Reply from the authors

Wewish to thankDr Eso and colleagues for their insightful comments regarding our paper. Below are our responses to their comments. We explain the method of the microsatellite instability (MSI) analysis below. The MSI analysis was performed at a central, single laboratory with CLIA certification and CAP accreditation. Tumor and normal DNA from formalin‐fixed paraffin‐ embedded (FFPE) tissues were analyzed using the MSI test kit (FALCO), which includes fluorescent‐labelled primers for coamplification of five mononucleotide markers (NR‐ 21, BAT‐25, MONO‐27, NR‐24, and BAT‐26). WhenMSI was judged in onlyMSI‐High (MSI‐H) tumor tissue, MSI was judged as MSI‐H when another peak was found outside the quasi‐monomorphic variation range (QMVR). However, when tumor and normal DNA from FFPE tissues are analyzed, it is not necessary that another peak is found outside the QMVR. In theMSI test kit (FALCO), MSI was judged by comparison of alleles in tumor and normal DNAwhen tumor and normal DNA from FFPE tissues were analyzed. In this case, the presence of new alleles in tumor DNA that are not present in normal DNA indicates MSI. (The QMVRmethod is applied when only tumor DNA is used.) Tumors exhibitingMSI at two ormoremarkers were classified as MSI‐H. There are other methods that can be used to measure MSI, such as immunostaining for mismatch repair proteins or next‐generation sequencing‐based testing. Although there are no data on the incidence of hepatocellular carcinoma (HCC) assessed using either MSI‐H immunostaining of mismatch repair proteins or next‐generation sequencing‐based testing, it has been reported that concordance between polymerase chain reaction data and next‐generation sequencing‐based testing was 94% (17/18). Pembrolizumab was approved in Japan for the treatment of advanced or recurrent solid MSI‐H tumors, evaluated by the MSI test kit (FALCO), a companion diagnostic test that identifies patients whose tumor has progressed after systemic chemotherapy and are poor candidates for other treatment options. Our two patients were judged as having MSI‐H tumors by FALCO because two or more markers showed instability. One of these patients classified as MSI‐H by FALCO with an advanced HCC showed a complete response to pembrolizumab. We thank Dr Eso and colleagues for their interest in our report. In the future era of immune checkpoint inhibitors we hope to determine the factors that affect the response of immune checkpoint inhibitors.

[1]  M. Imamura,et al.  Incidence of microsatellite instability‐high hepatocellular carcinoma among Japanese patients and response to pembrolizumab , 2020, Hepatology research : the official journal of the Japan Society of Hepatology.