Initial assessment of a cancer genomic profile test for patients with metastatic breast cancer: a retrospective study


 Comprehensive cancer genomic profile (CGP) tests are being implemented under Japanese universal health insurance system. However, the clinical usefulness of CGP test for breast cancer patients has not been evaluated. Of the 310 patients who underwent CGP testing at our institution between November 2019 and April 2021, 35 patients with metastatic breast cancer whose treatment strategy was discussed by our molecular tumor board within the study period were investigated after exclusion of 2 cases that could not be analyzed. The turn-around time, drug accessibility, and germline identification detection were evaluated. The subtype was luminal in 20 patients (57.1%), triple-negative in 12 patients (34.3%), and luminal-HER2 in 3 patients (8.6%). Actionable gene mutations were detected in 30 patients (85.7%), and 7 patients (20.0%) were recommended for clinical trial participation, with the drug administered to 2 patients (5.7%). Three patients (8.6%) died due to disease progression before the test results were disclosed. We report the results of an initial assessment of the utility of CGP testing for patients with metastatic breast cancer under Japanese universal health insurance system. Conducting CGP tests at a more appropriate time could provide patients with greater benefit from treatments based on their specific gene mutations.


Introduction
It is estimated that approximately one million people develop cancer each year, and nearly one in every two people develop cancer during their lifetime (1). Cancer genomic medicine, which enables more effective and e cient diagnosis, treatment, and prevention, is receiving increasing interest. In the United States, then-President Barack Obama announced the "Precision Medicine Initiative" in his State of the Union address in January 2015 (2), and in January 2016, the Vice President led the launch of the "Cancer Moonshot" project, which aimed to develop new diagnostic and treatment methods to overcome cancer (3). In the UK, Genomics England, established by the UK Department of Health in 2013, is implementing the "100,000 Genomes Project" to analyze the whole genome of patients with cancer and rare diseases. In December 2018, Genomics England announced the completion of the 100,000 Genomes Project analysis (4). In addition, in October of the same year, the UK Minister of Health announced the goal of conducting one million whole-genome analyses over the next 5 years.
In Japan, two types of "cancer genomic pro le (CGP) tests" (OncoGuide NCC Oncopanel System and FoundationOne CDx Cancer Genome Pro le) entered full-scale application under the universal health insurance system in June 2019 (5)(6)(7)(8). In accordance with the Clinical Practice Guidance for Next-Generation Sequencing in Cancer Diagnosis and Treatment (edition 2.1), the gene panel tests covered by insurance include those for patients with solid tumors for whom no standard treatment is available or for whom standard treatment has been completed (including those who are expected to complete such treatment) (8-10). In addition, for the purpose of consolidating precision oncology treatment with quality control and assurance in designated hospitals, the Ministry of Health, Labour and Welfare (MHLW) originally designated 12 cancer genomic medicine core hospitals, 33 hub hospitals, and 180 cooperative hospitals in April 2021 (8, 11). CGP tests use next-generation sequencing and other technologies to analyze a large number of cancerrelated genes simultaneously, which is expected to assist in determining treatment strategies, thereby improving cancer treatment outcomes. In the SAFIR01 study, 423 patients with metastatic breast cancer underwent gene panel testing, which identi ed actionable mutations in 195 patients (46%). The mutations commonly identi ed included PIK3CA mutation (25%), CCND1 ampli cation (18%), and FGFR1 ampli cation (12%), followed by AKT1 mutation, EGFR ampli cation, and MDM2 ampli cation. A total of 55 of these patients (13%) were treated based on identi ed gene mutations (12).
The TOP-GEAR (Trial of Onco-Panel for Gene-pro ling to Estimate both Adverse events and Response) project, a study using CGP testing, reported patient access to therapeutic agents after standard treatment in Japan. From May 2016 to May 2017, approximately half of the patients who participated in the project and were able to undergo genetic analysis were found to have abnormalities in actionable genes, and 25 patients (13.4%) received therapeutic drugs tailored to their individual gene mutations, of which only one involved breast cancer (13).
The CGP tests are being implemented in Japan under the universal health insurance program, but many issues remain to be resolved, such as the limited number of implementation targets after standard treatment, the complexity of the national insurance requirements, management of the molecular tumor board (expert panel), and drug accessibility. Therefore, the clinical usefulness of CGP testing for breast cancer patients has not been evaluated. The purpose of the present study was to evaluate these issues at our institute under Japanese universal health insurance system.

Patients
Of the 310 patients who underwent CGP testing at our institution between November 2019 and April 2021, those with metastatic breast cancer whose treatment plan was discussed by the molecular tumor board within the study period were retrospectively studied, except for patients whose analysis failed. In Japan, insurance coverage for CGP tests is restricted to patients with advanced solid tumors exhibiting disease progression during standard therapy or patients for whom there are no appropriate standard treatments, including patients with rare cancers and carcinomas of unknown primary origin.
The study was approved by the Institutional Review Board of our institution (2021-1195), and data were collected in compliance with the ethical requirements of our institution.

Pathological assessment
Immunohistochemical subtypes were determined based on the primary tumor. Tumor sections were stained with hematoxylin-eosin (HE) and immunohistochemically examined for estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2) expression. Immunohistochemical assessment of ER and PgR expression was performed using antibodies against ER, clone 1D5 (Dako Japan Inc., Tokyo, Japan), and for PgR, clone PgR636 (Dako Japan Inc.). Positive reactions for ER and PgR were de ned as nuclear staining in ≥10% of cancer cells, and negative reactions were de ned as staining in <10% of cancer cells. Hormone receptor positivity was de ned as positivity in ER and/or PgR staining. HER2 positivity was de ned as HER2 protein 3+ or HER2 gene ampli cation, according to the ASCO/CAP guidelines (14).

CGP test
The FoundationOne CDx Cancer Genomic Pro le was used in this study (5). The FoundationOne CDx Cancer Genomic Pro le (Foundation Medicine) is a CGP platform that applies next-generation sequencing to in vitro diagnostics with a hybrid capture-based target enrichment approach and wholegenome shotgun library construction in order to identify gene substitutions, insertions and deletions (indels), copy number alterations, and select rearrangements. The FoundationOne CDx Cancer Genomic Pro le detects alterations in a total of 324 genes, including all coding exons of 309 cancer-related genes (substitutions, insertions and deletions, copy number alterations) and select intronic regions of 36 commonly rearranged genes. In addition, the FoundationOne CDx Cancer Genomic Pro le simultaneously pro les for tumor mutation burden (TMB) as well as microsatellite instability (MSI) status.
The rates of CGP testing success, turn-around time (TAT), drug accessibility, and secondary nding detection were evaluated.
The pathologist selected the representative block from the patient's stored formalin-xed para n embedded (FFPE) specimens and estimated the tumor content in the HE specimen. In addition, 10 to 25 slices of 5µm were prepared to achieve a tumor volume of at least 25 mm 2 square, which is the volume required for analysis, and submitted for CGP test.

Molecular tumor board (Expert Panel)
The molecular tumor board of our institute discussed the reports from CGP tests (FoundationOne CDx) in conjunction with the Center for Cancer Genomics and Advanced Therapeutics (C-CAT) and generated nal reports for the patients. The molecular tumor board consists of physicians specializing in cancer pharmacotherapy; physicians with expertise in genetic medicine; genetic counselors; pathologists; physicians with expertise in molecular genetics and cancer genomic medicine; and those with expertise in bioinformatics. The evidence levels for therapeutic e cacy were categorized as A to F, following the JSMO/JSCO/JCA guideline (10). The actionable gene mutations were de ned as following, predictive evidence level D and more (10) and high-tumor mutation burden (after approval of pembrolizumb by FDA).

Patient characteristics
The CONSORT diagram of the study is shown in Figure 1. Characteristics of the 35 patients investigated in detail are listed in Table 1. All evaluated patients had invasive breast cancer. The median age of the patients was 56 years (range 32-81 years). Evaluation of histological type as established by needle biopsy or surgical specimen showed that more than half of the patients had invasive ductal carcinoma (54.3%). There were 7 speci c types, including 4 cases of invasive lobular carcinoma (11.4%) and one case (2.9%) each of spindle cell carcinoma, mucinous carcinoma, and metaplastic carcinoma. The subtype was luminal in 20 patients (57.1%), triple-negative in 12 patients (34.3%), and luminal-HER2 in 3 patients (8.6%). The median number of pretreatment regimens, including regimens currently being treated, was 5 (range 1-16). In 5 cases, CGP testing was performed during the initial treatment of metastatic breast cancer, and 4 patients (11.4%) had ≥10 pretreatment regimens.
The specimens submitted for CGP testing included 23 primary breast sites (65.7%) and 12 biopsies from metastatic sites (34.3%). The biopsy specimens included one case of pleural uid cell block.

Type and frequency of identi ed genetic variants
The gene mutations and ampli cations detected in this study are shown in Figure 2. As reported previously, TP53 and PIK3CA mutations were found in more than 40% of the cases. BRCA1/2 and RAD21C mutations were also found in approximately 30% of cases in this study.

Turn-around time
Page 9/17 The median turn-around time from obtaining consent to the molecular tumor board was 44 days (range 29-93 days), and from obtaining consent to explaining the CGP test results was 56 days (range 42-105 days).

Drug accessibility rate
Among the 35 patients investigated in detail, the molecular tumor board recommended treatment according to the gene mutation for 7 patients (20%), but only 2 patients (5.7%) participated in clinical trials, as 5 patients were unable to undergo treatment for their mutations due to disease progression (Fig. 4). The 7 cases in which the molecular tumor board recommended treatment according to the gene mutation are shown in Table 2.

Germline ndings
There were 9 cases in which germline pathological variants were suspected, among which 6 cases had known pathologic variants of BRCA1/2 before the CGP test (Table 3). In the CGP test, RAD51C, BRCA1, and PALB2 were identi ed as newly suspected germline variants in one case each. In a patient with triplenegative breast cancer suspected of having germline variants in RAD51C, con rmatory testing by speci c site analysis revealed pathological variants in RAD51C. Patients with suspected germline variants in BRCA1 were con rmed negative by BRCA analysis.

Discussion
We report here for the rst time the initial assessment of the utility of CGP testing for patients with metastatic breast cancer under Japanese universal health insurance system. In the rst 18 months after implementation of reimbursement for CGP tests, 310 CGP tests were conducted in our institute during the study period. Thirty-seven patients with metastatic breast cancer were tested, followed by pancreatic cancer and colorectal cancer. Actionable gene mutations were detected in 30 patients (85.7%), and participation in a clinical trial was recommended to 7 patients (20.0%). The drug was administered to only 2 patients (5.7%), as 3 patients (8.6%) had newly suspected germline pathological variants, and the results were subject to disclosure.
As for the current status of CGP testing under universal health insurance in Japan, 805 patients underwent CGP testing between June and October 2019, and it was reported that 10.9% of these patients were then treated based on their gene mutations (15). Furthermore, according to a report from 11 core hospitals for cancer genomic medicine, among the 747 cases in which CGP tests were conducted (from June 2019 to January 2020), 28 patients (3.7%) received genome-matched treatment (16). However, of these 28 patients treated according to their gene mutations, only 2 patients had breast cancer, and both were treated with an mTOR inhibitor for a PIK3CA mutation in that study (16). As such, under national health insurance in Japan, only approximately 10% of the patients who underwent CGP testing received a therapeutic drug targeted to their gene mutations.
This study identi ed several important issues regarding CGP testing under Japanese universal insurance system. The rst issue is the low rate of drug accessibility due to disease progression after CGP testing.
The second issue is that approximately 8% of patients died due to disease progression before the test results were disclosed; thus, the results of the CGP tests could not be explained.
Possible causes of these issues include the indication and o cial pricing system of reimbursement of CGP testing covered by Japanese universal health insurance system. The indication for CGP testing covered by insurance in Japan is restricted to patients with advanced solid tumors exhibiting disease progression during standard therapy (including those expected to be completed) or patients for whom there are no appropriate standard treatments, including those with rare cancers and carcinoma of unknown primary origin (8-10). Furthermore, reimbursement for the cost of a CGP test is 560,000 yen, paid in two steps. The rst reimbursement is 80,000 yen after applying for the informed consent for CGP testing and preparation of tumor samples. The second reimbursement of 480,000 yen is paid when the patient receives an explanation of the CGP test results after assessment by the molecular tumor board.
In our study, new treatment options, such as those available in clinical trials, were recommended to 7 patients (20.0%) by the molecular tumor board. However, 5 (14.3%) of these patients were unable to undergo genome-matched treatment due to disease progression after the CGP test. Although the de nition of the standard treatment for metastatic breast cancer prior to CGP testing remains controversial, the treatments strongly recommended in the guidelines of the Japanese Breast Cancer Society are considered candidates in Japan (17). However, the patient may be eligible for CGP testing without all standard therapies, depending on the e cacy and safety of previous therapies, the patient's general condition, and patient preference in consideration of the 6-to 8-week turnaround time from submission of tumor tissue to return of analysis results. Therefore, our results suggest that CGP testing conducted at the appropriate time and based on the e cacy of previous therapy, the patient's general condition, and patient preference might improve the drug accessibility rate.
As mentioned above, CGP testing is currently restricted to patients who nished standard therapies in order to avoid unnecessary investigations and reduce the burden for the molecular tumor board. However, patients who nished standard therapies for metastatic or recurrent cancer tend to have poor prognosis due to disease progression. Therefore, it is important to perform the CGP test at the diagnosis of metastatic breast cancer. Also, changes to the insurance system should be considered, in addition to increasing the number of clinical trials and promoting efforts to standardize quality while reducing the burden on the molecular tumor board.
In summary, it has been almost 2 years since CGP testing covered by the universal health insurance system was rst introduced in Japan, and implementation of cancer genomic medicine is progressing. We reported here the initial assessment of CGP testing for patients with metastatic breast cancer and revealed that the percentage of patients who could reach clinical trials is low. In our sample group, a number of patients died before the test results were disclosed, although more than 80% of the patients had actionable mutations. In order to make CGP testing clinically valuable for patients with metastatic breast cancer, it will be necessary to solve problems associated with the current program of insurance coverage for CGP testing in Japan.

Conclusion
We reported here the initial assessment of the utility of CGP testing for patients with metastatic breast cancer. In our institute, 5.7% of patients underwent genome-matched treatment, and 8.5% were found to have newly suspected germline variations. Conducting CGP tests at a more appropriate time could provide patients with greater bene t from treatments based on their speci c gene mutations.

Declarations
Ethics approval and consent to participate Availability of data and materials The datasets collected during and/or analyzed during the current study are available from the corresponding author on reasonable request.