Homologous Recombination Deficiency as a Novel Therapeutic Target in Various Solid Tumors


 Background: Homologous recombination deficiency (HRD) is related to tumorigenesis. Currently, the possibility of HRD as a prognostic biomarker to immune check point inhibitors is unknown.Methods: The status of homologous recombination deficiency (HRD) was assessed with the next-generation sequencing (NGS) TruSightTM Oncology 500 assay in 501 patients with advanced solid tumor including gastrointestinal (GI), genitourinary (GU), or rare cancer. Results: Among the 501 patients, HRD was observed as follows: 74.7% (347/501) patients; GU cancer (92.0%, 23 of 25), colorectal cancer (CRC) (86.1%, 130 of 151), hepatocellular carcinoma (HCC) (83.3%, 10 of 12), pancreatic cancer (PC) (76.2%, 32 of 42), biliary tract cancer (BTC) (75.0%, 36 of 48), sarcoma (65.0%, 39 of 60), melanoma (52.4%, 11 of 21), other GI cancers (50.0%, 11 of 22), and rare cancer (50.0%, 2 of 4). Sixty-five of the 501 patients had received immune checkpoint inhibitors (ICIs) during the course of the disease. Tumor types of 65 patients treated with ICIs are as follows: melanoma (95.2%, 20 of 21), HCC (33.3%, 4 of 12), rare cancer (25.0%, 1 of 4), GC (12.2%, 14 of 116), BTC (10.4%, 5 of 48), and sarcoma (5.0%, 3 of 60). Patients without HRD exhibited an objective response rate (ORR) of 33.3% (4 of 12), and patients with HRD exhibited an ORR of 34.0% (18 of 53). There was no significant difference in ORR between patients with and without HRD (p = 0.967). Progression-free survival (PFS) was 6.5 months (95% CI: 0.000 – 16.175) in patients without HRD and 4.1 months (95% CI: 2.062 – 6.138) in patients with HRD, revealing no statistical significance (p = 0.441). Conclusion: Herein, we reported the status of HRD using a cancer-panel for various solid tumor patients in routine clinical practice and demonstrated that HRD as a single biomarker was not sufficient to predict efficacy of ICIs in solid tumor patients.


Background
After immune checkpoint inhibitors (ICIs) were introduced for treatment of solid tumors, they exhibited improved survival and treatment outcomes compared to traditional non-immune anti-cancer therapies, especially for patients with advanced melanoma, non-small-cell lung cancer (NSCLC), urothelial cancer (UC), renal cell carcinoma (RCC), or other cancer types [1][2][3][4][5][6][7]. However, only some patients achieved a response to ICIs. This indicates the need for further development of immune-relevant biomarkers to identify patients who might bene t from immunotherapy.
The DNA damage repair (DDR) system is essential to maintain the integrity of the genome in organisms.
Genomic alteration due to failure to repair DDR causes tumor initiation. The homologous recombination (HR) pathway has a substantial in uence on genomic integrity and germline mutations in this pathway and is related to tumorigenesis [8][9][10]. HR is one of the major repair mechanisms of DNA double-strand breaks. Homologous recombination de ciency (HRD) is a DNA repair de ciency related to tumorigenesis and causes increased sensitivity to platinum-based chemotherapy and PARP inhibitors [11]. The concept of therapy-directed HRD is approved in ovarian and breast cancers. The mutation in the HR pathway related to BRCA1/2 was used to predict better objective response rates to platinum-based chemotherapy in advanced triple-negative breast cancer [12].
Recently, targeted cancer gene panel assay or NGS for HRD have been performed in clinical settings.
These panels assess genomic pro les including Tumor Mutational Burden (TMB), Microsatellite Instability (MSI), and HRD. To date, the clinical signi cance of gene mutations related to HRD has not been studied well across various solid tumors. Herein, we analyzed the status of HRD using cancer panels for various solid tumor patients in routine clinical practice and determined the value of HRD as a biomarker of response to ICIs.

Patients
Patients with pathologic con rmation of advanced gastrointestinal, GU, or rare cancers at Samsung Medical Center between Oct 2019 and Mar 2020 (n = 501), were prospectively tested for molecular aberrations, including TMB, with the TruSight™ Oncology 500 assay. All study participants provided written informed consent before study entry. The following clinicopathologic characteristics were collected for all patients: age, sex, primary tumor site, number of metastatic sites, site of metastasis, treatment, and survival. The study protocol was approved (#2020-11-151) by the Institutional Review Board of Samsung Medical Center (Seoul, Korea) and was conducted in accordance with the ethical principles of the Declaration of Helsinki and the Korea Good Clinical Practice guidelines. All patients provided written informed consent before enrollment. Written informed consent included disclosure of information, competency to make a decision, and voluntary nature of the decision for the purpose, bene t, and potential risk of this study.

Tumor samples
Samples for analysis were collected from 501 solid tumors and prepared as formalin-xed para nembedded (FFPE) material. The samples were gathered through biopsy at diagnosis, surgical specimen, or repeat biopsy at the time of disease progression; all were obtained before immunotherapy. The types of samples used in the analysis were as follows: biopsied samples (n = 320, 63.9%) and surgically resected samples (n = 181, 36.1%).
TruSight™ oncology 500assay Mann-Whitney test was used to compare the difference between HRD and non-HRD. Kaplan-Meier estimates and log rank tests were used in analysis of all time to event variables, and 95% con dence interval for the median time to event was computed. Table 1 presents the clinical characteristics of the 501 patients included in this study. The median age of the patients was 59.7 years (range, 21-86), and the majority were male (60.3%). The median age of males was 61 years, while that of female was 58 years. The most frequent tumor type was colorectal cancer (n = 151, 30.1%), followed by gastric cancer (n = 116, 23.2%), sarcoma (n = 60, 12.0%), pancreatic cancer (n = 42, 8.4%), genitourinary (GU) cancer (n = 25, 5.0%), other gastrointestinal (GI) tract cancer (n = 22, 4.4%), melanoma (n = 21, 4.2%), hepatocellular carcinoma (HCC) (n = 12, 2.4%), and rare cancer (n = 4, 0.8%). Among the 501 patients, 65 had been treated with immune checkpoint inhibitors (ICIs). Figure 1 shows the distribution of TMB, MSI, and HR de ciencies. All seven patients with MSI were TMB-high and HR-de cient. On the other hand, of 375 patients with HR de ciency, only 54 were con rmed to be TMBhigh or MSI.  Figure 1 presents the distribution relationship with other biomarkers. All MSI were TMB high and HR de ciency. However, some TMB high have no HR de ciency. Figure 2 shows the percentage of con rmed HRD for each tumor type listed in order of high frequency rate. The tumor with the highest frequency of HRD was GU cancer with 92.0% and the lowest frequency was other GI tract cancer (AOV cancer, appendiceal cancer, cecal cancer, duodenal cancer, and GIST) and rare cancer at 50.0%.  (Fig. 3).

Patient characteristics
Additionally, we conducted univariate and multivariate analyses for PFS after ICIs (Table 3). Univariate analysis revealed that smoking, HRD, and tumor mutational burden (TMB) were signi cant prognostic factors for PFS after ICIs. However, in multivariate analysis, TMB was the only meaningful prognostic factor. , and rare cancer (50.0%, 2 of 4). In 65 patients treated with ICIs, there were no signi cant differences for ORR and PFS between patients with and without HRD (p = 0.967 and p = 0.441, respectively). These ndings suggest that HRD as a single biomarker is not su cient to predict the e cacy of ICIs in solid tumor patients.
We analyzed HRD in 501 tumor samples using the NGS panel. The overall frequency of HRD was 74.7% (347/501). This nding was not consistent with other studies about HRD. A previous study reported that the prevalence of HR-DDR mutations was 17.4% in multiple tumor types [13]. This discordance might be caused by different studied genes including different NGS panels and different genes de ning HRD. Detection of HRD by the NGS panel has limitations. There is no established de nition to assess HRD. Therefore, there are many different results among published papers about the prevalence of HRD.
Furthermore, this difference might be caused by discrepancy between measurement of HRD with whole exome sequencing and NGS panels. Progression-free survival (PFS) after ICIs according to HRD status (n = 65)