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Enhancer signatures stratify and predict outcomes of non-functional pancreatic neuroendocrine tumors


Most pancreatic neuroendocrine tumors (PNETs) do not produce excess hormones and are therefore considered ‘non-functional’1,2,3. As clinical behaviors vary widely and distant metastases are eventually lethal2,4, biological classifications might guide treatment. Using enhancer maps to infer gene regulatory programs, we find that non-functional PNETs fall into two major subtypes, with epigenomes and transcriptomes that partially resemble islet α- and β-cells. Transcription factors ARX and PDX1 specify these normal cells, respectively5,6, and 84% of 142 non-functional PNETs expressed one or the other factor, occasionally both. Among 103 cases, distant relapses occurred almost exclusively in patients with ARX+PDX1 tumors and, within this subtype, in cases with alternative lengthening of telomeres. These markedly different outcomes belied similar clinical presentations and histology and, in one cohort, occurred irrespective of MEN1 mutation. This robust molecular stratification provides insight into cell lineage correlates of non-functional PNETs, accurately predicts disease course and can inform postoperative clinical decisions.

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Data availability

All relevant data are included in the manuscript and/or in its supplementary information files. ChIP-seq and RNA-seq data have been deposited in the National Center Biotechnology Information’s GEO under GSE116356. Other original data that support the findings of this study have been uploaded as Source Data.


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The present study has been supported by the Neuroendocrine Tumor Research Foundation (R.A.S., B.E.B., M.H.K. and D.C.C.), the SPORE program in gastrointestinal cancers (P50CA127003—National Cancer Institute, R.A.S.), the North American Neuroendocrine Tumor Society (C.M.H.) and a grant (no. PI18-01604 to P.C.) from Instituto de Salud Carlos III of the Spanish Economy and Competitiveness Ministry.

C.R.C. Pieterman, B. Havekes, A.R. Hermus, O.M. Dekkers, W.W. de Herder, M.L. Drent, A.N.A. van der Horst-Schrivers and P.H. Bisschop contributed to the Dutch MEN1 Study Group database and tissue repository. We thank J. Chan for critical reading of the manuscript.

Author information

P.C., Y.D., C.B.E., E.S., D.C.C., B.E.B. and R.A.S. designed the study. P.C. performed the experiments. Y.D. performed the computational analyses. P.C., L.A.A.B. and V.D. analyzed immunohistochemistry data. C.B.E., M.B., E.G., H.J.W., N.S., A.F.-T. and H.W.L. coordinated ChIP- and RNA-seq efforts. K.M.A.D., E.B.C., L.A.A.B., F.H.M.M., G.D.V., M.R.V., C.F.-d.C., C.F., T.A., A.D.S., E.S., M.H.K. and D.C.C. obtained and curated tissue collections and clinical data. P.C. and K.M.A.D. analyzed clinical data. M.K.G. and C.M.H. performed and scored telomere-specific FISH for ALT. B.E.B. and R.A.S. supervised the study. Y.D., P.C. and R.A.S. wrote the first manuscript draft. K.M.A.D., V.D., M.H.K., D.C.C., B.E.B. and R.A.S. revised the paper.

Correspondence to Yotam Drier or Bradley E. Bernstein or Ramesh A. Shivdasani.

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The authors declare no competing interests.

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Peer review information: J. Carmona was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 PNET subtypes are associated with distinct enhancers of lineage-restricted TFs.

a, H3K27ac, H3K4me2 and mRNA data tracks at ARX and PDX1 in all eight PNETs from the discovery set and from two samples of normal islets of Langerhans (Isl). ChIP-seq signals are scaled by promoter-based DESeq2 normalization (see Methods) and mRNA read counts are normalized by total read numbers (y axis represents 0–2 fragments per million reads). b, Distributions of ARX and PDX1 mRNA levels in A- and B-type PNETs. c, Pearson’s correlations of H3K27ac signals at PNET type A/α-cell and type B/β-cell enhancers in all 21 tumors from the discovery and validation cohorts (n = 8 and n = 13 biologically independent samples, respectively).

Extended Data Fig. 2 ARX and PDX1 immunostain in human normal islets and PNETs.

a, Double immunofluorescence for PDX1 (red) and ARX (green) in normal islets (marked by dashed white outlines). Scale bar, 50 μm. The results, representing hundreds of islets, verify antibody specificity, lineage-restricted expression and cell distributions: abundant PDX1+ β-cells scattered across islets and fewer ARX+ α-cells enriched in the islet periphery. b, Top: ARX and PDX1 IHC selectively mark endocrine α- and β-cells, respectively, in normal human islets. Many exocrine and ductal cells also express PDX1, as is well known24. The results represent hundreds of normal islets from multiple individuals, which revealed no ARX+ PDX1+ DP cells. Thus, although described in rodent embryos24, such cells are absent or extremely rare in the adult human pancreas. Bottom: IHC for ARX in a representative PNET and surrounding normal cells on TMAs from the Dutch cohort. The area boxed in the left image is magnified on the right. ARX+ cells dominate in the tumor and mark invasive foci (arrows). c, Range of IHC signal strength in ARX+ PNETs (+weak, ++ moderate, +++ strong), contrasted with uniformly robust PDX1 staining. Images are examples selected from 34 ARX+ and 31 PDX+ cases (Fig. 3b). Scale bars, 50 μm.

Extended Data Fig. 3 Additional IHC and enhancer characterization of PNETs.

a, Double immunofluorescence of representative ARX+ (type A, n = 34 biologically independent samples) and PDX1+ (type B, n = 31 biologically independent samples) tumors (T) adjacent to normal islets (N), showing selective detection of ARX (green) and PDX1 (red), respectively. Lack of antibody cross-reactivity controls for ARX and PDX1 co-staining (Fig. 3c) in DP tumors. b, SST expression in normal islets (δ-cells) and absence in all 77 Dutch PNETs, including the representative DN tumor (n = 6 biologically independent samples) shown here. c, IHC results for ARX and PDX1 shown alongside H3K27ac FiT-seq data from the same samples in three of the four cases (one of each subtype) from the discovery cohort where both FFPE and frozen samples were available.

Extended Data Fig. 4 Other endocrine-specific loci in PNETs.

a, H3K27ac, H3K4me2 and mRNA data tracks from all eight PNETs in the discovery set and from two normal islet samples at loci that control early pancreas ontogeny: NEUROG3 and PAX4. Histone marks and RNA-seq data are scaled as in Extended Data Fig. 1a. b, IHC for NEUROG3 in rare normal islets (dashed outlines), showing scarce NEUROG3+ endocrine cells (arrows). Hundreds of normal islets and all 19 biologically independent PNETs represented on one TMA (one example is shown) lacked expression. c, H3K27ac, H3K4me2 and mRNA data tracks from all eight PNETs in the discovery set and from two normal islet samples at loci that control terminal endocrine cell maturation, MAFA and FFAR1. Histone marks and RNA-seq data are scaled as in Extended Data Fig. 1a. A single outlier showed strong H3K27ac and mRNA at FFAR1.

Extended Data Fig 5 Differentiation status of PNETs.

a, Correlations of mRNA profiles in individual PNETs with those of pancreatic endocrine progenitor and mature cells37. x axis: Spearman’s correlations between log2(TPM + 1) values of each tumor and the average log2(TPM + 1) values of mature and progenitor populations.

Extended Data Fig 6 Association of PNET subtypes with ALT status.

a,c, Tumor size in all PNET subtypes in the Dutch (a) (n = 56 independent tumors) and the MGH (c) (n = 61 independent tumors) cohorts. Bars represent mean ± s.d. P values for differences in size of primary ARX+ and PDX1+ tumors determined by the two-sided Mann–Whitney U-test. b,d, Analyses of recurrence-free survival in the Dutch (b) (n = 30 cases) and MGH (d) (n = 35 cases) cohorts when ARX+ and PDX1+ tumors were considered separately, ungrouped from DP and DN tumors. P values and HRs were determined using two-sided log-rank and Mantel–Haenszel tests, respectively. e,f, Representative (e) (1 example each from 25 independent ALT+ and 87 independent ALT cases) and aggregate (f) (n = 112 biologically independent cases) results of telomere-specific FISH in cases classified as positive or negative for ALT. The statistical test was two-sided. g, Kaplan–Meier analysis of disease-free survival in all 112 cases with ALT data from both cohorts, without consideration of PNET subtype. Source data

Supplementary information

Reporting Summary

Supplememtary Tables

Supplementary Tables 1–6

Source data

Source Data Fig. 3

Unprocessed tissue microarray (TMA) scans with scoring key

Source Data Fig. 4

Statistical source data

Source Data Fig. 3, Fig. 4 and Extended Data Fig. 6

Statistical source data

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Fig. 1: Distinctive PNET enhancer profiles.
Fig. 2: PNET subtypes represent distinct endocrine lineages.
Fig. 3: ARX and PDX1 immunostains distinguish PNET subtypes.
Fig. 4: Different prognosis of PNET subtypes.
Extended Data Fig. 1: PNET subtypes are associated with distinct enhancers of lineage-restricted TFs.
Extended Data Fig. 2: ARX and PDX1 immunostain in human normal islets and PNETs.
Extended Data Fig. 3: Additional IHC and enhancer characterization of PNETs.
Extended Data Fig. 4: Other endocrine-specific loci in PNETs.
Extended Data Fig 5: Differentiation status of PNETs.
Extended Data Fig 6: Association of PNET subtypes with ALT status.