Clinical responses to PD-1 inhibition and their molecular characterization in six patients with mismatch repair-deficient metastatic cancer of the digestive system

Purpose Immune checkpoint inhibitors have shown efficacy in patients with microsatellite instability-high/mismatch repair-deficient (MSI-H/dMMR) gastrointestinal (GI) cancers. However, depth and duration of clinical response is not uniform. We assessed tumor mutation burden (TMB) as a response marker in patients with GI cancers treated with immune checkpoint inhibitors. Methods Detailed clinical and response data were collected from six patients with metastatic MSI-H/dMMR GI cancers treated with immune checkpoint inhibitors. Efficacy was assessed by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Tumors and matched normal tissue were profiled by targeted next generation sequencing (127 gene panel, size 0.8 Mb). Impact of included mutation types, germline filtering methodology and different variant allele frequency thresholds on TMB estimation was assessed. Results Objective radiographic responses were observed in all six patients, and complete response was achieved in two of the six patients. Responses were durable (minimum 25 months). TMB estimates were clearly above the two recently reported cut-offs for metastatic colorectal cancer of 12 or 37 mutations per megabase for five of six patients, respectively, while one patient had borderline TMB elevation. TMB did not show an association with extent and duration of response but was influenced by included mutation types, germline filtering method and variant allele frequency threshold. Conclusion Our case series confirms the clinical benefit of immune checkpoint blockade in patients with metastatic MSI-H/dMMR GI cancers and illustrates the vulnerability of TMB as predictive marker in a subset of patients. Electronic supplementary material The online version of this article (10.1007/s00432-020-03335-2) contains supplementary material, which is available to authorized users.


Detailed report of clinical response data for individual patients
Patient 1, a 58-year-old male with adenocarcinoma of the cecum, underwent right hemicolectomy shortly after being first diagnosed in April 2015. Surgery turned out to be highly complex and led to incomplete tumor resection (R2). Histopathologic assessment showed a signet ring cell carcinoma with involvement of lymphatic vessels and venules as well as eleven out of fourteen lymph nodes positive for tumor cells.
The molecular genetic analysis showed KRAS mutation in exon 4. In June 2015, this patient started palliative first-line treatment with FOLFIRI plus anti-VEGF antibody bevacizumab and showed partial response in the performed imaging controls. In February 2016, therapy was switched to maintenance with 5-FU/FA and bevacizumab due to stable disease but then had to be escalated again to FOLFIRI plus bevacizumab in August 2016. Three months later, the CT scan showed massive progression of his abdominal lymph node metastasis with additional suspected peritoneal carcinomatosis, which led to initiation of checkpoint inhibition with anti-PD-L1 antibody pembrolizumab in common dose of 2 mg/kg body weight every three weeks.
The imaging controls every three months showed initially partial remission of the documented target lesions under PD-L1 inhibition and since January 2017 stable disease. The level of tumor marker CEA (carcinoembryonic antigen) decreased since beginning pembrolizumab from 1198 µg/l to 3 µg/l in May 2017 (normal range 0 to 5 µg/l). No further increase of tumor marker was detected. In January 2018, the therapy interval was stretched to four weeks. Unfortunately, immunotherapy had to be discontinued in March 2019 due to progressive disease. The CT scan showed an abdominal lymph node bulk between the mesenteric vessels with suspected malignant ascites. The patient was admitted to hospital for pain management and evaluation of further treatment. A salvage therapy with FOLFOX did not lead to clinical or radiological benefit. The patient died in May 2019 in the hospital due to massive progression of his tumor disease.
2 Patient 2, a 48-year old female, was diagnosed with adenocarcinoma of the splenic flexure in March 2016. The performed clinical staging showed no further tumor involvement and the patient underwent left hemicolectomy shortly after. As a consequence of the post-operative pathological staging with tumor growth through the visceral peritoneum (pT4a) but without lymph node metastases and without lymphatic or blood vessel infiltration, the patient received adjuvant treatment with capecitabine as monotherapy. Only three months after beginning of adjuvant treatment, the imaging control showed suspected abdominal lymph nodes next to the mesenterium and the left kidney, and one liver metastasis up to 3 cm of diameter.
The initiated treatment with PD-1 inhibitor nivolumab led to rapid shrinkage of the target lesions in the imaging controls. The liver metastasis showed cystic and necrotic transformation and no further tumor activity was noted since the second CT scan in November 2016; the abdominal lymph node metastases decreased to a size <1 cm. As a possible side effect of the immunotherapy, a mediastinal mass was discovered in November 2016, which was diagnosed as reactive thymic hyperplasia after checkpoint inhibitor therapy. No clinical symptoms associated to the thymic enlargement were reported by the patient. Also, no other side effects or therapy-specific toxicities were observed. Due to the excellent response, the checkpoint inhibition therapy was discontinued in March 2017 with ongoing remission in the latest imaging control in June 2019.
Patient 3, a 48-year-old male, was diagnosed with carcinoma of the cecum in September 2015 and received right hemicolectomy. Due to locally advanced stage IIIB (pT4a, N1 (1/25), L1, G3), adjuvant chemotherapy with FOLFOX was started in October 2015 and completed without major toxicities six months later. The first year of follow-up showed no tumor-associated events, but in July 2017 a new rectal tumor with measured size of 3.9 × 3.5 cm as well as locoregional suspected lymph nodes were found in the MRI scan. In the tumor conference, a rectal metastasis of the initial tumor disease or a metachronous secondary malignancy were discussed as differential diagnosis. In this case, an innovative therapeutic approach with neoadjuvant simultaneous radio-immunotherapy with radiation of the tumor area and the pelvic 3 lymph vessels plus nivolumab every two weeks was chosen as a potentially curative treatment. The patient underwent the treatment without major side effects with application of complete radiation dose of 50.4 Gy and also without any dose adjustments of nivolumab. After four weeks of simultaneous radio-immunotherapy, nivolumab was administered for another eight weeks alone until November 2017. The first follow-up with MRI and rectal endosonography after end of radioimmunotherapy showed a complete remission of the tumor manifestation. Therefore, normal range 0 to 37 kU/l) and 19 µg/l (CEA; normal range 0 to 5 µg/l) to a minimum of 50 kU/l and 1 µg/l in July 2018. Since then, a slight increase of CA19-9 level to recently 91 kU/l has been observed without any clinical or radiological signs of tumor progression. Hence, immunotherapy is continued. showed partial remission after cumulative eight cycles of pembrolizumab. After tumor conference, the patient was admitted to surgery for a potentially secondary curative approach. In March 2018, an explorative laparotomy was conducted. The intraoperative sonography did not confirm any suspect of intrahepatic metastases, 6 thus a local lymphadenectomy of suspected interaortocaval lymph nodes and in the area of the hepatic portal system was performed, including cholecystectomy due to macroscopic tumor suspicion. The histopathological analysis showed complete pathological response without evidence of any vital tumor cells. The patient recovered from surgery without complications and has been followed up every three months, yet without any sign of tumor recurrence.

Details on bioinformatic analysis and variant calling
Initial variant calling was done using GensearchNGS (version 1.6.84, Phenosystems, Wallonia, Belgium) with the following filter criteria: minimum (tumor allele) frequency 3%, minimum coverage 140, minimum variant reads 2, ignore 5 bases from read borders, minimum alignment quality 20, minimum base quality 20, masked with ROI file xgen-pan-cancer-targets.bed. Next, technical filtering was done using the strand bias filters, VarBalance and PosBalance, i.e., forward/reverse read balance (1 is best, 0 is worst) >0, the alternative allele frequency filter >0.10 to minimize FFPE artifacts, and the gene name filter to exclude CRIPAK which attracts multiple mapping reads. In analogy to the settings used for PCR-based amplicon sequencing was curated by excluding common polymorphisms that had a population frequency >1% in the Ensembl database as used by GenSearchNGS. Recurrent bioinformatic artifacts or non-pathogenic variants were taken to be those variants with a frequency of at least 4 of 55 in our local database. The resulting technically filtered variants were exported in vcf format and imported into the Agilent Alissa Interpret platform for annotation (COSMIC, gnomAD) and subsequent TMB calculation.