Favorable response to immunotherapy in a pancreatic neuroendocrine tumor with temozolomide-induced high tumor mutational burden

1 INTRODUCTION

Pancreatic neuroendocrine neoplasms (PanNENs) are morphologically grouped into well-differentiated neuroendocrine tumors (PanNETs) and poorly-differentiated neuroendocrine carcinomas (PanNECs) [1]. NETs are graded as G1, G2, or G3 based on their proliferative activity [1]. Besides immunohistochemistry (IHC) tests, genetic tests also play important roles in PanNENs diagnosis [2].

Treatments for unresectable or metastatic PanNENs depend on the tumor pathology [3], while the treatment for well-differentiated PanNET G3 follows that of PanNET G2, i.e. use of the capecitabine/temozolomide (CAPTEM) regimen [4], and second-line treatments including everolimus [5] and sunitinib [6]. For poorly-differentiated PanNET G3, platinum-based chemotherapy is often preferred [7]. Currently, immunotherapy is not yet approved for PanNETs as clinical trial data have suggested an unpromising response [8], but recently a 75% objective response rate was reported in NET with high tumor mutational burden (TMB) (≥ 9.9 mutations/Mb) [9].

Here, we present the case of a patient diagnosed with metastatic PanNET G3 who had characteristic gene mutations along multiple lines of treatments.

2 CASE REPORT

A 49-year-old female (Figure 1A) (BMI, body mass index; 16.82) presenting epigastric pain underwent magnetic resonance imaging, which showed a tumor at the pancreatic tail, with enlarged regional lymph nodes, and tumor thrombosis within the portal and splenic vein (Figure 1B). An ultrasound-guided biopsy of the pancreatic tumor revealed a well-differentiated NET (Figure 2A) with high proliferation (Ki-67 index: 35%+; Supplementary Table S1). ⁶⁸Ga-DOTATATE-PET/CT showed a high expression of the somatostatin receptor (SSTR) in all lesions. After a multi-disciplinary team discussion, the patient was diagnosed with PanNET G3 on January 22, 2018. Comorbidity included hyperlipidemia and was treated with atorvastatin.

First-line treatment with six cycles of CAPTEM plus somatostatin analogs (SSA) (capecitabine 1 g Qd 1.5 g Qn, d1-14; TMZ 0.26 g Qd d10-14; SSA 30 mg d1 Q28d) led to a partial response (PR) and relief of symptoms. Adverse events included grade 2 leukopenia and thrombocytopenia. Following that, 8 cycles of capecitabine plus SSA were administered as maintenance therapy and follow-up CT scans revealed stable disease (Figure 1C). However, after the tenth cycle of maintenance treatment, multiple new liver lesions were discovered ranging from 5 mm to 28 mm (Figure 1D). Given the favorable response of the first-line treatment, two cycles of CAPTEM plus SSA were re-challenged, but both ⁶⁸Ga-DOTATATE and ¹⁸FDG-PET/CT showed progressive disease (PD) (Figure 1E). An ultrasound-guided biopsy of the liver lesions was performed and showed higher proliferation (Ki-67 index: 80%+; Supplementary Table S1) compared to the primary PanNET (Figure 2B). Since the pancreatic lesion remained stable, re-biopsy was avoided to reduce unnecessary injuries.

Tissue samples of the primary PanNET, liver metastasis and plasma samples collected at baseline and after PD (Figure 1A) were subjected to targeted next-generation sequencing (NGS; Nanjing Geneseeq Technology Inc., Nanjing, Jiangsu, China) for 425 cancer-relevant genes, as described previously [10]. Circulating tumor DNA (ctDNA) mutation positivity increased upon PD (Figure 3A), and the TMB in PD plasma and liver metastasis samples increased dramatically (Figure 1A), and was found to be associated with the alkylation-induced mutational signature, Signature 11 (Figure 3B), similar to a previous report [11]. However, the mutation signature could not be identified in baseline samples due to the low mutation number (< 5 mutations). Moreover, a germline heterozygous nonsense mutation of MUTYH (p.W156*) was identified by NGS in all plasma collected samples (Supplementary Table S2). Fraction and allele-specific copy number estimates from tumor sequencing [12] analysis showed that loss of heterozygosity (LOH) was common in the primary PanNET, including chromosome 1p, where the MUTYH gene is located (Figure 3C). However, due to the low amount of ctDNA in the baseline plasma sample, we could not detect any mutations above the 0.5% cut-off adopted for the analysis, which was determined by the applied targeted NGS.

As liver metastasis was the major tumor burden, tumor arterial embolization (TAE) was applied to the liver lesions (Figure 1F). The patient then received toripalimab (Shanghai Junshi Bioscience Co., Ltd in China), a programmed death receptor-1 (PD-1) monoclonal antibody, 240 mg every 3 weeks, and achieved a PR (Figure 1G) by the follow-up visit in December 2019. The TMB (Figure 1A) and mutational allele frequency (AF; Figure 3A) were dramatically reduced after immunotherapy (median AF of PD plasma vs ICI plasma: 5.49% vs 0.35%). The anti-PD-1 treatment is still ongoing and has been administrated for 17 cycles. As of the last follow-up on September 3, 2020 (BMI 17.58), no significant adverse effects were observed and CT scans revealed stable diseases at the primary PanNET and liver metastases (Figure 1H).

3 DISCUSSION

Pathological diagnosis of the primary PanNET in this case report was challenging as it contained features of both NETs and NECs. The evidence of PanNET includes pathological morphology, Ki-67 index, SSTR2 expression, and the presence of ATRX mutation. However, the IHC and NGS results revealed the loss of RB1 in the primary pancreatic tumor and liver metastasis, which is a feature of PanNECs [2]. To our knowledge, this is the first case reporting the co-existence of mutations in the ATRX and RB1 genes, which are routinely used to distinguish PanNETs and PanNECs However, the liver lesion was poorly differentiated with a high Ki-67 index that resembled NECs, suggesting a possible but rare transformation from NET G3 to NEC [1].

NGS analysis contributed to the pathogenic diagnosis of this case, as we identified an inactivating germline mutation in the base-excision-repair gene MUTYH which was reported as a novel pathogenesis in PanNETs [14]. However, whether this monoallelic germline MUTYH mutation is the trigger for the genome-wide LOH remains to be determined. To illustrate the biological mechanisms causing high TMB in PD samples, we performed mutational signature analysis based on the combinations of characteristic mutation types. Signature 11, which is associated with alkylation due to TMZ treatment, may have contributed the most to the elevated TMB in this case [15]. However, the high TMB might also be attributable to other unknown mechanisms as we only performed mutational signature analysis for the samples with TMB > 10 mutations/Mb to ensure reliability. Due to different DNA extraction techniques and insufficient ctDNA release, the discordance between tissue TMB and blood TMB was reported [13] and could explain the inconsistent TMB values detected in the liver tissue and PD plasma.

In conclusion, we described a PanNET G3 case, with increasing TMB induced by TMZ-related alkylation, which presented a favorable clinical response to anti-PD-1 treatment. Based on the described observations, we suggest that the use of ICIs should be further explored as a treatment option for PanNET patients with increased TMB following TMZ treatment.

DECLARATIONS