Clinical utility of comprehensive genomic profiling in Japan: Result of PROFILE-F study.

INTRODUCTION: Clinical sequencing has provided molecular and therapeutic insights into the field of clinical oncology. However, despite its significance, its clinical utility in Japanese patients remains unknown. Here, we examined the clinical utility of tissue-based clinical sequencing with FoundationOne® CDx and FoundationOne® Heme. Between August 2018 and August 2019, 130 Japanese pretreated patients with advanced solid tumors were tested with FoundationOne® CDx or FoundationOne® Heme.
RESULTS: The median age of 130 patients was 60.5 years (range: 3 to 84 years), and among them, 64 were males and 66 were females. Major cancer types were gastrointestinal cancer (23 cases) and hepatic, biliary, and pancreatic cancer (21 cases). A molecular tumor board had been completed on all 130 cases by October 31, 2019. The median number of gene alterations detected by Foundation testing, excluding variants of unknown significance (VUS) was 4 (ranged 0 to 21) per case. Of the 130 cases, one or more alterations were found in 123 cases (94.6%), and in 114 cases (87.7%), actionable alterations with candidates for therapeutic agents were found. In 29 (22.3%) of them, treatment corresponding to the gene alteration was performed. Regarding secondary findings, 13 cases (10%) had an alteration suspected of a hereditary tumor. Of the 13 cases, only one case received a definite diagnosis of hereditary tumor.
CONCLUSIONS: Our study showed that clinical sequencing might be useful for detecting gene alterations in various cancer types and exploring treatment options. However, many issues still need to be improved.

Introduction

In recent years, next-generation sequencing (NGS), which is a technology for analyzing the base sequence of DNA in a short time, has made it possible to examine a large amount of genomic information at once. Conventionally, cancer drug therapy has been selected based on organ of the primary tumor. However, it has been found that gene alterations in cancer cells can effect biology and treatment, even in cancers originating from the same organ. Therefore, cross-organ cancer genomic medicine is receiving a great deal of attention, in which cancer-related genes are comprehensively analyzed using a next-generation sequencer and a therapeutic drug is selected based on the results. Comprehensive examination of cancer-related genes from cancer tissues or blood samples is called comprehensive genomic profiling (CGP). The CGP tests, FoundationOne® CDx cancer genome profiling, and OncoGuide™ NCC Oncopanel System have been covered by Public health insurance since June 2019 and are widely adapted in Japan.

The results of examining the clinical utility of CGP tests have been reported in Japan as well. In Japan, CGP can only be used at the time of completion or expected completion of standard treatment to receive the reimbursement of Public Health Insurance. Therefore, it is difficult to make simple comparisons with foreign literature, and accumulation of data in Japan is necessary. In addition, FoundationOne® CDx cannot determine whether the detected mutations are of germline origin, but we may detect genetic alterations associated with hereditary tumors as secondary findings. Few reports of secondary findings in CGP tests are available in Japan. Therefore, to examine the clinical utility of the CGP test, we initiated an observational study using FoundationOne® CDx or FoundationOne® Heme and investigated the effect of the CGP test results on the treatment of patients. We also investigated secondary findings and outcomes in these tests.

Methods

Patients

We retrospectively reviewed 130 patients with advanced solid tumors who either progressed on, or were finishing standard systemic therapy. These patients underwent FoundationOne® CDx or FoundationOne® Heme between August 2018 and August 2019 under PROFILE-F study. The PROFILE-F study was approved by the institutional review board of Tokyo Medical and Dental University (TMDU; G2018-002) and registered in University Hospital Medical Information Network (UMIN; UMIN000028439).

Sequencing and detection of genomic variances

Patients underwent clinical-grade CUA-approved next-generation sequencing that investigates the entire coding DNA sequencing of 324 genes with FoundationOne® CDx and DNA sequencing of 406 genes and RNA sequencing of 265 genes with FoundationOne® Heme. Tumors were assessed for genomic aberrations, including insertions, deletion, base substitutions, copy number alterations, and fusions/ rearrangements. The methods for this type of comprehensive genomic profiling have been previously published, and extensive methods can be found elsewhere [1, 2].

Definition of actionability

Actionable alteration is defined as a genomic alteration that satisfies the following conditions: 1) mechanistically, the gene is associated with cancer and has the data indicating therapeutic efficacy; and 2) a drug is available for human use either in an antibody or a small molecule compound with low IC 50 concentration [3, 4].

Molecular tumor board

After genomic test results, each case was discussed at the molecular tumor board (MTB) with specialists, such as medical oncologists, pathologists, radiologists, bioinformaticians, genetic counselors, clinical research coordinators, and treating physicians. These members deliberated actionable genomic alterations and treatment options based on the patient’s medical history, treatment history, family history, imaging findings, histopathological findings, and genetic test results.

Results

Patients and characteristics

Between August 2018 and August 2019, 130 patients with advanced solid tumors who progressed with or were finishing standard systemic therapy or with rare cancers participated in the PROFILE-F study and underwent FoundationOne® CDx or FoundationOne® Heme. The median age of 130 patients at specimen exam date was 60.5 years (range: 3 to 84 years), and among them, 64 were males and 66 were females (Table 1). There were 16 patients (12.3%) with no previous chemotherapy by the date of test submission, 27 patients (20.8%) with one line and 87 patients (66.7%) with two or more lines. A total of 28 diverse cancer types were observed in 130 patients (Table 1). All patient populations were Japanese. The breakdown of FoundationOne® CDx and FoundationOne® Heme is shown in Table 2.

Table 1

Patients characteristics.

Characteristics	No. of patients (%)
Age at specimen exam date, years
Median(range)	60.5	(3–84)
Gender
Male	64	(49.2%)
Female	66	(50.8%)
Line of previous chemotherapy
0	16	(12.3%)
1	27	(20.8%)
≥2	87	(66.9%)
Type of cancer
Neuroendocrine tumor	14	(10.8%)
Pancreas cancer	12	(9.2%)
Breast cancer	12	(9.2%)
Colorectal cancer	11	(8.5%)
Head and Neck cancer	8	(6.2%)
Sarcoma	8	(6.2%)
Esophagus cancer	7	(5.4%)
CUP	7	(5.4%)
Biliary cancer	6	(4.6%)
NSCLC	6	(4.6%)
Ovary cancer	5	(3.8%)
Uterus cancer	5	(3.8%)
Urologic cancer	3	(2.3%)
Stomach cancer	2	(1.5%)
Liver cancer	2	(1.5%)
SCLC	2	(1.5%)
Other *	20	(15.4%)

Abbreviations: CUP, Cancer of unknown primary origin; NSCLC, Non-Small Cell Lung Cancer; SCLC, Small Cell Lung Cancer.

* Other cancers include hemangiopericytoma, chordoma, adrenal carcinoma, thymic carcinoma, peripheral schwannoma, peritoneal mesothelioma, nephroblastoma, neuroblastoma, extramammary Paget’s disease, chondrosarcoma, allantoic carcinoma, and primary intraosseous carcinoma.

Table 2

Types of tests.

Types of tests	No. of specimens submitted (%)
Foundation One® CDx	139(87.4%)
Foundation One® Heme	20(12.6%)
all specimens	159(100.0%) *

*There is a discrepancy between the number of specimens and the number of patients, including patients who submitted several tests for failure reports, patients who submitted both FoundationOne® CDx and FoundationOne® Heme, and patients who submitted specimens from multiple sites (primary and metastatic).

The common alterations and differences by each cancer type

The most commonly altered genes excluding VUS in all cancer types are shown in Fig 1A. Only alterations seen in at least 5% of patients were shown. The three most frequent alterations observed were TP53 (n = 72, 55.4%), CDKN2A (n = 29, 22.3%), KRAS (24, n = 18.5%). As shown in Fig 1B–1E, the most frequent gene alterations observed varied by tumor types.

Fig 1

The most common alterations in all cancer types (N = 130).

A. The most commonly altered genes in all cancer types, seen in at least 5% of patients, were shown. The three most frequent alterations observed were TP53 (n = 72, 55.4%), CDKN2A (n = 29, 22.3%) and KRAS (24, n = 18.5%). B. The most common alterations in Neuroendocrine tumor (N = 14). C. The most common alterations in Pancreas cancer (N = 12). D. The most common alterations in Breast cancer (N = 12). E. The most common alterations in Colorectal cancer (N = 11).

Alterations and actionability

Table 3 shows the alteration and actionability in 130 patients with each cancer type, while Fig 2 displays the flowchart of actionability of 130 patients. Overall, 123 (94.6%) of 130 patients had detectable alteration(s). Of the 130 patients, 114 (87.7%) had at least one actionable alteration. The median number of alterations (except VUS) per patient of all cancer types was 4 (range 0–21), and the median number of actionable alterations was 3 (range 0–12). The cancer types with the highest median number of actionable alterations were NSCLC (non-small cell lung cancer) (median: 5.5; range 1–8), esophagus caner (median: 5; range 2–12), ovary cancer (median: 5; range 1–5), and CUP (cancer of unknown primary origin) (median: 5; range 2–10). Meanwhile, the cancer type with the lowest median number of actionable alterations was neuroendocrine tumor (median: 0; range 0–6). We added Fig 3 showing evidence level defined by C-CAT (The Center for Cancer Genomics and Advanced Therapeutics) [5]. Of 130 patients, 29 (22.3%) received the treatment corresponding to the gene alteration. The cancer type with the highest rate of patients who received the treatment corresponding to the gene alteration was SCLC (small cell lung cancer) (1 in 2 or 50.0%). Table 4 shows the extended information in 130 patients with each cancer type. We also obtained the results of tumor alteration burden (TMB) and microsatellite status (MS). The median number of TMB was 4 (range 0–34). The cancer type with the highest median number of TMB was CUP (median:14; range 3–29). Only 1 patient who was diagnosed with cancer of unknown primary origin reaped the result of MS-high.

Table 3

Alterations and actionability in 130 patients with each cancer types.

Cancer types	No. of individuals	No. of individuals with detectable alteration(s) (%)	No. of individuals with ≥1 alterations (%)	Median no. of alterations (range)	No. of individuals with ≥1 actionable alterations (%)	Median no. of actionable alteration(s) (range)	No. of individuals who received molecular-targeted therapy (%)
Neuroendocrine tumor	14	14(100%)	8(57.1%)	1(0–8)	7(50.0%)	0(0–6)	1(7.1%)
Pancreas cancer	12	12(100%)	12(100%)	3.5(2–9)	8(66.7%)	2.5(1–6)	3(25.0%)
Breast cancer	12	12(100%)	12(100%)	5(1–14)	12(100%)	4(1–7)	3(25.0%)
Colorectal cancer	11	10(90.9%)	10(90.9%)	6.5(3–16)	10(90.9%)	4.5(2–8)	4(36.4%)
Head and Neck cancer	8	8(100%)	8(100%)	4.5(1–11)	8(100%)	4(1–6)	2(25.0%)
Sarcoma	8	8(100%)	8(100%)	5(2–9)	8(100%)	2.5(2–6)	2(25.0%)
Esophagus cancer	7	7(100%)	7(100%)	6(2–21)	7(100%)	5(2–12)	1(14.3%)
CUP	7	7(100%)	7(100%)	8(3–13)	7(100%)	5(2–10)	3(42.9%)
Biliary cancer	6	6(100%)	6(100%)	2.5(1–6)	6(100%)	2(1–4)	0(0%)
NSCLC	6	4(66.7%)	4(66.7%)	6.5(2–10)	4(66.7%)	5.5(1–8)	1(16.7%)
Ovary cancer	5	5(100%)	5(100%)	6(1–8)	5(100%)	5(1–5)	1(20.0%)
Uterus cancer	5	5(100%)	5(100%)	2(1–7)	5(100%)	1(1–6)	2(40.0%)
Urologic cancer	3	3(100%)	3(100%)	2(2–8)	3(100%)	2(1–8)	0(0%)
Stomach cancer	2	2(100%)	2(100%)	5(3–7)	2(100%)	2(2)	0(0%)
Liver cancer	2	2(100%)	2(100%)	4.5(4–5)	2(100%)	4(4)	0(0%)
SCLC	2	1(50%)	1(50.0%)	3(3)	1(50.0%)	2(2)	1(50.0%)
Other	20	17(85%)	17(85.0%)	2(1–11)	15(75.0%)	2(0–9)	5(25.0%)
All	130	123(94.6%)	117(90.0%)	4(0–21)	114(87.7%)	3(0–12)	29(22.3%)

*without VUS (variants of unknown significance).

Fig 2

Flowchart of actionability of 130 patients.

123 (94.6%) of 130 patients had detectable alteration(s). There were 114 (87.7%) patients with at least one actionable alteration. 29 (22.3%) patients received the treatment corresponding to the gene alteration.

Fig 3

Levels of evidence defined by C-CAT.

Alterations with evidence level D or higher were detected in 94 (72.3%) of 130 patients.

Table 4

Extended information in 130 patients with each cancer types.

Cancer types	No. of individuals	Median TMB* (range)	No. of individuals with TMB ≥10 (%)	No. of individuals with MS**-High (%)	No. of individuals with ≥1 clinical trial options (%)
Neuroendocrine tumor	14	1(0–6)	0(0%)	0(0%)	6(42.9%)
Pancreas cancer	12	3(0–6)	0(0%)	0(0%)	8(66.7%)
Breast cancer	12	2.5(0–9)	0(0%)	0(0%)	11(91.7%)
Colorectal cancer	11	3.5(0–11)	1(9.1%)	0(0%)	10(90.9%)
Head and Neck cancer	8	5.5(1–34)	1(12.5%)	0(0%)	8(100%)
Sarcoma	8	2.5(1–13)	2(25.0%)	0(0%)	6(75.0%)
Esophagus cancer	7	4(3–14)	1(14.3%)	0(0%)	6(85.7%)
CUP	7	14(3–29)	4(57.1%)	1(14.3%)	5(71.4%)
Biliary cancer	6	4(3–9)	0(0%)	0(0%)	4(66.7%)
NSCLC	6	2.5(0–10)	1(16.7%)	0(0%)	4(66.7%)
Ovary cancer	5	4(0–14)	1(20.0%)	0(0%)	5(100%)
Uterus cancer	5	3(3–8)	0(0%)	0(0%)	4(80.0%)
Urologic cancer	3	3(0–23)	1(33.3%)	0(0%)	3(100%)
Stomach cancer	2	5(5)	0(0%)	0(0%)	1(50.0%)
Liver cancer	2	4.5(4–5)	0(0%)	0(0%)	2(100%)
SCLC	2	9(9)	0(0%)	0(0%)	0(0%)
Other	20	3.5(0–9)	0(0%)	0(0%)	10(50.0%)
All	130	4(0–34)	12(9.2%)	1(0.8%)	97(74.6%)

*Tumor mutation burden: without cannot determined patients.

**Microsatellite status.

Treatment corresponding to the gene alteration

Of 130 patients, 29 (22.3%) were treated based on comprehensive genomic profiling. We divided the patients according to the type of therapeutic drug used (Table 5). The three categories used are approved drug (Approved), investigational drug (Investigational), and off-label use (Off-label). Among the treatments that patients received, approved drug was 51.7% (15 out of 29), investigational drug was 31.0% (9 out of 29), and off-label use was 17.2% (5 out of 29). Among patients who received approved treatment, 5 patients received immune checkpoint inhibitors, nivolumab, or pembrolizumab (Nos. 9, 14, 15, 24, and 28), and 5 patients received PARP inhibitors or platinum-based anticancer agents for homologous recombination repair-related gene alterations (Nos. 10, 12, 13, 18, and 29). The best responses in patients who received approved treatment were CR 6.7%, PR 33.3%, SD 13.3%, PD 26.7%, and N.D. 20.0%. Among patients who received investigational drugs, 3 took a combination of pertuzumab and trastuzumab for ERBB2 amplification (Nos. 23, 26, and 27) and 1 received olaparib for ATM alteration (No. 25). The administration of these investigational drugs were conducted at our own facility. Since other investigational drugs were conducted at other institutions, the patients were referred to them accordingly. No. 20 patient was a case with EGFR uncommon alterations which were not detected by the initial PNA LNA PCR-Clamp method, but were able to be detected by FoudantionOne® CDx [6]. Although not included in Table 5, there was a case in which NGS influenced the treatment strategy. The patient had been treated for pathologically diagnosed primary intrahepatic cholangiocarcinoma before NGS. But She had a history of pancreatic cancer surgery, and the NGS results of the pancreatic resection specimen and liver resection specimen matched, so the MTB discussion changed the diagnosis to liver metastasis of pancreatic cancer. She responded to a pancreatic cancer regimen.

Table 5

Patients who received the treatment corresponding to the gene alteration.

No.	diagnosis	Age (years)	Gender	Lines of previous CTx	Targeted gene aberration	Drug	Category
1	Breast cancer	60	F	6	CCND1 amplification	Palbociclib	Approved
2	Breast cancer	43	F	11	PIK3CA N345K, AKT1 amplification	Everolimus	Approved
3	Breast cancer	43	F	6	FGFR1 amplification	Combination of TAS-117 and TAS120	Investigational
4	Colorectal cancer	65	M	7	APC R232*	Wnt inhibitor	Investigational
5	Colorectal cancer	70	F	5	PIK3CA E545K	mTOR inhibitor	Investigational
6	Colorectal cancer	71	F	4	FLT3 amplification	Regorafenib	Approved
7	Small intestinal cancer	66	M	2	APC E1379*, APC K534*, APC splice site 835-8A>G	β-catenin inhibitor	Investigational
8	Esophageal cancer	64	M	0	TMB high	Pembrolizumab	Off-label
9	Sarcoma of the esophagus	63	F	0	TMB high	Nivolumab	Approved
10	Uterine sarcoma	58	F	3	RAD51B loss	IP(Ifomide, CDDP and Mesna)	Approved
11	Cervical cancer	55	F	4	ARID1A E1647*	ATR inhibitor	Investigational
12	Cervical cancer	79	F	1	BRCA1 S153fs*5	1) CBDCA, 2) Olaparib	Approved
13	Ovarian cancer	72	F	4	BRCA2 R2318*	CDDP	Approved
14	Tongue cancer	63	M	2	PD-L1 TPS70%†	Nivolumab	Approved
15	Maxillary cancer	50	M	0	TMB high	Nivolumab	Approved
16	Pancreatic cancer	64	M	3	PIK3CA H1047R	Copanlicib	Off-label
17	Pancreatic cancer	43	M	3	KRAS G12D	Combination of Trametinib and Hydroxychlorquine	Off-label
18	Pancreatic cancer	72	F	3	ATM R2993*	FOLFOX	Approved
19	Small-cell lung cancer	63	M	1	TMB 9 muts/Mb	Nivolumab	Off-lavel
20	Lung adenocarcinoma	70	F	12	EGFR G719D, EGFR E709A§	Afatinib	Approved
21	Duodenal neuroendocrine tumor	58	F	6	BRCA1 rearrangement	Olaparib	Off-label
22	Hemangiopericytoma	56	F	0	NAB2-STAT6 fusion	Pazopanib	Approved
23	Urachal cancer	40	M	1	ERBB2 amplification	Combination of Pertuzumab and Trastuzumab	Investigational
24	Malignant peripheral nerve sheath tumor	63	F	1	TMB 9 muts/Mb, MSH6 N1307fs*9	Pembrolizumab	Approved
25	Nephroblastoma	9	M	4	ATM K2749I(VUS)||	Olaparib	Investigational
26	Extramammary Paget’s disease	55	M	1	ERBB2 amplification	Combination of Pertuzumab and Trastuzumab	Investigational
27	Cancer of unknown primary origin	70	F	1	ERBB2 amplification	Combination of Pertuzumab and Trastuzumab	Investigational
28	Cancer of unknown primary origin	58	M	1	MSI high, TMB high	Pembrolizumab	Approved
29	Cancer of unknown primary origin	72	F	1	RAD51D K91fs*13	Olaparib	Approved

TMB more than 10 muts/Mb is defined as TMB-high.

†: PD-L1 TPS was measured as an optional service of FoundationOne® CDx.

§: A case with EGFR uncommon alterations which were not detected by the initial PNA LNA PCR-Clamp method, but were able to be detected by FoudantionOne® CDx. [6].

||: Although reported as VUS in the FoundationOne® Heme report, a preclinical study has shown the sensitivity of PARP inhibitor to this alteration (ATM p.K2749I) [7].

Secondary findings

In this study, we detected somatic alterations in tumor tissue to explore cancer treatment options. However, as a result of discussions at the molecular tumor board based on factors like detected alterations, family history, and age of onset, there were 13 patients (10%) suspected of having a hereditary tumor as secondary findings (Fig 4). Of the 13 patients, 4 (3.1%) underwent a test for a definite diagnosis of hereditary tumor. Only 1 patient (0.8%) reached a definite diagnosis for it. Fig 4 also shows the reason patients suspected of having a hereditary tumor were not tested for definite diagnosis for hereditary tumor. Four patients had not yet received genetic counseling, 3 died before genetic counseling, 1 disagreed to know suspected hereditary tumors, and 1 refused a definitive test after genetic counseling.

Fig 4

Outcome of patients with suspected secondary findings.

Of the 130 patients, 13 (10%) were suspected of having a hereditary tumor. Only 1 patient (0.8%) reached a definite diagnosis for it.

Discussion

Recently, tumor agonistic genomic medicine, which uses somatic or germline genetic alterations to guide decisions about treatment choice, has been attracting attention. Many institutions are investigating the clinical utility of the Comprehensive Genomic Profiling (CGP) test, which is indispensable in cancer genomic medicine. One of the indicators of clinical utility of the CGP test is whether patients actually received the treatment recommended by the CGP test. In the present study, we investigated the impact of the CGP tests, FoundationOne® CDx or FoundationOne® Heme, on patients’ treatment choices.

The clinical utility of CGP testing has been reported previously in Europe and the U.S. Sohal DPS et al. reported that 11% (24 of 223) of patients received the recommended treatment based on the CGP test using the FoundationOne platform [8]. Hirshfield KM et al. reported that the rate of clinical intervention based on the CGP test using the FoundationOne platform was 35% (31 of 92), including genetically guided therapy, diagnostic modification, and trigger for germline genetic testing [9]. In the NCI-MATCH trial, a clinical trial in which subjects’ tumor samples are screened with a CGP test and subjects with potentially targetable genetic alterations are entered into a clinical trial corresponding to that genetic alteration, the percentage of patients assigned to treatment was 17.8% [10].

In Japan, CGP can only be used at the time of completion or expected completion of standard treatment to receive the reimbursement of Public Health Insurance [5]. Therefore, it is difficult to make simple comparisons with foreign literature, and accumulation of data in Japan is necessary. In Japan, OncoGuide™ NCC Oncopanel System and FoundationOne® CDx cancer genome profiling are currently reimbursed by Public Health Insurance. Other research-based NGS (e.g., PleSSision-160, CANCERPLEX, OncoPrime) results have been reported in Japan [11-14]. Hayashi et al. reported that of 20 pancreatic cancer patients who underwent a targeted amplicon exome sequencing for 160 cancer-related genes (PleSSision-160), 100%(20/20) had actionable gene alterations, 35%(7/20) had druggable alterations detected, and only 10%(2/20) could be treated with therapeutic agents based on the results of genomic testing [11]. Saotome et al. reported that among the ovarian tumor patients who underwent PleSSision-160, actionable alterations were detected in 90.9%(80/88) and druggable alterations were detected in 40.9%(36/88) [12]. Kou et al. reported that among 85 patients with cancers of unknown primary site, rare tumors, or any solid tumors that were refractory to standard chemotherapy who underwent an NGS‐based multiplex gene assay (OncoPrime), 69 patients had potentially actionable alterations detected. 9(13.0%) of 69 patients received a subsequent therapy based on the NGS assay results [14]. According to a report by Sunami et al. using the reimbursed OncoGuide™ NCC Oncopanel System, of 230 patients with advanced solid tumors, 111 (59.4%) of which harbored actionable gene alterations and twenty-five (13.3%) cases have since received molecular-targeted therapy according to their gene alterations [15]. Takeda et al reported that among the 175 patients who underwent FoundationOne® CDx, 174 had at least one known or likely pathogenic gene alteration, and 24 of these patients (14%) received corresponding targeted therapy [16]. In our study, 22.3% of patients received treatment based on CGP results, which is almost the same as other Japanese reports. We defined actionable alteration as a genomic alteration that satisfies the following conditions: 1) mechanistically, the gene is associated with cancer and has the data indicating therapeutic efficacy; and 2) a drug is available for human use either in an antibody or a small molecule compound with low IC 50 concentration [3, 4]. But this is an area of on-going debate. It is difficult to compare the number of actionable alterations with other Japanese reports. So we added Fig 3 showing evidence level defined by C-CAT (The Center for Cancer Genomics and Advanced Therapeutics) [5]. Alterations with evidence level D or higher were detected in 94 (72.3%) of 130 patients. The reasons for not receiving molecular targeted therapy despite the detection of actionable mutations were as follows: 1) investigational drugs were only available overseas or in distant parts of Japan, 2) clinical trial recruitment had already ended, 3) comorbidity or poor PS prevented participation in the trial, or 4) the disease had progressed to the point where treatment was not indicated. 1) and 2) suggests that there are inter-facility and inter-regional disparities in access to investigational drugs. Correcting the disparities in obtaining information and referrals for investigational drugs is desirable. Cases such as 3) and 4) may be influenced by the fact that CGP in Japan Public Health Insurance can be used only at the time of completion or prospective completion of standard treatment. If the CGP could be performed from the start of cancer treatment, it might lead to better treatment choices.

We experienced a patient whose diagnosis was changed in the MTB (called expert panel in Japan) discussion due to the CGP results and who was treated based on the new diagnosis. The clinical usefulness of the CGP test may be enhanced by a comprehensive discussion of the optimal treatment for the patient based on the CGP test results in the MTB. In Japan, an expert panel is required as a condition of insurance treatment, and this is expected to boost the usefulness of the CGP test. However, expert panels in Japan differ from facility to facility, and standardization is an issue to be addressed in the future [17].

Hirshfield et al. reported that tumor sequencing results can be a trigger for germline testing [9]. In a previous report, hereditary tumors were suspected in five (6.2%) of 80 patients, three of whom underwent definitive testing for hereditary tumors, and two (2.5%) of whom were confirmed [14]. In the present study, we experienced a case in which the results of CGP testing to explore treatment options led to the diagnosis of hereditary tumors. However, although hereditary tumors were suspected in 13 (10%) out of 130 patients, only 4 (3.1%) patients underwent tests for confirmatory germline testing. This may be since hereditary tumors are still not widely recognized in Japan, or the priority to explore hereditary possibility was lower than pursuing the treatment for the patient. The diagnosis of hereditary tumors can not only lead to the treatment of the patient, but also to the prevention and early detection of cancer in the next generation. Taking all these into consideration, spreading awareness and accurate knowledge about hereditary tumors should be considered as important aspects of cancer treatment.

There are several limitations in our study. First, it was difficult to make statistical comparisons with the foreign literature due to the nature of the Public Health insurance system in Japan. Second, the definition of actionable alteration is still controversial, and we were unable to compare the actionable alteration rate with other Japanese literature. Third, because this was a retrospective study, we were not able to follow the progress of some patients who had been treated at other hospitals.

Conclusion

This study showed that CGP tests might be useful for detecting gene alterations in various cancer types and exploring treatment options. However, many issues still require improvement, including better access to investigational and off-label use drugs, standardization of MTB, and understanding of hereditary tumors.

All Patients data.

This table contains all patient characteristics and genetic information.

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3 Dec 2021

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"Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

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We will update your Data Availability statement to reflect the information you provide in your cover letter.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments to the Author

The authors reported the clinical utility of CGP test in Japan using commercial targeted sequencing system, and they concluded it might be useful for detecting gene alterations in various cancer types and exploring treatment options.

Major comments:

1. The definition of actionability in this report is ambiguous. Please redefine the actionability using evidence levels in accordance with previously reported guidance.

2. Please mention the status of genomic medicine in Japan more exactly. For example, detection rate of actionable genomic alteration and implementation rate of genotype-matched treatment in other platforms such as OncoPrime, P5, PleSSision, CANCERPLEX, CLHURC and so on.

3. VUS should not be contained in Fig1A-G.

4. Please mention the limitation of this study.

5. The authors concluded that many issues still need to be improved in conclusion of the abstract. However, those many issues are not referred in this paper. Please discuss the problems of genomic medicine in Japan more.

6. Please attach all genomic information data of the patients in Supplement files.

Minor comments:

1. Please classify ‘Gastrointestinal cancer’, ‘Hepatic Biliary and Pancreatic cancer’, ‘Lung Cancer,’ ‘Gynecologic cancer’ into esophagus, stomach, colorectal, liver, biliary, pancreas, NSCLC, SCLC, ovary, uterus.

2. Patient No.19 should be removed from the lists of patients who received the treatment corresponding to the gene alteration in Table 3. This is not the case of genotype-matched treatment.

3. What is the definition of TMB-High in this report?

Reviewer #2: Overall, this study’s goal is to describe the utility of Clinical genomic sequencing in Japan, a country that historically puts significant limitations on who can receive NGS sequencing. As a consequence, limited data is available from this country regarding NGS clinical utility.

I think the data they have collected could be useful to have in the literature, but as of now, the authors present the data purely in an observational format without providing any context or statistical comparisons about how their data in Japanese populations compare to previously published Foundation CDx retrospective studies.

Given the only novelty of this paper would be how numbers of clinically relevant alterations compare in populations in Japan vs other populations, statistical comparisons between this study's population and others are important to include as part of the results in order to provide context.

I’m unsure about the use of the word “Actionability” in the context of their document. I believe the right phrase may instead be “Clinical Utility”. In my thought process, “actionability” would imply an alteration that would change the treatment options available to a patient. If possible, It would be helpful to report which of the alterations reported in Foundation Dx fall under “actionable”. Based on the frequency reported (including an average of 3 “actionable” alterations/patient) I have to assume many of these alterations are only clinically significant in their prognostic abilities, not in their ability to open therapeutic options. Examples of these are TP53 or KRAS, which are clinically useful from a prognostic standpoint, but I feel calling them “actionable” may be disingenuous. However, I do not feel strongly about this semantic distinction if it doesn't bother the editor or other reviewers.

From a recommendation standpoint, if they are able to provide statistical context for how the results in their population compare with previous Foundation CDx reports in USA and Europe, including alteration prevalence and power to detect statistical differences, I think the paper is worth adding to the literature via PLOSOne publication.

(more detailed comments in the attached document).

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Hideyuki Hayashi

Reviewer #2: Yes: Travis Zack

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: comments.docx

Click here for additional data file.

27 Feb 2022

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 
https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 
https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Reply: I have checked and corrected the above matters.

2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

Reply: I confirm and correct the above statement.

3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

"Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Reply: I upload our minimal underlying data set as Supporting Information files.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Reply: I correct the above matter.

Reviewer #1

The authors reported the clinical utility of CGP test in Japan using commercial targeted sequencing system, and they concluded it might be useful for detecting gene alterations in various cancer types and exploring treatment options.

Reply: We thank the Reviewer 1’s favorable comments.

Major comments:

1. The definition of actionability in this report is ambiguous. Please redefine the actionability using evidence levels in accordance with previously reported guidance.

Reply: We thank reviewer’s comment regarding actionability. This is an area of on-going debate. We added Fig 3 showing evidence level defined by C-CAT (The Center for Cancer Genomics and Advanced Therapeutics) and elaborated in discussion.

2. Please mention the status of genomic medicine in Japan more exactly. For example, detection rate of actionable genomic alteration and implementation rate of genotype-matched treatment in other platforms such as OncoPrime, P5, PleSSision, CANCERPLEX, CLHURC and so on.

Reply: We summarized recent reports of clinical utility of CGP in Japan and commented in discussion.

3. VUS should not be contained in Fig1A-G.

Reply: We revised the figure excluding VUSs.

4. Please mention the limitation of this study.

Reply: We thank the reviewer to remind us of including the limitation of the study. We added limitation in discussion.

5. The authors concluded that many issues still need to be improved in conclusion of the abstract. However, those many issues are not referred in this paper. Please discuss the problems of genomic medicine in Japan more.

Reply: We thank this reviewer for his remarks. We added the problems of genomic medicine in Japan in discussion.

6. Please attach all genomic information data of the patients in Supplement files.

Reply: We added Supporting Information files that includes key genomic data used in this study.

Minor comments:

1. Please classify ‘Gastrointestinal cancer’, ‘Hepatic Biliary and Pancreatic cancer’, ‘Lung Cancer,’ ‘Gynecologic cancer’ into esophagus, stomach, colorectal, liver, biliary, pancreas, NSCLC, SCLC, ovary, uterus.

Reply: Thank you for your suggestion. We revised figures with more detailed classifications.

2. Patient No.19 should be removed from the lists of patients who received the treatment corresponding to the gene alteration in Table 3. This is not the case of genotype-matched treatment.

Reply: Thank you for your comment. We removed the patient No. 19 from the table 3 and revised result and discussion sections.

3. What is the definition of TMB-High in this report?

Reply: TMB more than 10 muts/Mb is defined as TMB-high. In case No. 19, the patient had TMB 9 muts/Mb (intermediate), but since there were no other genetic alterations to target for treatment, after discussion among the experts in the field, it was decided to target TMB for treatment. Case No. 24 showed a mutation in the MSH6 gene. The TMB was 9, which is not in the TMB-High category by definition, but together with the MSH6 mutation, it was considered to be a target for treatment. Therefore, we have modified the expression TMB-High in Table 3, No.19 and No.24.

Reviewer #2

Overall, this study’s goal is to describe the utility of Clinical genomic sequencing in Japan, a country that historically puts significant limitations on who can receive NGS sequencing. As a consequence, limited data is available from this country regarding NGS clinical utility.

I think the data they have collected could be useful to have in the literature, but as of now, the authors present the data purely in an observational format without providing any context or statistical comparisons about how their data in Japanese populations compare to previously published Foundation CDx retrospective studies.

Given the only novelty of this paper would be how numbers of clinically relevant alterations compare in populations in Japan vs other populations, statistical comparisons between this study's population and others are important to include as part of the results in order to provide context.

Reply: Thank you for your suggestion. We tried statistical comparison to foreign literatures. But it was difficult. We added this in limitation.

I’m unsure about the use of the word “Actionability” in the context of their document. I believe the right phrase may instead be “Clinical Utility”. In my thought process, “actionability” would imply an alteration that would change the treatment options available to a patient. If possible, It would be helpful to report which of the alterations reported in Foundation Dx fall under “actionable”.

Based on the frequency reported (including an average of 3 “actionable” alterations/patient) I have to assume many of these alterations are only clinically significant in their prognostic abilities, not in their ability to open therapeutic options. Examples of these are TP53 or KRAS, which are clinically useful from a prognostic standpoint, but I feel calling them “actionable” may be disingenuous. However, I do not feel strongly about this semantic distinction if it doesn't bother the editor or other reviewers.

Reply: Thank you for your comments. We explain the definition of actionable in discussion.

From a recommendation standpoint, if they are able to provide statistical context for how the results in their population compare with previous Foundation CDx reports in USA and Europe, including alteration prevalence and power to detect statistical differences, I think the paper is worth adding to the literature via PLOSOne publication. (more detailed comments in the attached document).

Reply: Thank you for your suggestion. We tried statistical comparison to foreign literatures. But it was difficult. We added it in limitation.

We also thank the review #2 for suggested changes in the manuscripts. We included these suggestions in the revised manuscript.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

15 Mar 2022

Clinical utility of Comprehensive Genomic Profiling in Japan: Result of PROFILE-F study.

PONE-D-21-35160R1

Dear Dr. Aoyagi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Hyunseok Kang, MD, MPH

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors reported the clinical utility of CGP test in Japan using commercial targeted sequencing system, and they concluded it might be useful for detecting gene alterations in various cancer types and exploring treatment options. The authors replayed to all of my comment.

Reviewer #2: I appreciate authors detailed in addressing concerns I brought up and for the diligence in adding the context of previous NGS clinical sequencing reports from other populations in how they compare to this particular study.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Hideyuki Hayashi

Reviewer #2: No

23 Mar 2022

PONE-D-21-35160R1

Clinical utility of Comprehensive Genomic Profiling in Japan: Result of PROFILE-F study.

Dear Dr. Aoyagi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

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on behalf of

Dr. Hyunseok Kang

Academic Editor

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