1. Introduction
Cancer is the leading cause of death worldwide. Although the development of anticancer drugs for advanced cancer has been remarkable, the life expectancy of patients with advanced cancer is still only a few years. However, the recent progress of molecular techniques enabled multi-omics analysis to enhance our understanding cancer biology. In particular, next-generation sequencing (NGS) technology has revolutionized the simultaneous assessment of hundreds or even thousands of genes, not only in clinical research but also in clinical care [
1,
2]. Recently, it was reported that treatment with molecular targeted therapies based on comprehensive genomic profiling (CGP) significantly improved survival outcomes for patients with pancreatic cancer compared with patients who received non-molecularly matched therapies [
3]. Given this result, the National Comprehensive Cancer Network (NCCN) guideline stated that not only genetic testing for inherited mutations, but also CGP via FDA-approved and/or validated next-generation sequencing (NGS) based assays were recommended for patients with locally advanced/metastatic pancreatic cancer [
4]. Although there are not many molecular biomarkers that lead to effective treatment, the introduction of precision oncology can have a substantial effect on survival in patients with various solid tumors.
CGP tests, which promote genotype-matched therapy for advanced cancer, were covered by national healthcare insurance by The Ministry of Health, Labor, and Welfare (MHLW) in 2019, and they are available in daily routine clinical care in Japan. Currently, three types of CGP tests, two tissue-based tests, FoundationOne
® CDx and NCC Oncopanel [
5], and one blood-based test, FoundationOne
® Liquid CDx, are available. These tests have two functions. One is as companion diagnostics for the use of certain drugs, and the other is for CGP. However, unfortunately, the CGP test can currently only be used once in a patient’s lifetime due to restrictions on reimbursement. Therefore, it is important to avoid test failure because several clinical factors can easily lead to sequencing failure.
Even when the tests are successfully conducted, the ratio of patients who can proceed to matched therapies based on CGP is still low. The Center for Cancer Genomics and Advanced Therapeutics (C-CAT) reported only 8.1% of patients received matched therapies via CGP. To maximize the utility of CGP, it is essential not only to develop new drugs, but also to establish new biomarkers. The development of cancer immunotherapy, represented by PD-1/PD-L1 blockade, is emerging regardless of cancer type. Currently, patients with most cancer types can receive cancer immunotherapy, but the objective response rate depends on the cancer type. While PD-L1 expression is the most common biomarker for predicting responses to PD-1/PD-L1 blockade therapy, it alone is insufficient for selecting patients who benefit from immunotherapy [
6,
7]. Patients with microsatellite instability-high (MSI-high) and/or tumor mutational burden-high (TMB-high) are expected to have good responses, but biomarkers of cancer immunotherapy are still not well known [
8,
9,
10,
11,
12,
13].
Homologous recombination (HR) is one of the essential processes for the repair of DNA double-strand breaks.
BRCA1 and
BRCA2 genes are representative genes involved in the HR process. There are several key components for HR other than
BRCA1/2, which are the so-called HR-repair genes (HRR genes), and some HRR gene alterations in germlines cause hereditary cancer syndromes. Recent clinical trials showed that the loss of function of HRR genes in cancer (homologous recombination deficiency: HRD) can be a therapeutic target of poly (ADP-ribose) polymerase (PARP) inhibitors by synthetic lethality [
14,
15].
ARID1A is one of the SWI/SNF chromatin remodeling complex subunits.
ARID1A acts as a tumor suppressor and is frequently mutated in various cancer types. Recent research showed that
ARID1A has important roles in the DNA damage response. ARID1A-containing SWI/SNF complexes allow DNA repair proteins to access DNA damage sites and help repair double-strand breaks (DSB) via non-homologous end joining (NHEJ) and HR [
16,
17]. Furthermore, Shen and colleagues reported that
ARID1A also interacts with
MSH2, which is one of the mismatch repair genes (MMR genes), and recruits MSH2 to chromatin during DNA replication.
ARID1A deficiency compromises MMR and leads to increased mutagenesis [
18]. It is not well known whether HRR gene alterations can be the target of anti-cancer drugs other than PARP inhibitors.
In the present study, the results of CGP conducted as clinical research in our institute were reviewed. First, the aim was to develop a precision oncology system to complete CGP and benefit patients with unresectable/metastatic disease with the CGP results. The molecular biomarkers that predict the effectiveness of cancer immunotherapy were also explored, and HRR gene alterations can be proposed as biomarkers of cancer immunotherapy (UMIN000050577).
4. Discussion
CGP is becoming essential in medical oncology for diagnosis, classification, and choice of treatment, serving as an important tool for clinical decision support. Although the national health insurance system in Japan reimbursed CGP in clinical care, CGP is available in limited dedicated hospitals and not all patients are tested at this time. The MHLW designated 12 Core Hospitals for Cancer Genomic Medicine and more than 100 Cooperative Hospitals for Cancer Genomic Medicine in September 2022. Now is a transitional period for CGP to become common in Japan. We should maximize the usefulness of CGP for patients without effective choices of treatment.
FoudationOne
® CDx is one of the NGS-based CGP tests utilized globally, and its clinical validation data is extensively documented elsewhere [
25]. This test is exclusively performed by Foundation Medicine as a central laboratory testing service, adhering to the regulations of the College of American Pathologists (CAP) and Clinical Laboratory Improvement Amendments (CLIA). The concordance between FoundationOne
® CDx and the validated orthogonal comparator assays has been assessed, and the positive percent agreement (PPA) is reported to range from 89.4% to 100%, depending on biomarkers. The limit of detection (LoD) was determined based on either allele frequency or tumor purity. The LoD allele fraction for SUBs and indels was in the range of 2.0% to 12.74%, and the LoD tumor purity for copy number alterations and genomic rearrangements was in the range of 1.8% to 30%. The reproducibility of calling genomic alterations exceeded 99%. In the present study, the FoudationOne
® CDx results of 220 cancer patients were retrospectively reviewed, and an attempt was made to identify a new predictive biomarker for medical treatment. Problems in obtaining adequate test results were also investigated.
The present study showed the association between HRR gene alterations and TMB-high. As described in the introduction, HRR gene alterations can cause mutagenesis in several ways.
CDK12 is one of the representative HRR genes, and Wu and colleagues reported that
CDK12-mutant prostate cancer produces neo-fusion genes, and these neo-fusion genes can be neo-antigens for the immune system [
26]. A small cohort of
CDK12-mutant prostate cancer cases suggested a minor but positive effect of PD-1 blockade therapy [
27]. However, neo-fusion cannot be detected unless RNA sequencing is performed. Therefore, neo-fusion assessment is not available because current commercial CGP tests in Japan are basically based on DNA sequencing technologies. Although there are qualitative differences between single nucleotide variants and neo-fusion, the present study supports HRR gene-mutant cancer also showing TMB-high, and HRR mutants can be the target of PD-1/PD-L1 blockade therapy. Of HRR genes,
ARID1A was found to be frequently mutated in the present dataset and the MSK-IMPACT dataset. Consistent with the present results, some studies reported that HRR genes or
ARID1A alteration can be a biomarker that predicts sensitivity to PD-1/PD-L1 blockade therapy [
28,
29].
Whether HRR gene alterations are a true predictive biomarker for immunotherapy can depend on the cancer type. For example, Hugo reported that PD-1 blockade was more effective for
BRCA2-mutant melanoma [
30], but avelumab did not show a clinically meaningful effect for advanced ovarian carcinoma (JAVELIN Ovarian 100 trial), despite the fact that HRD is closely involved in the development of ovarian cancer [
31]. Although subgroup analysis by genotype has not yet been conducted in the JAVELIN Ovarian 100 study, further analysis could provide insight into whether HRR mutants are a true target of PD-1/PD-L1 blockade therapy.
Currently, PARP inhibition is one of the established treatment options for BRCA-mutant cancers, but these cancers can overcome PARP inhibitors via “BRCA reversion”. BRCA reversion restores BRCA function by secondary mutations that skip the stop codon or deleterious mutations [
32,
33]. Adding PD-1/PD-L1 blockade therapy to PARP inhibitor therapy can regulate tumor activity even after the tumor overcomes PARP inhibition. Combination therapies of a PARP inhibitor and a PD-1/PD-L1 blockade are currently being evaluated in several clinical studies, and some of them are reporting hopeful results [
34,
35].
The present study also suggests several problems to solve in the CGP process, including sample quality and the management of PGPVs. Generally, the performance of diagnostic tests can be influenced by the method, region, and time period [
36]. Although FoudationOne
® CDx centralizes testing to maintain quality, it’s important to acknowledge that pre-analytical factors can impact testing success [
37]. In our practice, tumor content affected testing success or failure the most. In addition, pre-operative chemotherapy tended to cause testing failure, though not significantly. Currently, liquid biopsy, represented by FoundationOne
® Liquid CDx, is also available in clinical care. However, there are some pitfalls in liquid biopsy. Liquid biopsy is less sensitive in detecting genomic alterations, especially when the amount of circulating tumor DNA (ctDNA) is low. The amount of ctDNA depends on tumor load, tumor type, and metastasis site [
38,
39,
40,
41]. Tissue-based testing is still the standard method for CGP rather than liquid-based tests. We should try to obtain sufficient biopsy samples before conducting pre-operative chemotherapy.
Identifying pathogenic germline variants (PGVs) can inform future cancer risks, surveillance, and prevention options for patients and their family members. From this point of view, the Core Hospitals for Cancer Genomic Medicine in Japan are mandated to establish a genetic counseling system to enhance public health. The present study found 43 cases harboring PGPVs, and genetic counseling was recommended to the attending physicians and patients. However, only four patients had genetic counseling despite these promotions. The National Health Interview Survey conducted in the United States has revealed that both the region and time period significantly impacted the rate of genetic testing among individuals with risk factors for Lynch syndrome [
42]. The low rate of genetic counseling in the present study can be attributed to limited awareness and a lack of attitudes toward genetic testing among patients [
43]. Enhancing understanding of genetic counseling through education for patients and healthcare providers is imperative. It has gradually become common to receive genetic counseling and testing during cancer treatment in Japan, in contrast to the period of the present study, as treatment with PARP inhibitors has gained popularity. Additionally, the costs for identifying PGVs may serve as one of the reasons discouraging individuals from genetic counseling. The national health insurance system in Japan allows cancer patients to receive treatment without a high financial burden, but this public insurance system does not cover diagnosis and surveillance for most hereditary cancer syndromes except for hereditary breast and ovarian cancer syndrome. Therefore, patients with these syndromes must cover all costs for the care of hereditary cancer syndromes in most cases. This national health insurance system is still insufficient for early cancer detection in hereditary cancer syndrome patients in Japan.
The present study has highlighted various problems and limitations associated with CGP in Japan. However, the ongoing advancements in technology are expected to move precision medicine forward. Current CGP in clinical care is restricted to DNA analysis for select genes and is not able to reveal multi-omics processes involved in cancers, such as methylation, cytokines, and protein expression. Several recent clinical trials have sought to enhance outcomes for cancer patients by combining DNA, RNA, and DNA methylation analyses [
1,
44,
45]. In addition, the analysis of cytokines and immune cells is becoming crucial for predicting the efficacy of immunotherapy [
2,
46,
47]. The future of precision medicine demands a shift towards multi-omics approaches that go beyond DNA sequencing, that will bring a more comprehensive understanding of the biological processes underlying cancer and offer improved outcomes for patients.