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Abstract

Background:

Germline pathogenic variants (GPV) in MUTYH are rare in patients with pancreatic neuroendocrine tumors (PanNET).

Objectives:

We aimed to characterize PanNET patients with GPV and/or somatic pathogenic variants (SPV) in MUTYH.

Design:

Retrospective multicenter cohort.

Methods:

Patients with PanNET harboring MUTYH pathogenic variants (MUTYH+) were identified from centers in Brazil, Europe, and the USA. Data were extracted from medical records. Germline or somatic variants were evaluated by targeted sequencing of cancer-associated genes. The primary endpoint was to clinically characterize these patients and the secondary endpoint was overall survival (OS). Aggressive behavior was defined as rapid progression, transformation to neuroendocrine carcinoma (NEC)-like histology and/or OS of <2 years from first-line treatment. All cases were diagnosed by expert pathologists.

Results:

In total, 23 patients with MUTYH+-associated PanNET were identified (17 germline; 4 somatic). Median age was 48 years (22–72), 65% were male, 12 (52%) had first-degree family history of cancer, and 70% had synchronic metastatic disease. Tumors were of G1, G2, and G3 in 17%, 52%, and 31%, of cases, respectively, and median KI-67 was 12% (2%–80%). Seven of 11 (64%) G1/G2 PanNET with tumor biopsy upon progression evolved to G3 (3 NEC-like). Median OS of 19 patients with metastases was 4.6 years (95% confidence interval (CI): 2.4–6.8), with 12 (63%) cases demonstrating aggressive behavior. MUTYH p.Gly396Asp and p.Tyr179Cys were the most frequent variants (mostly GPV). Of 13 tumors arising in patients with germline MUTYH mutations and tested for loss of heterozygosis of the wild allele, 6 (50%) exhibited a second hit event in MUTYH, with 4 demonstrating aggressive behavior. Tumor mutation burden (TMB) was low in the six treatment-naïve PanNET (TMB: 0–9) with this information, but three of seven tumors molecularly profiled after alkylating drugs and/or radioligand therapy had high TMB (TMB: 97, 65, and 728).

Conclusion:

MUTYH mutation-associated PanNET (germline and/or somatic) is associated with aggressive and high-grade disease when metastatic.

Keywords

germline mutation MUTYH neuroendocrine tumors pancreatic

Introduction

Pancreatic neuroendocrine tumors (PanNET) are rare neoplasms associated with heterogeneous symptoms, behavior, and evolution. When metastatic, median overall survival (OS) varies significantly, depending on several factors, including tumor grade. For instance, patients with grade 1 or 2 metastatic PanNET have a median OS of 5 years,¹ while those with grade 3 PanNET, typically survive for a median of 2–3 years.² Yet, even within the tumor grade subgroups, heterogeneous outcomes are observed, encouraging the investigation of further prognostic biomarkers.

Molecular profiling studies in PanNET have identified somatic pathogenic variants (SPV) in genes involved in four main pathways: chromatin remodeling, DNA damage repair, activation of mammalian target of rapamycin (mTOR) signaling, and telomere maintenance.^3,4 ATRX/DAXX and MEN1, genes involved in chromatin remodeling, are the most common somatic mutations (40%–50% of sporadic PanNET), followed by alterations in genes involved in the mTOR pathway (14%–25%).^3,5 Additionally, whole genome sequencing has revealed that up to 17% of patients with PanNET also harbor germline pathogenic variants (GPV), including newly identified variants in DNA repair genes MUTYH, CHEK2, and BRCA2.^4–6 However, given their rarity, the clinical implications of such GPV have not been fully evaluated. SPV in MUTYH appear to be relatively rare, but anecdotally, there is a perception of higher aggressiveness among cases of MUTYH mutated advanced PanNET (SPV and/or GPV).

MUTYH is a base excision repair gene, involved in the protection of DNA from mutations caused by oxidative stress. Germline biallelic inactivation of MUTYH causes the autosomal recessive MUTYH-associated colorectal polyposis syndrome and is associated with somatic G:C > T:A transversions in the APC gene, the driver of colorectal polyps and adenocarcinoma.⁷ There is also an increased risk of duodenal, ovarian, and bladder cancers in biallelic carriers,⁸ while monoallelic GPV carriers may have increased risks for gastric, hepatobiliary, and endometrial cancer.^9,10 Recently, it has been demonstrated that MUTYH deficiency caused by GPV also occurs in PanNET,⁴ but how this affects the biological behavior of these tumors remains undetermined.

Here, we have undertaken a multicenter and retrospective cohort of patients with PanNET and GPV and/or SPV in MUTYH with the objective to evaluate their clinical characteristics and outcomes.

Methods

Patients with histologically proven diagnosis of well-differentiated PanNET of any grade and stage, and a GPV or SPV in MUTYH (MUTYH+) were retrospectively identified from institutional databases of centers in Brazil, Europe, and USA. All cases were classified by pathologists who are experts in the diagnosis of neuroendocrine neoplasms (NEN). The reasons underlying the identification of MUTYH variants were: (1) consecutive unselected cases (AC Camargo Cancer Center, Haukeland University Hospital, Memorial Sloan Kettering Cancer Center, and University of California), meaning that all patients with a diagnosis of PanNET were enrolled in research projects of germline testing, regardless of family or personal history of cancer; or (2) patients with aggressive PaNET, where the attending physician ordered an NGS somatic panel to guide treatment decisions on targeted therapies (Moffitt Cancer Center and one case from AC Camargo Cancer Center). Neuroendocrine carcinomas (NEC) at diagnosis were excluded. Most commonly, GPV were identified from screening of the consecutive and unselected cases with neuroendocrine tumors (e.g.,), and SPV were identified through tumor comprehensive genomic profiling of aggressive cases (Table 1).

Table 1.

Overview of MUTYH GPV and/or SPV in PanNET cases with multigene germline testing and/or tumor comprehensive genomic profiling.

Consecutively screened unselected patients tested
Center	Population screened	Number of PanNET cases tested for GPV ± SPV: present/absent	SPV tested in GPV carriers:present/absent
ACC, Brazil	108: cases of NEN younger than 50 years	43: 5/38	3: 1 (VAF 86%)/2
HUS, Norway	240 consecutive cases of G3 NEN tested for GPV	21: 1/20	Not done
MSK, USA	312 consecutive PanNET	312: 4/305	Not done
UCSF, USA	165 consecutive G3 NEN	26: 2 GPV/23 1 SPV (VAF 24%)/23	1: 1 (VAF unknown)/0
Total	402	13	–
Cases selected based on aggressive disease or other clinical indication
Center	Cases	SPV tested	GPV tested in those with SPV	GPV and SPV present
Moffitt, USA	3 PanNET cases	3	1	1 (VAF 69.3%) 1 SPV (VAF 20%) 1 SPV (VAF 82%; germline suspected)
ACC, Brazil	1 PanNET case	1 (VAF 5%)	1	0 (GPV not found)
Cases selected based on different research projects—SPV and/or GVP tested
Center	Cases	SPV testedGPV tested	GPV tested in those with SPV;SPV tested in those with GPV	GPV and SPV present
VR, Italy	103 Not consecutive NEN	4 (VAF 51%, 46%; 76% (germline suspected), 67% (germline suspected)) 2	Not done Not done	– –

ACC, AC Camargo Cancer Center, São Paulo, Brazil. HUS, Haukeland University Hospital, Bergen, Norway. MSK, Memorial Sloan Kettering Cancer Center, New York City, USA. Moffitt, Moffitt Cancer Center, Tampa, USA. UCSF, University of California San Francisco, San Francisco, USA. VR, University and Hospital Trust of Verona, Italy.

GPV, germline pathogenic variants; NEN, neuroendocrine neoplasm; PanNET, pancreatic neuroendocrine tumor; SPV, somatic pathogenic variants; VAF, variant allele frequency.

Because we were interested in evaluating the clinical course in patients with MUTYH+ PanNET, we classified the clinical behavior of each case as either aggressive or indolent. A case was deemed aggressive if it was rapidly progressive (at diagnosis or during the disease course), required treatment with chemotherapy, had NEC-like behavior, or survival of less than 2 years from diagnosis of metastatic disease. These criteria were agreed upon by all of the authors, all with clinical experience in the treatment of NET patients.

The following clinical characteristics and treatment-related outcomes were extracted from medical records: age and tumor stage at diagnosis, sex, race, tumor grade (by the World Health Organization 2019 classification)¹¹ and histological differentiation at diagnosis, and upon progression on cancer-directed treatments (if this information was available), tumor functionality, family and personal history of cancer, performance status, sites of metastases and tracer uptake on somatostatin-receptor imaging and/or ¹⁸F-FDG-PET, type of treatments, response and duration of response, dates of therapies, and date of death or last follow-up.

Multigene germline testing and tumor comprehensive genomic profiling were done in the context of research or standard of care using a variety of commercial or institutional platforms. All panels included genetic sequencing of MUTYH. Detailed information on MUTYH analyzes and tumor biomarkers were gathered, if available. For GPV and SPV carriers, we collected data on reference transcripts, variant allele frequency (VAF) in germline and somatic testing, microsatellite instability status, and tumor mutational burden (TMB) status. Occurrence of double hit in MUTYH was considered when the VAF indicated loss of heterozygosity (LOH) of the wild-type allele in the tumors (VAF >65%).¹²

Statistics and endpoints

Our primary endpoint was to clinically characterize MUTYH+ (SPV and/or GPV) PanNET cases. The secondary endpoint was OS from first-line systemic therapy. Descriptive statistics were used to summarize the population characteristics. OS was estimated by the Kaplan–Meier method and calculated from the date of diagnosis of metastatic disease (date of biopsy or radiological evidence of metastases, whichever came first) until death from any cause. The follow-up time was estimated by the Reverse Kaplan–Meier method. All analyses were performed using the STATA version 17, StataCorp LLC.

Ethical aspects

Each institution had ethical approval for their cases and/or chart review.

The EQUATOR guideline was followed to report our results.

Results

Clinical characteristics

Considering the centers who performed germline testing for consecutive patients, 12 (2.9%) out of 402 PanNET patients harbored a MUTYH GPV and one, a SPV. Ten additional selected cases were identified by somatic next-generation sequencing tests which were performed for clinical reasons or availability of research projects (Table 1).

Overall, our population is composed of 23 cases, with 17 patients presenting a PanNET in the setting of a confirmed (N = 14) of very likely (N = 3; VAF in the tumor >65%) MUTYH GPV.

Median age of MUTYH+ PanNET patients was 48 years, 65% were male, 8% had a personal history of cancer, 65% had family members with cancer, and 70% of patients were metastatic at diagnosis (Table 2). Seven patients were diagnosed with localized PanNET: one patient presented metastatic disease after 17 months from surgical resection of the primary tumor, and another, 24 months after the initial diagnosis; 5 (22%) patients remain recurrence-free at a median follow-up of 20 months.

Table 2.

Baseline characteristics of patients.

Variable	MUTYH mut PanNET (N = 23, 100%)
Age (median, range)	48 (22–72)
Male sex	15 (65%)
Race Caucasian	22 (96%)
Initial KI-67 (median, range)	12 (2–80)
Initial tumor grade
G3 NET^a	7 (31%)
G2	12 (52%)
G1	4 (17%)
G1/2 NET which became clinically aggressive	9/16^b (56%)
Hormone secretion	6 (26%)
Gastrin	2 (9%)
Insulin	1 (4%)
VIP	1 (4%)
ACTH	1 (4%)
Gastrin + glucagon	1 (4%)
Stage IV at diagnosis	16 (70%)
Liver metastases^c	17 (74%)
Bone metastases^c	6 (26%)
Family history of cancer	15 (65%)
Personal history of cancer	2 (8%) Skin and atypical lung carcinoid

Two patients with G3 NET were considered ambiguous morphology G3 NET-NEC (#7 MCC and #8 MCC on Table 3).

From 16 G1/2 NET, 9 became clinically aggressive (rapidly progressive). Of these, 8 had tumor biopsy upon progression. Seven cases remained indolent and did not perform another tumor biopsy. One patient with initial G2 NET showed an aggressive progression of disease but did not undergo a biopsy to investigate if the tumor evolved to G3 NET.

Liver and/or bone metastases could be present at diagnosis or emerge during disease course.

ACTH, adrenocorticotropic hormone; PanNET, pancreatic neuroendocrine tumor; VIP, vasoactive intestinal peptide.

Nearly one-third (31%) had initial grade 3 NET (two of them with ambiguous G3 NET/NEC histology) and 56% of initially grade 1 or 2 tumors became clinically aggressive NET. Of 11 patients with grade 1 or 2 initial PanNET who had tumor biopsy performed upon progression, 7 (64%) evolved to G3 (3 NEC-like).

Twenty-six percent of patients had functioning PanNET (including 13% non-functional at diagnosis which became functioning). Two patients had gastrinomas at diagnosis and one had an ACTHoma at diagnosis; the other cases developed hormone-secretion upon tumor progression: one had a non-functioning G3 (KI-67 25%) PanNET which became an insulinoma after progression on first-line lanreotide; one patient had a non-functioning G2 (KI-67 5%) PanNET which became a VIPoma after progression on second-line everolimus, and one G2 PanNET became a gastrin and glucagon-secreting G3 NET (ambiguous histology NEC-like features) after progression on second-line GEMOX, 18 months after the initial diagnosis.

Genomic alterations

Genomic characterization of 13 of the 23 patients showed that the MUTYH p.Gly396Asp pathogenic variant was the most frequent (30%; seven patients, with four cases with a GPV only, and two with both GVP and SPV), followed by p.Tyr179Cys (four patients, all GPV). Among six patients tested for both GPV and SPV, three presented both variants. Three additional cases were considered to harbor a double hit event in MUTYH in their PanNET because of a VAF >65%. Eighteen of 23 patients had tumors evaluated for microsatellite stability, with all being MSS.

Thirteen patients had tumors molecularly profiled either before any systemic treatment (N = 5) or upon progression on alkylating agents and/or a peptide-receptor radionuclide therapy (PRRT; N = 8). Overall, the most common alterations were found in genes associated with chromatin remodeling (ATRX, DAXX, ARID1A), histone methyltransferases (MEN1), and mTOR pathway (PTEN, TSC2). TMB varied from zero to more than 700 mutations per megabase, depending on the timing of NGS: treatment-naïve PanNET harbored exclusively low TMB, while hypermutation was exclusively identified in tumors profiled after treatment with alkylating agents and/or PRRT, and presented defects in mismatch repair genes (MLH1, MSH2, MSH6; Table 3).

Table 3.

Genomic and clinical characterization of patients with PanNET and GPV and/or SPV in MUTYH.

Case	Sex/age at diagnosis (years)	Initial grade (KI-67%)	Functionality	Family and personal history of cancer	MUTYH mutant	Germline Zygosity	Genetic alteration in MUTYH	Double hit MUTYH	Somatic mutation VAF (%)	MSI status	Other genomic alterations; timing of somatic NGS	Initial stage/first-line treatment	Treatments and outcomes	Aggressive behavior?
#1 ACC	F/38	2 (5)	Initially nonfunctioning; became VIPoma	Father (skin unk histology at unk age); Mother (thyroid skin unk histology at unk age); none	G	HT	p.Gln29Ter	NA	NA	NA	NA	4/SSA	Initially G2 NET; became aggressive when VIPoma/SSA for 12 m, then discontinued due to PD; 2 L everolimus for 14 m with partial response; upon PD, became VIPoma; SSA reintroduced with clinical benefit, after 6 m increased dose to 60 mg; Lu¹⁷⁷ with limited benefit and death.	Yes. Became aggressive upon progression on everolimus, transforming into a VIPoma which was refractory to PRRT and SSA. Lived for 4 years–18 m after VIPoma diagnosis.
#2 ACC	M/22	3 (23)	ACTHoma	Sister (breast at 28 yo—same MUTYH GPV but LOH negative in breast cancer); maternal grandmother (breast at old age); none	G	HT	c.504+19_504+31del (p.spl?)	NA	NA	NA	NA	4/PE	PD upfront with PE; 2 L AF, clinical benefit for 12 m; 3 L Lu¹⁷⁷ then PD and death.	Yes. Clinically aggressive since diagnosis, with severe Cushing syndrome and upfront progression with platin-based chemo and PRRT. Lived for 3 years since metastatic.
#3 ACC	F/42	3 (25)	Initially nonfunctioning; became Insulinoma	Father with MUTYH GPV (without colonic polyposis); mother (breast at 55 yo); paternal grandfather (prostate 63 yo); maternal grandfather (lung 82 yo); none	G	HT	p.Tyr179Cys	No	48 (tumor)	MSS/pMMR	NA	4/SSA	PD after 2 m with SSA becoming insulinoma; clinical benefit on 2 L everolimus for 2 years as of Dec/2024.	No.
#4 ACC	F/46	2 (12)	No	None	S	NA	c.36+1G>A (p.spl?)	No	5 (ctDNA) MUTYH not found in tumor	MSS/ pMMR	MLH1 loss, ATRX, CDKN1A; TMBh = 97 Mut/Mb Tumor tissue confirmed TMBh and SBS11 mutational signature in metastasis (post ALK, upon PD)	4/SSA	1 L with SSA for 12 m then oligoPD treated with 13 cycles of CAPTEM with major response → R0 resection of primary and liver mets. Surgical tumor KI-67 90% and TMBh in ctDNA. Four months later, rapid PD. Treated with 7 cycles of PE with SD. PD treated with FFX for 6 cycles with partial response but limiting tox. Treated with pembro with SD for 9 cycles then PD. Ipilimumab + nivolumab com complete response but G3 hepatitis. Still in remission after 18 m on nivolumab as of Dec./2024.	Yes. Became aggressive, rapidly progressive after CAPTEM, with NEC-like behavior.
#5 ACC	M/31	3 (70)	No	Maternal grandmother (kidney 60 yo)	G/S	HT	p.Ala357fs	Yes	49 (primary tumor) 86 (metastasis)	MSS/pMMR	MUTYH SBS36 mutational signature in metastasis (treatment naïve)	4/FFX	FFX for 9 m with partial response; Y-90 on liver lesions, with SD for 7 m; rechallenge with FFX for 4 m, then PD and death.	Yes. Very aggressive, rapidly progressive, and NEC-like behavior since diagnosis. Lived for 18 m.
#6 ACC	M/40	2 (19)	No	Father (colon at 60 y)	G	HT	p.Arg241Trp	No	NA	MSS	NA	2/SSA	Localized resected G2 NET at diagnosis. Liver mets 2 years after. 1 L SSA for 9 m with PD on Oct./2024.	Unk, short follow-up.
#7 MCC	M/38	3 (80) NEC-NET G3 ambiguous morphology	No	Mother (breast cancer 66 yo) and maternal grandmother (gastric cancer at unk age)	S	NA	p.Gln261	Yes	82 (tumor)	MSS/pMMR	DAXX, PIK3CA, RB1; TMB = 9 mut/mb (treatment naïve)	4/PE	PE with SD but no clinical benefit; then ALK and FU with PD upfront.	Yes. Very aggressive since diagnosis, progressive, and NEC-like behavior. Lived for 7 m.
#8 MCC	M/44	3 (25) NEC-NET G3 ambiguous morphology	No	Mother (breast cancer at 40 yo)	G/S	HT	p.Gly396Asp	Yes	69 (tumor)	MSS/pMMR	BCOR, DNMT3A, GHF, HNF1A, MAP3K13, MSH2, NF1, TP53; TMB = 6 mut/mb (treatment naïve)	2/Pembro	Localized resected G2 NET at diagnosis, developed metastasis after 17 m; PD upfront with pembrolizumab; RT for some lesions and lost FUP.	Yes. Aggressive since diagnosis, NEC-like behavior. Unk survival due to loss of follow-up.
#9 MCC	F/39	2 (5)	No	Two maternal aunts (breast cancer, 65 and 70 yo)	G/S	HT	p.Gly396Asp	No	20 (tumor)	MSS/pMMR	No genomic alterations; TMB = zero (treatment naïve)	4/SSA	PD upfront with SSA; SD with AF for 6 m then PD; 3 L Lenvatinib + pembrolizumab, with SD for 13 months; after PD, 4 L everolimus with SD for 7 m. Last FUP Nov./2023.	No.
#10 HUS	M/72	2 (NA)	NA	None	G	HT	p.Tyr176Cys	NA	NA	MSS/pMMR	NA	3/A	Initial G2 NET resected which evolved to G3 locally advanced disease. 1 L with ALK with SD for 12 m.	Yes. Aggressive, rapidly progressive from diagnosis of advanced disease. Lived for 15 m.
#11 UCSF	M/65	1 (1)	No	None	G/S	HT	p.Gly396Asp	Yes	NA	NA	KRAS, MEN1, NF1, VHL, PTEN; TMB = NA (post ALK, upon PD)	4/SSA	G1 NET at diagnosis. Liver biopsy 4 y after diagnosis demonstrated G3 PanNET. PD upfront with SSA; AF on 2 L with clinical benefit for 5 m; TACE on some lesions; everolimus with SD.	Yes. Aggressive, with NEC-like behavior. Lived for 4.4 years when lost follow-up.
#12 UCSF	F/54	2 (12)	No	Father (colon cancer unk age)	G/S	HT	p.Pro402Leu	NA	NA	NA	DAXX, TSC2, SETD2; TMB = NA (post-PRRT)	4/Lu¹⁷⁷	G2 NET at diagnosis. Liver biopsy 3 y later showed progression to G3 with NEC-like morphology; Lu¹⁷⁷ with SD and PD after 12 m; started AF with PD upfront; SD with PE for 9 m; after PD, used everolimus, CP, and anti-PD-1 with PD upfront on all these treatments.	Yes. Initially indolent but then aggressive became G3 NET with NEC-like behavior. Lived for 4.8 years–only 4 months after G3/NEC diagnosis.
#13 UCSF	M/56	2 (4.5)	Gastrinoma	Personal history of skin cancer (unk histology)	S	NA	c.129C>T	No	24 (tumor)	MSS/pMMR	MEN1, TP53, CDKN2B, EGFR, MSH2; TMB = 728 mut/mb (post-ALK)	4/SSA	SSA for 5 m; after PD, showed clinical benefit with AF for 18 m, then PD with TMBh and aggressive course with PD upfront on pembrolizumab.	Yes. Initially indolent. Became aggressive after CAPTEM, rapidly progressive, TMBh refractory to immunotherapy. Lived for 2.7 years–14 months from aggressive transformation.
#14 MSK	M/55	2 (15)	Gastrinoma	Mother (breast cancer, 78 yo), maternal aunt (breast cancer, 80 yo), maternal 1st cousin (prostate cancer, unk age), paternal aunt (breast cancer, 58 yo), paternal uncle (stomach, lung cancer, lymphoma 84 yo), paternal 1st cousin (pancreas adenocarcinoma, 70 yo)	G	NA	p.Tyr179Cys	NA	NA	MSS/pMMR	NA	4/SSA	SSA for 20 m with clinical benefit, plus TAE; after PD, 2 L with everolimus, with clinical benefit for almost 4 y; after PD, AF for 28 m with clinical benefit; after PD, Lu¹⁷⁷ for 30 m; after PD, Lenvatinib for 32 m. Alive for 15 years as of Feb./2024.	No
#15 MSK	M/61	2 (20)	Initially nonfunctioning; became gastrinoma, glucagonoma	Father (prostate cancer, 70 yo), sister (breast cancer, 42 yo)	G	NA	p.Gly396Asp	NA	NA	MSS/pMMR	NA	4/AF	AF for 10 m with clinical benefit; after PD, GEMOX for 9 m associated with HAE, with clinical benefit. 18 m after diagnosis, a new hepatic biopsy showed a acinar-NEC histology and the tumor started to secrete gastrin and glucagon when SSA and TACE were co-administered.	Yes. Aggressive after CAPTEM, with NEC-like behavior and secretion of gastrin and glucagon. Lived for 2.2 years.
#16 MSK	M/44	2 (10)	No	None	G	NA	p.Tyr179Cys	NA	NA	MSS/pMMR	NA	2	Localized disease; pancreatectomy with adjuvant CAPOX. Alive without recurrence for 19 months as of Jul./2024	No
#17 MSK	M/69	1 (1)	No	Mother (colon cancer, 62 yo)	G	NA	Deletion of exons 4–16	NA	NA	MSS/pMMR	NA	2	Localized disease, pancreatectomy. Alive without recurrence for 21 months as of Jul./2024	No
#18 VR	F/46	1 (2)	No	None	S	HT	p.A385PfsTer23	No	51 (ctDNA)	MSS/pMMR	Only VUS TMB = zero (ctDNA; post PRRT)	4/Lu¹⁷⁷	SD with Lu¹⁷⁷ for 2.2 years as of July/2024.	No
#19 VR	F/53	2 (20)	No	None	S	HT	Splice site	Yes	67 (tumor)	MSS/pMMR	DAXX, MEN1, BLM, NF1, MSH6, CHEK2; TMB = 65.9 mut/mb (post-ALK and PRRT)	4/AF	SD with AF for 8m; after PD, Lu¹⁷⁷ with SD for 12 m; after PD, AFP for 4 m; after PD, started Ipi + Nivo with complete response for 14 m as of July/2024. Alive for 5.4 years as of July/2024.	Yes. Became aggressive, with TMBh, and rapidly progressive after alkylating agent and PRRT but achieved complete remission with immunotherapy.
#20 VR	M/57	3 (25)	No	Unk	G/S	HT	p.Tyr179Cys	Yes	76 (tumor)	MSS/pMMR	PTEN, DAXX, IKZF1; TMB = 10 mut/mb (post PRRT and platin)	4/AFP	AFP with partial response for 25 m; after PD, Lu¹⁷⁷ for 13 m with partial response; after PD, sunitinib for 13 m with SD; after PD, ALK for 4 m. Lived for 4.6 years.	No
#21 VR	M/59	2 (8)	No	Father (colorectal cancer, unk age)	S	HT	p.Gly396Asp	No	46 (tumor)	MSS/pMMR	ARID1A, DAXX, EP300; TMB = NA (post PRRT)	3	Neoadjuvant FOLFOX/CAPOX, followed by surgical treatment. Alive without recurrence for 6.2 years as of July/2024	No
#22 VR	M/48	2 (3)	No	Paternal uncle (pancreatic adenocarcinoma 75 yo). Patient without other tumors but carrier of TSS and familial adenomatous polyposis	G	HT	p.Gly396Asp	NA	NA	NA	NA	4/Lu¹⁷⁷	1L Lu¹⁷⁷ with partial response for 41 m; after PD, 2 L everolimus with SD for 17 m as of July/2024.	No
#23 VR	F/65	1 (2)	No	Father (head and neck, 42 yo), Paternal brother in law (prostate cancer, 50 yo), paternal sister in law (renal cancer (40 yo); atypical lung carcinoid KI-67 5%.	G	HT	p.Gly396Asp	No	51 (ctDNA)	MSS/pMMR	CHEK2; TMB = 1 mut/mb (treatment naïve)	1/SSA	Only surgery of the primary NET in Feb. 2024	No

AF, alkylating agent + fluoropyrimidine: capecitabine + temozolomide (CAPTEM) or dacarbazine + 5-fluorouracil; AFP, dacarbazine + capecitabine + cisplatin; ALK, alkylating agent (monotherapy): dacarbazine or temozolomide; CP, carboplatin + paclitaxel; F, female; FFX, FOLFIRINOX; FU, fluoropyrimidine; FUP, follow up; G, germline; GO, gemcitabine + oxaliplatin; GPV, germline pathogenic variants; HAE, hepatic arterial embolization; HT, heterozygosity; Ipi + nivo, ipilimumab + nivolumab; LOH, loss of heterozygosis; Lu¹⁷⁷, Lutetium¹⁷⁷; M, male; MSI, microsatellite instable/deficient mismatch repair; MSS/pMMR, microsatellite stable/proficient mismatch repair; NA, not accessed; NEC, neuroendocrine carcinoma; NET, neuroendocrine tumor; NGS, next generation sequencing; PanNET, pancreatic neuroendocrine tumor; pembro, pembrolizumab; PD, progression of disease; PE, cisplatin + etoposide; PRRT, peptide-receptor radionuclide therapy; RT, radiotherapy; S, somatic; SD, stable disease; SPV, somatic pathogenic variants; SSA, somatostatin analog; Sun, sunitinib; TACE, transarterial chemoembolization; TAE, transarterial embolization; TMB, tumor mutation burden; tox, toxicity; VAF, variant allele frequency; unk, unknown; VUS, variant of unknown significance; yo, years old.

Clinical outcomes

Seven patients presented with initial localized disease, two had a later recurrence. Nineteen patients had metastatic disease. The median follow-up of all cases was 11.6 years. Systemic treatments varied and included somatostatin analogs, cisplatin-based chemotherapy regimens, alkylating-based therapies, mTOR inhibitor, and PRRT. Somatostatin analogs were the most frequent initial treatment (44%), followed by alkylating-based therapies (CAPTEM or dacarbazine plus 5-fluorouracil; 17%).

Median OS from first-line treatment of 19 metastatic patients was 4.6 years (95% CI: 2.4–6.8). Among these 19 metastatic patients, 12 (63%) exhibited aggressive evolution of their PanNET, either at diagnosis (N = 5) or upon progression (N = 7) with G3/NEC-like behavior (Table 3). Of six patients who harbored a second hit event in MUTYH in their tumors, five presented aggressive disease: three at diagnosis and one, upon progression on alkylating drug. One patient with a SPV with VAF of 76% had an indolent PanNET. One patient with LOH presented the characteristic MUTYH SBS36 signature (defined by an excess of G:C → T:A transversions). He was a 31-year-old male with a nonfunctioning aggressive G2 PanNET at diagnosis (initial KI-67 was 12% in hepatic bulky metastases) and with a GPV (p.Ala357fs). He initially had tumor response to Folfirinox, lasting for 9 months, but developed progressive disease and died 18 months from diagnosis. In his treatment-naïve primary PanNET, a heterozygotic SPV (p.Ala357fs, VAF 49%) was identified, and interestingly, we detected LOH of the MUTYH wild-type allele (VAF 86%) in his synchronic metastatic PanNET, with the SBS36 mutational signature.

Six patients received immune checkpoint inhibitors. One patient had a PanNET with low TMB and experienced upfront progression with pembrolizumab; another patient with a low TMB had disease controlled for 13 months with pembrolizumab combined with lenvatinib. A patient whose TMB was unknown had upfront NET progression with pembrolizumab. Three patients were treated with immune checkpoint inhibitors when their tumors became hypermutated, and two had tumor response. These two patients experienced complete and ongoing radiological response (lasting for 14 and 24 months so far) with nivolumab and ipilimumab. The patient with a TMB of 97 had a MUTYH SPV with VAF of 5% in ctDNA (#4); tumor tissue sequencing confirmed hypermutation and demonstrated an SBS11 genetic signature (alkylating-induced), but did not evidence MUTYH SPV. The SPV found ctDNA was likely subclonal. She initially had minor tumor response with pembrolizumab for nearly 6 months, when the tumor progressed; she then presented complete remission with nivolumab and ipilimumab for 18 months so far. The patient with a TMB of 65 and complete response with nivolumab and ipilimumab presented a double hit of MUTYH in the tumor (#19). The other patient (#13) with hypermutated PanNET had a MUTYH SPV with a VAF of 24% (germline testing was not performed) and developed a TMB of 728 after 18 months of CAPTEM; the tumor was unresponsive to pembrolizumab.

Discussion

In this international multicenter retrospective series, we report the first clinical characterization of patients with PanNET and pathogenic variants of MUTYH. Their frequency seems rare, most patients were young, male, and had metastatic disease at diagnosis. MUTYH p.Gly396Asp and p.Tyr179Cys were the most common variants, all being GPV. Of six patients who harbored a second hit event in MUTYH in their tumors, five presented aggressive disease. Overall, nearly two-thirds of metastatic patients demonstrated an aggressive course over time, with NEC-like behavior and rapidly progressive disease; 64% of patients with initial G1–2 PanNET with biopsies upon progression developed G3. TMB high was identified in three out of seven cases pretreated with alkylating agents and/or PRRT. Of these, two experienced complete disease remission with nivolumab and ipilimumab.

Scarpa et al.⁴ found 11 germline variants in MUTYH, of which 5 were considered GPV. A novel G:C > T:A somatic mutational signature was discovered and named MUTYH signature (SBS36) after validation in PanNET and an MUTYH-associated polyposis-related tumor, all tumor samples with biallelic inactivation in tumor tissues. Among consecutive Brazilian patients with early-onset NEN, nearly 16% presented a GPV in a cancer predisposing gene, with the most commonly affected gene being MUTYH in four cases.⁶ In a large pan-solid tumor cohort of over 229,000 solid tumors, MUTYH alterations were found in 2.8% of tumors, 55% of them predicted to be germline.¹³ Recently, two studies also found monoallelic GPV in this gene in patients with PanNET: 3 out of 112 cases in one cohort (two cases with p.Tyr165Cys and one with p.Thr474Profs*3 (ENST00000372115.3)), with all three cases annotated as “biallelic” in SignalDB, likely with LOH,¹⁴ and two cases with MUTYH GPV out of 88 cases in another study.⁵ In our cohort, MUTYH mutation p.Gly396Asp, a known pathogenic variant, was the most frequently identified, and this variant has also been associated with small bowel NET.^15,16 We also found a low frequency (2.9%) of MUTYH pathogenic variants among 402 screened patients.

The molecular profiling of treatment-naïve MUTYH+ PanNET in our study seems similar to sporadic PanNET,^3,4 with MEN1, DAXX, and ATRX mutations being the most commonly found. A recent study reported that MUTYH-altered versus -wild-type cancers had significantly higher TMB and more frequent alterations in KRAS G12C, but not in KRAS in general.¹³ In our study, most patients had a TMB below 10 before any treatment, but we found a high TMB in three patients after receiving alkylating agents.

Clinically, several characteristics of PanNET patients with MUTYH pathogenic variants seem peculiar. Median age was 48 years old, whereas the most recent epidemiology data report that nearly 90% of patients with PanNET are 45 years or more, with peak incidence among patients of 70–75 years.¹ While male patients usually comprise 50%–55% in overall PanNET cohorts, in our study, nearly two-thirds were male.¹ One-third of cases had an initial G3 tumor, which seems a high proportion compared to PanNET in general, where G3 is observed in about 12% of cases.¹⁷ The median initial KI-67 of 12% also seems higher than usually observed in PanNET, which is in the range of 5%.¹⁸ The majority of G1–2 NET developed into G3 NET upon progression which is highly unusual. Retrospective series of G1–2 PanNET patients with tumor biopsies performed at progression have reported median KI-67 index increase of 14%–45%.¹⁸ The largest series evaluating PanNET grade progression observed that only 12 (4.5%) out of 264 samples of initially G1–2 changed to a G3 PanNET.¹⁹

During the past two decades, the genetic profiles of PanNET have been investigated and researchers have tried to identify if genetic markers influenced prognosis. MEN1, DAXX, and ATRX mutations are the most commonly found in PanNET.^3,4 Yet, their correlation with patient prognosis has not been elucidated. Some authors have associated those mutations with improved OS in the metastatic setting.^3,5,20 Another retrospective study found the opposite: patients with PanNET carrying MEN1, DAXX, and ATRX mutations had worse clinical outcomes than those with tumors carrying the wild-type alleles of all three genes; the authors suggested that the presence of these mutations was associated with an alpha-cell signature, similarly to the alpha-cells of pancreatic islets, and related to a more aggressive behavior.²¹ BRAF and SETD2 alterations have also been associated with a more aggressive behavior in PanNET.⁵ Ours is the first study to suggest that either GVP and/or SPV of MUTYH predispose to worse prognosis in metastatic PanNET patients.

The majority (63%) of MUTYH+ PanNET had an aggressive course, with clinical NEC-like behavior (and NEC morphology when tumor samples were examined), either at diagnosis or after progression. While median OS of 4.6 years is similar to that reported in clinical trials²² and population-based studies¹ of G1–2 metastatic PanNET patients, 7 (58%) out of 12 cases classified as aggressive had initially indolent disease which later became aggressive during the disease course. This finding makes OS an inaccurate endpoint of our cohort. Also, the OS of a patient with G3 PanNET and MUTYH GPV in a large retrospective series of G3 NEN was shorter than the OS of G3 PanNET patients with other GPV.²³

Nearly 40% of tumors tested for LOH evidenced a possible second hit in the PanNET. Interestingly, four of six aggressive cases with GPV and tumor profiling had evidence of a second hit genomic event in MUTYH. Five out of six cases with a double hit event in MUTYH in their tumors presented aggressive disease. These findings suggest a possible specific driver mechanism in MUTYH pathogenic variants. It is possible that heterozygous MUTYH GPV cause some degree of DNA repair defects in PanNET, which are boosted by cytotoxic therapy, predisposing LOH of the wild alleles. A second hit event leads to cumulative genomic instability and tumor progression and could explain why patients with initially indolent PanNET developed aggressive disease after alkylating agents and/or PRRT.

Because of our small sample, it was not possible to properly evaluate response to different therapies. Yet, two complete responses to nivolumab and ipilimumab are unusual in metastatic PanNET. These two patients (#4 and #19) had hypermutated PanNET with mutations in mismatch repair genes, what may explain their excellent outcomes. Pembrolizumab was utilized in two cases with TMB high tumors but it was not effective. One patient had upfront tumor progression (#13); the other, with a MUTYH SPV (VAF 5%) found in ctDNA experienced stable/minor tumor shrinkage for about 6 months and achieved complete remission with nivolumab and ipilimumab (#4). In a recent case report, a Chinese PanNET patient with a MUTYH GPV (and likely with LOH of the wild allele in the tumor) also experienced a partial radiological response to a programmed death receptor-1 monoclonal antibody.²⁴ How the presence of MUTYH variants contributes to response to immunotherapies in PanNET remains undetermined.

Our study has limitations. The most relevant is the inherent bias associated with the retrospective nature of our study, what prevented an accurate estimate of the frequency of MUTYH GPV and/or SPV in patients with PanNET. Because of our small sample, variable intervals of imaging tests, and lack of central radiology review, it was not possible to estimate response rate and progression free survival of different therapies. Another important limitation was the unavailability of tumor tissues for all patients to investigate LOH of the MUTYH wild alleles. A central pathology revision of tumor samples was not performed but all cases were diagnosed by expert pathologist in NEN who work at NET reference centers.

Future research on the role of MUTYH in the PanNET should be sought. Our group will evaluate the natural history of MUTYH+ tumors, with subsequent tumor biopsies performed upon disease progression. Such studies could help us understand the molecular mechanisms leading to tumor aggressiveness and, in some cases, hypermutation. Another interesting area for research is how MGMT is expressed in PanNET with MUTYH mutations and how this influences responses to alkylating agents.

PanNET are a heterogeneous disease with variable clinical course, even within the tumor grade subgroups. Efforts are needed to identify prognostic molecular biomarkers to help predict disease aggressiveness and to guide more effective treatment for patients. Based on our findings, we recommend that patients with PanNET be evaluated for GPV and SPV, if possible. If a MUTYH GPV is found, genetic counseling of family members is advised and LOH of the wild allele in the tumor could be investigated. For confirmed double hit cases, close monitoring for the developing of aggressive disease is advised (e.g., clinical and radiological evaluation every 3 months). Additionally, molecular profiling of tumor biopsy collected upon progression on therapies could be informative of TMB. If TMB high is found, a combination of nivolumab and ipilimumab could be considered, if there is access to such therapies.

Conclusion

GPV in MUTYH PanNET seem rare and are more common in males and younger patients when compared with the overall PanNET population. Patients with PanNET and MUTYH GPV tend to have more aggressive and high-grade, NEC-like evolution, when metastatic. LOH of the wild allele seems to be associated with tumor aggressiveness.

Footnotes

Acknowledgements

None.

Declarations

ORCID iD

Rachel P. Riechelmann

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