response rate; MTD, maximum tolerated dose; PFS, progression-free survival.

MMRd solid tumors. Numerous ongoing studies are now is neither necessary nor suffi cient to drive ICB response, investigating activity of ICB agents in other DNA repair– and it is likely that distinct DNA lesions arising from dif- deficient settings, including tumors with mutations in ferent DNA repair–defi cient backgrounds may produce very BRCA1/2 or POLE. different immunologic effects. A more thorough under- The basis for many of these studies is the observation standing of the impact of genomic instability—in all its that DNA repair defi ciency often leads to increased muta- forms—on ICB response will require contributions from tional load and neoantigen burden, both of which have DNA repair experts, computational biologists, immunolo- been correlated with ICB response in a variety of settings. gists, and clinicians. However, the relationship between DNA repair and ICB Although genomic instability has a clear association with response is clearly more complex. High mutational burden ICB response in some settings, the most powerful tools for

july 2017 CANCER DISCOVERY | 687

REVIEW Mouw et al. predicting ICB response will likely integrate multiple types of 3. Burrell RA, McGranahan N, Bartek J, Swanton C. The causes and data from across platforms. For example, combining genomic consequences of genetic heterogeneity in cancer evolution. Nature instability markers such as mutational burden with tran- 2013;501:338–45. 4. Khanna A. DNA damage in cancer therapeutics: a boon or a curse? scriptional or IHC-based readouts of immune activity such Cancer Res 2015;75:2133–8. as tumor and stromal T-cell populations, IFNγ-related gene 5. Jeggo PA, Pearl LH, Carr AM. DNA repair, genome stability expression, and STING pathway activity may allow for devel- and cancer: a historical perspective. Nat Rev Cancer 2016;16: opment of “immunoscores” with improved sensitivity and 35–42. specificity (70). 6. Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, et al. DNA Ultimately, the most common clinical setting in which ICB damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 2005;434:864–70. agents may be used is in the up-front setting in combination 7. Burrell RA, McClelland SE, Endesfelder D, Groth P, Weller MC, with standard-of-care therapies, which often include DNA- Shaikh N, et al. Replication stress links structural and numerical damaging agents. Little is currently known regarding the cancer chromosomal instability. Nature 2013;494:492–6. mechanistic interactions between DNA-damaging agents and 8. Ceccaldi R, Rondinelli B, D’Andrea AD. Repair Pathway Choices ICB, and both immunosuppressive and immune-activating and Consequences at the Double-Strand Break. Trends Cell Biol properties of DNA-damaging agents have been described. 2016;26:52–64. Progress in this area will be driven both by empiric data from 9. Solimini NL, Xu Q, Mermel CH, Liang AC, Schlabach MR, Luo J, et al. Recurrent hemizygous deletions in cancers may optimize pro- ongoing and planned clinical trials as well as from carefully liferative potential. Science 2012;337:104–9. planned in vitro and in vivo studies designed to unravel the 10. Lawrence MS, Stojanov P, Mermel CH, Robinson JT, Garraway LA, complexities of the interaction between DNA damage and Golub TR, et al. Discovery and saturation analysis of cancer genes immune-modulating agents. across 21 tumour types. Nature 2014;505:495–501. 11. Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, Kros JM, Hainfellner JA, Mason W, Mariani L, et al. MGMT gene Disclosure of Potential Conflicts of Interest silencing and benefit from temozolomide in glioblastoma. N Engl J P.A. Konstantinopoulos is a consultant/advisory board member Med 2005;352:997–1003. for Pfizer, Merck, and Vertex. No potential conflicts of interest were 12. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn disclosed by the other authors. MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005;352:987–96. 13. Cancer Genome Atlas Research N, Kandoth C, Schultz N, Cherniack Grant Support AD, Akbani R, Liu Y, et al. Integrated genomic characterization of K.W. Mouw is funded by The American Society of Radiation endometrial carcinoma. Nature 2013;497:67–73. 14. Cancer Genome Atlas, N. Comprehensive molecular characteriza- Oncology (ASTRO), the Bladder Cancer Advocacy Network (BCAN), tion of human colon and rectal cancer. Nature 2012;487:330–7. and the KL2/Catalyst Medical Research Investigator Training award 15. Yamamoto H, Perez-Piteira J, Yoshida T, Terada M, Itoh F, Imai K, (an appointed KL2 award) from Harvard Catalyst, The Harvard et al. Gastric cancers of the microsatellite mutator phenotype dis- Clinical and Translational Science Center (National Center for play characteristic genetic and clinical features. Gastroenterology Research Resources and the National Center for Advancing Transla- 1999;116:1348–57. tional Sciences, National Institutes of Health Award KL2 TR001100). 16. Diaz-Padilla I, Romero N, Amir E, Matias-Guiu X, Vilar E, Muggia M.S. Goldberg is funded by the Ovarian Cancer Research Fund F, et al. Mismatch repair status and clinical outcome in endometrial Alliance (Liz Tilberis Scholar), the Melanoma Research Alliance cancer: a systematic review and meta-analysis. Crit Rev Oncol Hema- (Team Science Awards), and the Susan F. Smith Center for Women’s tol 2013;88:154–67. Cancers. P.A. Konstantinopoulos is funded by the Department of 17. Ribic CM, Sargent DJ, Moore MJ, Thibodeau SN, French AJ, Gold- Defense Ovarian Cancer Research Program (DOD OCRP), award berg RM, et al. Tumor microsatellite-instability status as a predictor number W81XWH-15-1-0564. A.D. D’Andrea is funded by the Stand of benefit from fluorouracil-based adjuvant chemotherapy for colon Up To Cancer-Ovarian Cancer Research Fund-Ovarian Cancer cancer. N Engl J Med 2003;349:247–57. National Alliance-National Ovarian Cancer Coalition Dream 18. Rebbeck TR, Mitra N, Wan F, Sinilnikova OM, Healey S, McGuf- Team Translational Research Grant (Grant Number: SU2C-AACR- fog L, et al. Association of type and location of BRCA1 and DT16-15). Stand Up To Cancer is a program of the Entertainment BRCA2 mutations with risk of breast and ovarian cancer. JAMA Industry Foundation. Research grants are administered by the 2015;313:1347–61. American Association for Cancer Research, the Scientific Partner 19. King MC, Marks JH, Mandell JB, New York Breast Cancer Study, of SU2C. A.D. D’Andrea was also funded by grants from the U.S. G. Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2. Science 2003;302:643–6. National Institutes of Health (R01DK43889, R37HL052725, 20. Bolton KL, Chenevix-Trench G, Goh C, Sadetzki S, Ramus SJ, Kar- P01HL048546, P50CA168504), the Breast Cancer Research Founda- lan BY, et al. Association between BRCA1 and BRCA2 mutations tion, and the Ludwig Center at Harvard. and survival in women with invasive epithelial ovarian cancer. JAMA 2012;307:382–90. Received February 28, 2017; revised May 5, 2017; accepted May 18, 21. Lord CJ, Ashworth A. BRCAness revisited. Nat Rev Cancer 2016; 2017; published OnlineFirst June 19, 2017. 16:110–20. 22. Nik-Zainal S, Davies H, Staaf J, Ramakrishna M, Glodzik D, Zou X, et al. Landscape of somatic mutations in 560 breast cancer whole- genome sequences. Nature 2016;534:47–54. REFERENCES 23. Patch AM, Christie EL, Etemadmoghadam D, Garsed DW, George J, . 1 Hoeijmakers JH. DNA damage, aging, and cancer. N Engl J Med Fereday S, et al. Whole-genome characterization of chemoresistant 2009; 361:1475–85. ovarian cancer. Nature 2015;521:489–94. 2. Roos WP, Thomas AD, Kaina B. DNA damage and the balance 24. Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, between survival and death in cancer biology. Nat Rev Cancer 2016; et al. Specific killing of BRCA2-deficient tumours with inhibitors of 16:20–33. poly(ADP-ribose) polymerase. Nature 2005;434:913–7.

688 | CANCER DISCOVERY july 2017 www.aacrjournals.org

DNA Repair Biomarkers of Immunotherapy Response REVIEW

25. Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson 45. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, TB, et al. Targeting the DNA repair defect in BRCA mutant cells as et al. Safety and activity of anti-PD-L1 antibody in patients with a therapeutic strategy. Nature 2005;434:917–21. advanced cancer. N Engl J Med 2012;366:2455–65. 26. Scott CL, Swisher EM, Kaufmann SH. Poly (ADP-ribose) polymerase 46. Rosenberg JE, Hoffman-Censits J, Powles T, van der Heijden MS, inhibitors: recent advances and future development. J Clin Oncol Balar AV, Necchi A, et al. Atezolizumab in patients with locally 2015;33:1397–406. advanced and metastatic urothelial carcinoma who have progressed 27. Matulonis UA, Harter P, Gourley C, Friedlander M, Vergote I, following treatment with platinum-based chemotherapy: a single- Rustin G, et al. Olaparib maintenance therapy in patients with plat- arm, multicentre, phase 2 trial. Lancet 2016;387:1909–20. inum-sensitive, relapsed serous ovarian cancer and a BRCA muta- 47. Robert C, Long GV, Brady B, Dutriaux C, Maio M, Mortier L, et al. tion: Overall survival adjusted for postprogression poly(adenosine Nivolumab in previously untreated melanoma without BRAF muta- diphosphate ribose) polymerase inhibitor therapy. Cancer 2016;122: tion. N Engl J Med 2015;372:320–30. 1844–52. 48. Ansell SM, Lesokhin AM, Borrello I, Halwani A, Scott EC, Gutierrez 28. Stover EH, Konstantinopoulos PA, Matulonis UA, Swisher EM. M, et al. PD-1 blockade with nivolumab in relapsed or refractory Biomarkers of Response and Resistance to DNA Repair Targeted Hodgkin’s lymphoma. N Engl J Med 2015;372:311–9. Therapies. Clin Cancer Res 2016;22:5651–60. 49. Ferris RL, Blumenschein G Jr., Fayette J, Guigay J, Colevas AD, Lici- 29. O’Connor MJ. Targeting the DNA Damage Response in Cancer. Mol tra L, et al. Nivolumab for Recurrent Squamous-Cell Carcinoma of Cell 2015;60:547–60. the Head and Neck. N Engl J Med 2016;375:1856–67. 30. Brown JS, O’Carrigan B, Jackson SP, Yap TA. Targeting DNA Repair 50. Reck M, Rodriguez-Abreu D, Robinson AG, Hui R, Csoszi T, in Cancer: Beyond PARP Inhibitors. Cancer Discov 2017;7:20–37. Fulop A, et al. Pembrolizumab versus Chemotherapy for PD- 31. Liu D, Plimack ER, Hoffman-Censits J, Garraway LA, Bellmunt J, L1-Positive Non-Small-Cell Lung Cancer. N Engl J Med 2016;375: Van Allen E, et al. Clinical validation of chemotherapy response 1823–33. biomarker ERCC2 in muscle-invasive urothelial bladder carcinoma. 51. Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, Lao CD, JAMA Oncol 2016;2:1094–6. et al. Combined Nivolumab and Ipilimumab or Monotherapy in 32. Van Allen EM, Mouw KW, Kim P, Iyer G, Wagle N, Al-Ahmadie H, Untreated Melanoma. N Engl J Med 2015;373:23–34. et al. Somatic ERCC2 mutations correlate with cisplatin sensitiv- 52. Snyder A, Makarov V, Merghoub T, Yuan J, Zaretsky JM, Desrichard ity in muscle-invasive urothelial carcinoma. Cancer Discov 2014;4: A, et al. Genetic basis for clinical response to CTLA-4 blockade in 1140–53. melanoma. N Engl J Med 2014;371:2189–99. 33. Ceccaldi R, O’Connor KW, Mouw KW, Li AY, Matulonis UA, 53. Van Allen EM, Miao D, Schilling B, Shukla SA, Blank C, Zimmer L, D’Andrea AD, et al. A unique subset of epithelial ovarian cancers et al. Genomic correlates of response to CTLA-4 blockade in meta- with platinum sensitivity and PARP inhibitor resistance. Cancer Res static melanoma. Science 2015;350:207–11. 2015;75:628–34. 54. Rizvi NA, Hellmann MD, Snyder A, Kvistborg P, Makarov V, Havel 34. Dunn GP, Old LJ, Schreiber RD. The three Es of cancer immunoed- JJ, et al. Cancer immunology. Mutational landscape determines iting. Annu Rev Immunol 2004;22:329–60. sensitivity to PD-1 blockade in non-small cell lung cancer. Science 35. Atkins MB, Lotze MT, Dutcher JP, Fisher RI, Weiss G, Margolin K, 2015;348:124–8. et al. High-dose recombinant interleukin 2 therapy for patients with 55. Schumacher TN, Schreiber RD. Neoantigens in cancer immuno- metastatic melanoma: analysis of 270 patients treated between 1985 therapy. Science 2015;348:69–74. and 1993. J Clin Oncol 1999;17:2105–16. 56. Rajasagi M, Shukla SA, Fritsch EF, Keskin DB, DeLuca D, Carmona 36. Klapper JA, Downey SG, Smith FO, Yang JC, Hughes MS, Kammula E, et al. Systematic identification of personal tumor-specific neoan- US, et al. High-dose interleukin-2 for the treatment of metastatic tigens in chronic lymphocytic leukemia. Blood 2014;124:453–62. renal cell carcinoma: a retrospective analysis of response and sur- 57. Shukla SA, Rooney MS, Rajasagi M, Tiao G, Dixon PM, Lawrence vival in patients treated in the surgery branch at the National Can- MS, et al. Comprehensive analysis of cancer-associated somatic cer Institute between 1986 and 2006. Cancer 2008;113:293–301. mutations in class I HLA genes. Nat Biotechnol 2015;33:1152–8. 37. Sylvester RJ, van der Meijden AP, Witjes JA, Kurth K. Bacillus 58. Yadav M, Jhunjhunwala S, Phung QT, Lupardus P, Tanguay J, Bum- calmette-guerin versus chemotherapy for the intravesical treatment baca S, et al. Predicting immunogenic tumour mutations by com- of patients with carcinoma in situ of the bladder: a meta-analy- bining mass spectrometry and exome sequencing. Nature 2014;515: sis of the published results of randomized clinical trials. J Urol 572–6. 2005;174:86–91; discussion 91–2. 59. McGranahan N, Furness AJ, Rosenthal R, Ramskov S, Lyngaa R, 38. Topalian SL, Drake CG, Pardoll DM. Immune checkpoint blockade: Saini SK, et al. Clonal neoantigens elicit T cell immunoreactivity a common denominator approach to cancer therapy. Cancer Cell and sensitivity to immune checkpoint blockade. Science 2016;351: 2015;27:450–61. 1463–9. 39. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of 60. Anagnostou V, Smith KN, Forde PM, Niknafs N, Bhattacharya R, age. Nature 2011;480:480–9. White J, et al. Evolution of neoantigen landscape during immune 40. Hodi FS, O’Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen checkpoint blockade in non-small cell lung cancer. Cancer Discov JB, et al. Improved survival with ipilimumab in patients with meta- 2017;7:264–76. static melanoma. N Engl J Med 2010;363:711–23. 61. Schreiber RD, Old LJ, Smyth MJ. Cancer immunoediting: integrat- 41. Robert C, Thomas L, Bondarenko I, O’Day S, Weber J, Garbe C, et al. ing immunity’s roles in cancer suppression and promotion. Science Ipilimumab plus dacarbazine for previously untreated metastatic 2011;331:1565–70. melanoma. N Engl J Med 2011;364:2517–26. 62. Vanneman M, Dranoff G. Combining immunotherapy and targeted 42. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, therapies in cancer treatment. Nat Rev Cancer 2012;12:237–51. McDermott DF, et al. Safety, activity, and immune correlates of anti- 63. Grootjans J, Kaser A, Kaufman RJ, Blumberg RS. The unfolded PD-1 antibody in cancer. N Engl J Med 2012;366:2443–54. protein response in immunity and inflammation. Nat Rev Immunol 43. Brahmer JR, Drake CG, Wollner I, Powderly JD, Picus J, Sharfman 2016;16:469–84. WH, et al. Phase I study of single-agent anti-programmed death-1 64. Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert (MDX-1106) in refractory solid tumors: safety, clinical activity, L, et al. PD-1 blockade induces responses by inhibiting adaptive pharmacodynamics, and immunologic correlates. J Clin Oncol immune resistance. Nature 2014;515:568–71. 2010;28:3167–75. 65. Linnemann C, van Buuren MM, Bies L, Verdegaal EM, Schotte R, 44. Hamid O, Robert C, Daud A, Hodi FS, Hwu WJ, Kefford R, et al. Calis JJ, et al. High-throughput epitope discovery reveals frequent Safety and tumor responses with lambrolizumab (anti-PD-1) in recognition of neo-antigens by CD4+ T cells in human melanoma. melanoma. N Engl J Med 2013;369:134–44. Nat Med 2015;21:81–5.

july 2017 CANCER DISCOVERY | 689

REVIEW Mouw et al.

66. Tran E, Turcotte S, Gros A, Robbins PF, Lu YC, Dudley ME, et al. 85. Johanns TM, Miller CA, Dorward IG, Tsien C, Chang E, Perry A, Cancer immunotherapy based on mutation-specific CD4+ T cells in et al. Immunogenomics of hypermutated glioblastoma: a patient a patient with epithelial cancer. Science 2014;344:641–5. with germline POLE deficiency treated with checkpoint blockade 67. Kreiter S, Vormehr M, van de Roemer N, Diken M, Lower M, immunotherapy. Cancer Discov 2016;6:1230–36. Diekmann J, et al. Mutant MHC class II epitopes drive therapeutic 86. Bouffet E, Larouche V, Campbell BB, Merico D, de Borja R, Aronson immune responses to cancer. Nature 2015;520:692–6. M, et al. Immune checkpoint inhibition for hypermutant glioblas- 68. Rooney MS, Shukla SA, Wu CJ, Getz G, Hacohen N. Molecular and toma multiforme resulting from germline biallelic mismatch repair genetic properties of tumors associated with local immune cytolytic deficiency. J Clin Oncol 2016;34:2206–11. activity. Cell 2015;160:48–61. 87. Kelderman S, Schumacher TN, Kvistborg P. Mismatch repair-defi- 69. Li B, Li T, Pignon JC, Wang B, Wang J, Shukla SA, et al. Landscape cient cancers are targets for anti-PD-1 therapy. Cancer Cell 2015; of tumor-infiltrating T cell repertoire of human cancers. Nat Genet 28:11–3. 2016;48:725–32. 88. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce- 70. Galon J, Mlecnik B, Bindea G, Angell HK, Berger A, Lagorce C, et al. Pages C, Tosolini M, Camus M, Berger A, Wind P, et al. Type, den- Towards the introduction of the ‘Immunoscore’ in the classification sity, and location of immune cells within human colorectal tumors of malignant tumours. J Pathol 2014;232:199–209. predict clinical outcome. Science 2006;313:1960–4. 71. Li B, Severson E, Pignon JC, Zhao H, Li T, Novak J, et al. Comprehen- 89. Smyrk TC, Watson P, Kaul K, Lynch HT. Tumor-infiltrating lym- sive analyses of tumor immunity: implications for cancer immuno- phocytes are a marker for microsatellite instability in colorectal therapy. Genome Biol 2016;17:174. carcinoma. Cancer 2001;91:2417–22. 72. Alexandrov LB, Ju YS, Haase K, Van Loo P, Martincorena I, Nik- 90. Llosa NJ, Cruise M, Tam A, Wicks EC, Hechenbleikner EM, Taube Zainal S, et al. Mutational signatures associated with tobacco smok- JM, et al. The vigorous immune microenvironment of microsatellite ing in human cancer. Science 2016;354:618–622. instable colon cancer is balanced by multiple counter-inhibitory 73. Hugo W, Zaretsky JM, Sun L, Song C, Moreno BH, Hu-Lieskovan S, checkpoints. Cancer Discov 2015;5:43–51. et al. Genomic and transcriptomic features of response to anti-PD-1 91. Le DT, Uram JN, Wang H, Bartlett BR, Kemberling H, Eyring AD, therapy in metastatic melanoma. Cell 2016;165:35–44. et al. PD-1 blockade in tumors with mismatch-repair deficiency. 74. Strickland KC, Howitt BE, Shukla SA, Rodig S, Ritterhouse LL, Liu N Engl J Med 2015;372:2509–20. JF, et al. Association and prognostic significance of BRCA1/2-muta- 92. Lipson EJ, Sharfman WH, Drake CG, Wollner I, Taube JM, Anders tion status with neoantigen load, number of tumor-infiltrating RA, et al. Durable cancer regression off-treatment and effective rein- lymphocytes and expression of PD-1/PD-L1 in high grade serous duction therapy with an anti-PD-1 antibody. Clin Cancer Res 2013; ovarian cancer. Oncotarget 2016;7:13587–98. 19:462–8. 75. McAlpine JN, Porter H, Kobel M, Nelson BH, Prentice LM, Kallo- 93. Stadler ZK, Battaglin F, Middha S, Hechtman JF, Tran C, Cercek A, ger SE, et al. BRCA1 and BRCA2 mutations correlate with TP53 et al. Reliable detection of mismatch repair deficiency in colorectal abnormalities and presence of immune cell infiltrates in ovarian cancers using mutational load in next-generation sequencing pan- high-grade serous carcinoma. Mod Pathol 2012;25:740–50. els. J Clin Oncol 2016;34:2141–7. 76. Clarke B, Tinker AV, Lee CH, Subramanian S, van de Rijn M, Turbin 94. Campesato LF, Barroso-Sousa R, Jimenez L, Correa BR, Sabbaga J, D, et al. Intraepithelial T cells and prognosis in ovarian carcinoma: Hoff PM, et al. Comprehensive cancer-gene panels can be used to novel associations with stage, tumor type, and BRCA1 loss. Mod estimate mutational load and predict clinical benefit to PD-1 block- Pathol 2009;22:393–402. ade in clinical practice. Oncotarget 2015;6:34221–7. 77. Disis ML, Patel MR, Pant S, Hamilton EP, Lockhart AC, Kelly 95. Chatzinikolaou G, Karakasilioti I, Garinis GA. DNA damage and K, et al. Avelumab (MSB0010718C; anti-PD-L1) in patients with innate immunity: links and trade-offs. Trends Immunol 2014;35: recurrent/refractory ovarian cancer from the JAVELIN Solid Tumor 429–35. phase 1b trial: Safety and clinical activity. in American Society of 96. Barber GN. STING: infection, inflammation and cancer. Nat Rev Clinical Oncology (ASCO) Annual Meeting. 2016. Chicago, IL. Immunol 2015;15:760–70. 78. Colebatch A, Hitchins M, Williams R, Meagher A, Hawkins NJ, 97. Ablasser A, Goldeck M, Cavlar T, Deimling T, Witte G, Rohl I, et al. Ward RL. The role of MYH and microsatellite instability in the cGAS produces a 2′-5′-linked cyclic dinucleotide second messenger development of sporadic colorectal cancer. Br J Cancer 2006;95: that activates STING. Nature 2013;498:380–4. 1239–43. 98. Chen Q, Sun L, Chen ZJ. Regulation and function of the cGAS- 79. Rashid M, Fischer A, Wilson CH, Tiffen J, Rust AG, Stevens P, STING pathway of cytosolic DNA sensing. Nat Immunol 2016;17: et al. Adenoma development in familial adenomatous polyposis and 1142–9. MUTYH-associated polyposis: somatic landscape and driver genes. 99. Woo SR, Fuertes MB, Corrales L, Spranger S, Furdyna MJ, Leung J Pathol 2016;238:98–108. MY, et al. STING-dependent cytosolic DNA sensing mediates innate 80. Pilati C, Shinde J, Alexandrov LB, Assie G, Andre T, Helias-Rodze- immune recognition of immunogenic tumors. Immunity 2014;41: wicz Z, et al. Mutational signature analysis identifies MUTYH defi- 830–42. ciency in colorectal cancers and adrenocortical carcinomas. J Pathol 100. Klarquist J, Hennies CM, Lehn MA, Reboulet RA, Feau S, Janssen 2017;242:10–15. EM. STING-mediated DNA sensing promotes antitumor and auto- 81. Rayner E, van Gool IC, Palles C, Kearsey SE, Bosse T, Tomlinson I, immune responses to dying cells. J Immunol 2014;193:6124–34. et al. A panoply of errors: polymerase proofreading domain muta- 101. Corrales L, Glickman LH, McWhirter SM, Kanne DB, Sivick KE, tions in cancer. Nat Rev Cancer 2016;16:71–81. Katibah GE, et al. Direct Activation of STING in the tumor micro- 82. van Gool IC, Eggink FA, Freeman-Mills L, Stelloo E, Marchi E, de environment leads to potent and systemic tumor regression and Bruyn M, et al. POLE Proofreading Mutations Elicit an Antitumor immunity. Cell Rep 2015;11:1018–30. Immune Response in Endometrial Cancer. Clin Cancer Res 2015; 102. Mulligan JM, Hill LA, Deharo S, Irwin G, Boyle D, Keating KE, et al. 21:3347–55. Identification and validation of an anthracycline/cyclophospha- 83. Howitt BE, Shukla SA, Sholl LM, Ritterhouse LL, Watkins JC, Rodig mide-based chemotherapy response assay in breast cancer. J Natl S, et al. Association of Polymerase e-Mutated and Microsatellite- Cancer Inst 2014;106:djt335. Instable Endometrial Cancers With Neoantigen Load, Number 103. Parkes EE, Walker SM, Taggart LE, McCabe N, Knight LA, Wilkin- of Tumor-Infiltrating Lymphocytes, and Expression of PD-1 and son R, et al. Activation of STING-dependent innate immune signal- PD-L1. JAMA Oncol 2015;1:1319–23. ing By S-Phase-Specific DNA damage in breast cancer. J Natl Cancer 84. Church DN, Stelloo E, Nout RA, Valtcheva N, Depreeuw J, ter Haar Inst 2017;109. N, et al. Prognostic significance of POLE proofreading mutations in 104. Hartlova A, Erttmann SF, Raffi FA, Schmalz AM, Resch U, Anugula endometrial cancer. J Natl Cancer Inst 2015;107:402. S, et al. DNA damage primes the type I interferon system via the

690 | CANCER DISCOVERY july 2017 www.aacrjournals.org

DNA Repair Biomarkers of Immunotherapy Response REVIEW

cytosolic DNA sensor STING to promote anti-microbial innate 126. Diamond MS, Kinder M, Matsushita H, Mashayekhi M, Dunn GP, immunity. Immunity 2015;42:332–43. Archambault JM, et al. Type I interferon is selectively required by 105. Gaston J, Cheradame L, Yvonnet V, Deas O, Poupon MF, Judde JG, dendritic cells for immune rejection of tumors. J Exp Med 2011;208: et al. Intracellular STING inactivation sensitizes breast cancer cells 1989–2003. to genotoxic agents. Oncotarget 2016;7:77205–24. 127. Apetoh L, Ghiringhelli F, Tesniere A, Obeid M, Ortiz C, Criollo A, et al. 106. Brzostek-Racine S, Gordon C, Van Scoy S, Reich NC. The DNA dam- Toll-like receptor 4-dependent contribution of the immune system age response induces IFN. J Immunol 2011;187:5336–45. to anticancer chemotherapy and radiotherapy. Nat Med 2007;13: 107. Pepin G, Ferrand J, Honing K, Jayasekara WS, Cain JE, Behlke MA, 1050–9. et al. Cre-dependent DNA recombination activates a STING-depend- 128. Ghiringhelli F, Apetoh L, Tesniere A, Aymeric L, Ma Y, Ortiz C, et al. ent innate immune response. Nucleic Acids Res 2016;44:5356–64. Activation of the NLRP3 inflammasome in dendritic cells induces 108. Ahn J, Xia T, Konno H, Konno K, Ruiz P, Barber GN. Inflammation- IL-1beta-dependent adaptive immunity against tumors. Nat Med driven carcinogenesis is mediated through STING. Nat Commun 2009;15:1170–8. 2014;5:5166. 129. Emens LA, Middleton G. The interplay of immunotherapy and 109. Kondo T, Kobayashi J, Saitoh T, Maruyama K, Ishii KJ, Barber GN, chemotherapy: harnessing potential synergies. Cancer Immunol Res et al. DNA damage sensor MRE11 recognizes cytosolic double- 2015;3:436–43. stranded DNA and induces type I interferon by regulating STING 130. Demaria S, Volm MD, Shapiro RL, Yee HT, Oratz R, Formenti SC, trafficking. Proc Natl Acad Sci U S A 2013;110:2969–74. et al. Development of tumor-infiltrating lymphocytes in breast 110. Taube JM, Anders RA, Young GD, Xu H, Sharma R, McMiller TL, cancer after neoadjuvant paclitaxel chemotherapy. Clin Cancer Res et al. Colocalization of inflammatory response with B7-h1 expres- 2001;7:3025–30. sion in human melanocytic lesions supports an adaptive resistance 131. Casares N, Pequignot MO, Tesniere A, Ghiringhelli F, Roux S, mechanism of immune escape. Sci Transl Med 2012;4:127ra37. Chaput N, et al. Caspase-dependent immunogenicity of doxoru- 111. Demaria O, De Gassart A, Coso S, Gestermann N, Di Domizio J, bicin-induced tumor cell death. J Exp Med 2005;202:1691–701. Flatz L, et al. STING activation of tumor endothelial cells initiates 132. Sistigu A, Yamazaki T, Vacchelli E, Chaba K, Enot DP, Adam J, et al. spontaneous and therapeutic antitumor immunity. Proc Natl Acad Cancer cell-autonomous contribution of type I interferon signaling Sci U S A 2015;112:15408–13. to the efficacy of chemotherapy. Nat Med 2014;20:1301–9. 112. Tang CH, Zundell JA, Ranatunga S, Lin C, Nefedova Y, Del Valle JR. 133. Vacchelli E, Ma Y, Baracco EE, Sistigu A, Enot DP, Pietrocola F, Agonist-mediated activation of STING induces apoptosis in malig- et al. Chemotherapy-induced antitumor immunity requires formyl nant B cells. Cancer Res 2016;76:2137–52. peptide receptor 1. Science 2015;350:972–8. 113. Curran E, Chen X, Corrales L, Kline DE, Dubensky TW Jr., Dut- 134. Michaud M, Martins I, Sukkurwala AQ, Adjemian S, Ma Y, Pelle- tagupta P, et al. STING pathway activation stimulates potent immu- gatti P, et al. Autophagy-dependent anticancer immune responses nity against acute myeloid leukemia. Cell Rep 2016;15:2357–66. induced by chemotherapeutic agents in mice. Science 2011;334: 114. Davoli T, Uno H, Wooten EC, Elledge SJ. Tumor aneuploidy cor- 1573–7. relates with markers of immune evasion and with reduced response 135. Vereecque R, Saudemont A, Quesnel B. Cytosine arabinoside to immunotherapy. Science 2017;355:eaaf8399. induces costimulatory molecule expression in acute myeloid leuke- 115. Roh W, Chen PL, Reuben A, Spencer CN, Prieto PA, Miller JP, mia cells. Leukemia 2004;18:1223–30. et al. Integrated molecular analysis of tumor biopsies on sequential 136. Ghebeh H, Lehe C, Barhoush E, Al-Romaih K, Tulbah A, Al-Alwan CTLA-4 and PD-1 blockade reveals markers of response and resist- M, et al. Doxorubicin downregulates cell surface B7-H1 expression ance. Sci Transl Med 2017;9. pii: eaah3560. and upregulates its nuclear expression in breast cancer cells: role 116. Restifo NP, Smyth MJ, Snyder A. Acquired resistance to immuno- of B7-H1 as an anti-apoptotic molecule. Breast Cancer Res 2010; therapy and future challenges. Nat Rev Cancer 2016;16:121–6. 12:R48. 117. Spranger S, Spaapen RM, Zha Y, Williams J, Meng Y, Ha TT. Up- 137. Lutsiak ME, Semnani RT, De Pascalis R, Kashmiri SV, Schlom J, regulation of PD-L1, IDO, and T(regs) in the melanoma tumor Sabzevari H. Inhibition of CD4(+)25+ T regulatory cell function microenvironment is driven by CD8(+) T cells. Sci Transl Med 2013; implicated in enhanced immune response by low-dose cyclophos- 5:200ra116. phamide. Blood 2005;105:2862–8. 118. Kataoka K, Shiraishi Y, Takeda Y, Sakata S, Matsumoto M, Nagano 138. Beyer M, Kochanek M, Darabi K, Popov A, Jensen M, Endl E, et al. S, et al. Aberrant PD-L1 expression through 3′-UTR disruption in Reduced frequencies and suppressive function of CD4+CD25hi multiple cancers. Nature 2016;534:402–6. regulatory T cells in patients with chronic lymphocytic leukemia 119. Koyama S, Akbay EA, Li YY, Herter-Sprie GS, Buczkowski KA, Rich- after therapy with fludarabine. Blood 2005;106:2018–25. ards WG, et al. Adaptive resistance to therapeutic PD-1 blockade is 139. Zhang L, Dermawan K, Jin M, Liu R, Zheng H, Xu L, et al. Dif- associated with upregulation of alternative immune checkpoints. ferential impairment of regulatory T cells rather than effector Nat Commun 2016;7:10501. T cells by paclitaxel-based chemotherapy. Clin Immunol 2008;129: 120. Spranger S, Bao R, Gajewski TF. Melanoma-intrinsic beta-catenin 219–29. signalling prevents anti-tumour immunity. Nature 2015;523:231–5. 140. Vincent J, Mignot G, Chalmin F, Ladoire S, Bruchard M, Chevriaux 121. Shukla SA, Howitt BE, Wu CJ, Konstantinopoulos PA. Predicted A, et al. 5-Fluorouracil selectively kills tumor-associated myeloid- neoantigen load in non-hypermutated endometrial cancers: Cor- derived suppressor cells resulting in enhanced T cell-dependent relation with outcome and tumor-specific genomic alterations. antitumor immunity. Cancer Res 2010;70:3052–61. Gynecol Oncol Rep 2017;19:42–5. 141. Shurin GV, Tourkova IL, Kaneno R, Shurin MR. Chemotherapeutic 122. Peng W, Chen JQ, Liu C, Malu S, Creasy C, Tetzlaff MT, et al. Loss agents in noncytotoxic concentrations increase antigen presenta- of PTEN promotes resistance to T cell-mediated immunotherapy. tion by dendritic cells via an IL-12-dependent mechanism. J Immu- Cancer Discov 2016;6:202–16. nol 2009;183:137–44. 123. Wang W, Kryczek I, Dostal L, Lin H, Tan L, Zhao L, et al. Effector T 142. Nakahara T, Uchi H, Lesokhin AM, Avogadri F, Rizzuto GA, cells abrogate stroma-mediated chemoresistance in ovarian cancer. Hirschhorn-Cymerman D, et al. Cyclophosphamide enhances Cell 2016;165:1092–105. immunity by modulating the balance of dendritic cell subsets in 124. Zaretsky JM, Garcia-Diaz A, Shin DS, Escuin-Ordinas H, Hugo W, lymphoid organs. Blood 2010;115:4384–92. Hu-Lieskovan S, et al. Mutations associated with acquired resistance 143. Pfirschke C, Engblom C, Rickelt S, Cortez-Retamozo V, Garris C, to PD-1 blockade in melanoma. N Engl J Med 2016;375:819–29. Pucci F, et al. Immunogenic Chemotherapy Sensitizes Tumors to 125. Galluzzi L, Buque A, Kepp O, Zitvogel L, Kroemer G. Immuno- Checkpoint Blockade Therapy. Immunity 2016;44:343–54. genic cell death in cancer and infectious disease. Nat Rev Immunol 144. Wang L, Amoozgar Z, Huang J, Saleh MH, Xing D, Orsulic S, 2017;17:97–111. et al. Decitabine enhances lymphocyte migration and function and

july 2017 CANCER DISCOVERY | 691

REVIEW Mouw et al.

synergizes with CTLA-4 blockade in a murine ovarian cancer model. 162. Gameiro SR, Jammeh ML, Wattenberg MM, Tsang KY, Ferrone S, Cancer Immunol Res 2015;3:1030–41. Hodge JW. Radiation-induced immunogenic modulation of tumor 145. Lesterhuis WJ, Salmons J, Nowak AK, Rozali EN, Khong A, Dick enhances antigen processing and calreticulin exposure, resulting in IM, et al. Synergistic effect of CTLA-4 blockade and cancer chem- enhanced T-cell killing. Oncotarget 2014;5:403–16. otherapy in the induction of anti-tumor immunity. PLoS One 163. Garnett CT, Palena C, Chakraborty M, Tsang KY, Schlom J, Hodge 2013;8:e61895. JW. Sublethal irradiation of human tumor cells modulates phe- 146. Wu L, Yun Z, Tagawa T, Rey-McIntyre K, de Perrot M. CTLA-4 block- notype resulting in enhanced killing by cytotoxic T lymphocytes. ade expands infiltrating T cells and inhibits cancer cell repopulation Cancer Res 2004;64:7985–94. during the intervals of chemotherapy in murine mesothelioma. Mol 164. Chakraborty M, Abrams SI, Camphausen K, Liu K, Scott T, Cole- Cancer Ther 2012;11:1809–19. man CN, et al. Irradiation of tumor cells up-regulates Fas and 147. Maleki Vareki S, Chen D, Di Cresce C, Ferguson PJ, Figueredo R, enhances CTL lytic activity and CTL adoptive immunotherapy. Pampillo M, et al. IDO downregulation induces sensitivity to pem- J Immunol 2003;170:6338–47. etrexed, gemcitabine, FK866, and methoxyamine in human cancer 165. Mole RH. Whole body irradiation; radiobiology or medicine? Br J cells. PLoS One 2015;10:e0143435. Radiol 1953;26:234–41. 148. Atkins MB, Hsu J, Lee S, Cohen GI, Flaherty LE, Sosman JA, et al. 166. Kingsley DP. An interesting case of possible abscopal effect in malig- Phase III trial comparing concurrent biochemotherapy with cispl- nant melanoma. Br J Radiol 1975;48:863–6. atin, vinblastine, dacarbazine, interleukin-2, and interferon alfa-2b 167. Demaria S, Ng B, Devitt ML, Babb JS, Kawashima N, Liebes L, with cisplatin, vinblastine, and dacarbazine alone in patients with et al. Ionizing radiation inhibition of distant untreated tumors metastatic malignant melanoma (E3695): a trial coordinated by (abscopal effect) is immune mediated. Int J Radiat Oncol Biol Phys the Eastern Cooperative Oncology Group. J Clin Oncol 2008;26: 2004;58:862–70. 5748–54. 168. Golden EB, Chhabra A, Chachoua A, Adams S, Donach M, Fenton- 149. Keilholz U, Conradt C, Legha SS, Khayat D, Scheibenbogen C, Kerimian M, et al. Local radiotherapy and granulocyte-macrophage Thatcher N, et al. Results of interleukin-2-based treatment in colony-stimulating factor to generate abscopal responses in patients advanced melanoma: a case record-based analysis of 631 patients. with metastatic solid tumours: a proof-of-principle trial. Lancet J Clin Oncol 1998;16:2921–9. Oncol 2015;16:795–803. 150. Lynch TJ, Bondarenko I, Luft A, Serwatowski P, Barlesi F, Chacko 169. Postow MA, Callahan MK, Barker CA, Yamada Y, Yuan J, Kitano S, R, et al. Ipilimumab in combination with paclitaxel and carboplatin et al. Immunologic correlates of the abscopal effect in a patient with as first-line treatment in stage IIIB/IV non-small-cell lung can- melanoma. N Engl J Med 2012;366:925–31. cer: results from a randomized, double-blind, multicenter phase II 170. Golden EB, Demaria S, Schiff PB, Chachoua A, Formenti SC. study. J Clin Oncol 2012;30:2046–54. An abscopal response to radiation and ipilimumab in a patient 151. Reck M, Bondarenko I, Luft A, Serwatowski P, Barlesi F, Chacko R, with metastatic non-small cell lung cancer. Cancer Immunol Res et al. Ipilimumab in combination with paclitaxel and carboplatin as 2013;1:365–72. first-line therapy in extensive-disease-small-cell lung cancer: results 171. Chandra RA, Wilhite TJ, Balboni TA, Alexander BM, Spektor A, Ott from a randomized, double-blind, multicenter phase 2 trial. Ann PA, et al. A systematic evaluation of abscopal responses following Oncol 2013;24:75–83. radiotherapy in patients with metastatic melanoma treated with 152. Ward J. Nature of lesions formed by ionizing radiation, in DNA ipilimumab. Oncoimmunology 2015;4:e1046028. damage and repair. In: Nickoloff J, Hoekstra M, editors. Springer; 172. Deng L, Liang H, Burnette B, Beckett M, Darga T, Weichselbaum 1998. p. 65–84. RR, et al. Irradiation and anti-PD-L1 treatment synergistically pro- 153. Order SE. The effects of therapeutic irradiation on lymphocytes and mote antitumor immunity in mice. J Clin Invest 2014;124:687–95. immunity. Cancer 1977;39:737–43. 173. Dovedi SJ, Adlard AL, Lipowska-Bhalla G, McKenna C, Jones S, 154. Sharabi AB, Lim M, DeWeese TL, Drake CG. Radiation and check- Cheadle EJ, et al. Acquired resistance to fractionated radiother- point blockade immunotherapy: radiosensitisation and potential apy can be overcome by concurrent PD-L1 blockade. Cancer Res mechanisms of synergy. Lancet Oncol 2015;16:e498–509. 2014;74:5458–68. 155. Reits EA, Hodge JW, Herberts CA, Groothuis TA, Chakraborty 174. Twyman-Saint\sVictor C, Rech AJ, Maity A, Rengan R, Pauken KE, M, Wansley EK, et al. Radiation modulates the peptide repertoire, Stelekati E, et al. Radiation and dual checkpoint blockade acti- enhances MHC class I expression, and induces successful antitumor vate non-redundant immune mechanisms in cancer. Nature 2015; immunotherapy. J Exp Med 2006;203:1259–71. 520:373–7. 156. Wan S, Pestka S, Jubin RG, Lyu YL, Tsai YC, Liu LF. Chemothera- 175. Schaue D, Xie MW, Ratikan JA, McBride WH. Regulatory T cells in peutics and radiation stimulate MHC class I expression through radiotherapeutic responses. Front Oncol 2012;2:90. elevated interferon-beta signaling in breast cancer cells. PLoS One 176. Schaue D, Comin-Anduix B, Ribas A, Zhang L, Goodglick L, Sayre 2012;7:e32542. JW, et al. T-cell responses to survivin in cancer patients undergoing 157. Gupta A, Probst HC, Vuong V, Landshammer A, Muth S, Yagita H, radiation therapy. Clin Cancer Res 2008;14:4883–90. et al. Radiotherapy promotes tumor-specific effector CD8+ T cells 177. Young KH, Newell P, Cottam B, Friedman D, Savage T, Baird via dendritic cell activation. J Immunol 2012;189:558–66. JR, et al. TGFbeta inhibition prior to hypofractionated radiation 158. Hallahan DE, Spriggs DR, Beckett MA, Kufe DW, Weichselbaum RR. enhances efficacy in preclinical models. Cancer Immunol Res 2014; Increased tumor necrosis factor alpha mRNA after cellular exposure 2:1011–22. to ionizing radiation. Proc Natl Acad Sci U S A 1989;86:10104–7. 178. Herter-Sprie GS, Koyama S, Korideck H, Hai J, Deng J, Li YY, et al. 159. Sharabi AB, Nirschl CJ, Kochel CM, Nirschl TR, Francica BJ, Velarde Synergy of radiotherapy and PD-1 blockade in Kras-mutant lung E, et al. Stereotactic radiation therapy augments antigen-specific cancer. JCI Insight 2016;1:e87415. PD-1-mediated antitumor immune responses via cross-presentation 179. Kang J, Demaria S, Formenti S. Current clinical trials testing the of tumor antigen. Cancer Immunol Res 2015;3:345–55. combination of immunotherapy with radiotherapy. J Immunother 160. Matsumura S, Wang B, Kawashima N, Braunstein S, Badura M, Cancer 2016;4:51. Cameron TO, et al. Radiation-induced CXCL16 release by breast 180. Demaria S, Formenti SC. Radiation as an immunological adju- cancer cells attracts effector T cells. J Immunol 2008;181:3099–107. vant: current evidence on dose and fractionation. Front Oncol 161. Hallahan D, Kuchibhotla J, Wyble C. Cell adhesion molecules medi- 2012;2:153. ate radiation-induced leukocyte adhesion to the vascular endothe- 181. Huang J, Wang L, Cong Z, Amoozgar Z, Kiner E, Xing D, et al. The lium. Cancer Res 1996;56:5150–5. PARP1 inhibitor BMN 673 exhibits immunoregulatory effects in a

692 | CANCER DISCOVERY july 2017 www.aacrjournals.org

DNA Repair Biomarkers of Immunotherapy Response REVIEW

Brca1(–/–) murine model of ovarian cancer. Biochem Biophys Res cer-associated immunosuppression. Clin Cancer Res 2017. DOI: Commun 2015;463:551–6. 10.1158/1078-0432.CCR-16-3215. 182. Higuchi T, Flies DB, Marjon NA, Mantia-Smaldone G, Ronner L, 184. Yap TA, Krebs MG, Postel-Vinay S, Bang YJ, El-Khoueiry A, Abida Gimotty PA, et al. CTLA-4 blockade synergizes therapeutically with W, et al. Phase I modular study of AZD6738, a novel oral, potent PARP inhibition in BRCA1-deficient ovarian cancer. Cancer Immu- and selective ataxia telangiectasia Rad3-related (ATR) inhibitor in nol Res 2015;3:1257–68. combination (combo) with carboplatin, olaparib or durvalumab 183. Jiao S, Xia W, Yamaguchi H, Wei Y, Chen MK, Hsu JM, et al. in patients (pts) with advanced cancers, in EORTC-NCI-AACR PARP inhibitor upregulates PD-L1 expression and enhances can- Molecular Targets and Cancer Therapeutics. 2016: Munich DE.

july 2017 CANCER DISCOVERY | 693

DNA Damage and Repair Biomarkers of Immunotherapy Response

Kent W. Mouw, Michael S. Goldberg, Panagiotis A. Konstantinopoulos, et al.

Cancer Discov 2017;7:675-693. Published OnlineFirst June 19, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/2159-8290.CD-17-0226

Cited articles This article cites 179 articles, 67 of which you can access for free at: http://cancerdiscovery.aacrjournals.org/content/7/7/675.full#ref-list-1

Citing articles This article has been cited by 41 HighWire-hosted articles. Access the articles at: http://cancerdiscovery.aacrjournals.org/content/7/7/675.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerdiscovery.aacrjournals.org/content/7/7/675. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.