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. 2020 Apr 28;12(1):38.
doi: 10.1186/s13073-020-00730-9.

Centromeric cohesion failure invokes a conserved choreography of chromosomal mis-segregations in pancreatic neuroendocrine tumor

Rene Quevedo  1   2 , Anna Spreafico  1   3 , Jeff Bruce  1 , Arnavaz Danesh  1 , Samah El Ghamrasni  1 , Amanda Giesler  1 , Youstina Hanna  1 , Cherry Have  4 , Tiantian Li  1 , S Y Cindy Yang  1   2 , Tong Zhang  1   4 , Sylvia L Asa  4 , Benjamin Haibe-Kains  1   2   5   6 , Monika Krzyzanowska  1 , Adam C Smith  4   7 , Simron Singh  8 , Lillian L Siu  9   10 , Trevor J Pugh  11   12   13
Affiliations

Centromeric cohesion failure invokes a conserved choreography of chromosomal mis-segregations in pancreatic neuroendocrine tumor

Rene Quevedo et al. Genome Med. .

Abstract

Background: Pancreatic neuroendocrine tumors (PANETs) are rare, slow growing cancers that often present with local and distant metastasis upon detection. PANETS contain distinct karyotypes, epigenetic dysregulation, and recurrent mutations in MEN1, ATRX, and DAXX (MAD+); however, the molecular basis of disease progression remains uncharacterized.

Methods: We evaluated associations between aneuploidy and the MAD+ mutational state of 532 PANETs from 11 published genomic studies and 19 new cases using a combination of exome, targeted panel, shallow WGS, or RNA-seq. We mapped the molecular timing of MAD+ PANET progression using cellular fractions corrected for inferred tumor content.

Results: In 287 PANETs with mutational data, MAD+ tumors always exhibited a highly recurrent signature of loss of heterozygosity (LOH) and copy-number alterations affecting 11 chromosomes, typically followed by genome doubling upon metastasis. These LOH chromosomes substantially overlap with those that undergo non-random mis-segregation due to ectopic CENP-A localization to flanking centromeric regions in DAXX-depleted cell lines. Using expression data from 122 PANETs, we found decreased gene expression in the regions immediately adjacent to the centromere in MAD+ PANETs. Using 43 PANETs from AACR GENIE, we inferred this signature to be preceded by mutations in MEN1, ATRX, and DAXX. We conducted a meta-analysis on 226 PANETs from 8 CGH studies to show an association of this signature with metastatic incidence. Our study shows that MAD+ tumors are a genetically diverse and aggressive subtype of PANETs that display extensive chromosomal loss after MAD+ mutation, which is followed by genome doubling.

Conclusions: We propose an evolutionary model for a subset of aggressive PANETs that is initiated by mutation of MEN1, ATRX, and DAXX, resulting in defects in centromere cohesion from ectopic CENP-A deposition that leads to selective loss of chromosomes and the LOH phenotype seen in late-stage metastatic PANETs. These insights aid in disease risk stratification and nominate potential therapeutic vulnerabilities to treat this disease.

Keywords: Exome sequencing; Gene expression profiling; Genetic instability; Loss of heterozygosity; Molecular cytogenetics; Molecular timing; Pancreatic neuroendocrine tumors; Publicly available data; Whole-genome sequencing.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Loss of heterozygosity and copy-number profiles for PANET samples. Loss of heterozygosity profiles depicted as being copy-loss/haploid (blue), copy-neutral/diploid (purple), or copy-gain/triploid+ (red) for each PANET sample in the a exome, b whole-genome, and c AACR GENIE cohorts. Motif plots describe the most recurrent zygosity (d) or copy-number (e–g) states for each chromosome. MAD+ PANETs in the exome and whole-genome cohorts depict patterns of copy-neutral and copy-gain (e), while PANETs in the AACR GENIE cohort depict patterns of copy-loss and copy-gain for MAD+ samples (f) and copy-neutral for MAD− samples (g)
Fig. 2
Fig. 2
Ectopic CENP-A loading localizations in DAXX-depleted and wild-type SW403 colorectal cancer cell lines from the Nye et al. dataset. a Mis-segregated chromosomes identified by Worrall et al. through single-cell analysis, compared to the LOH chromosomes we define. b, c Chromosome-relative localization of CENP-A peaks that are found in either DAXX-depleted-only regions (b) or peaks that are found in both DAXX-depleted and control cells (c) for both the commonly mis-segregated and normal-segregation chromosomes as identified by Worrall et al. d Depiction of acquired, maintained, and lost CENP-A peaks when SW403 undergo DAXX depletion. e Correlation plots between mis-segregation fractions and size of the centromere (CEN), or the cytobands immediately flanking the centromere (periCEN) on the p-arm or q-arm. f Correlation plots between the number of CENP-A peaks and mis-segregation fractions
Fig. 3
Fig. 3
Gene expression recapitulating the copy-number signature of MAD+ PanNETs. a, b The copy-number signature was inferred from RNA-seq data from the Sadanandam (nMAD+ = 28, nMAD− = 47) and Chan (nMAD+ = 30, nMAD− = 17) datasets by calculating the z-score on a per-gene basis using MAD+ PANETs compared to MAD−. These plots visualize data from the a Sadanandam dataset and the b Chan dataset. c, d Regions of a chromosome arm that have elevated or repressed gene expression in MAD+ samples for the LOH chromosomes (red) relative to genes on heterozygous chromosomes (blue) are plotted against the fractional distance to the centromere (0 = at centromere boundary, 1 = chromosomal arm end). Three datasets are illustrated here: the Tang et al. dataset composed of 77 normal pancreatic islets (top), the Sadanandam PANET dataset (middle), and the Chan PANET dataset (bottom). d Distances between loess regression lines of LOH to heterozygous chromosomes where all 3 aforementioned datasets are overlapped on each other to better visualize overlapping and discordant regions relative to normal islet cells
Fig. 4
Fig. 4
Cancer chromosome fraction for MAD genes in the GENIE PANET samples. Estimations of the theoretical tumor allelic fraction for MEN1 (red diamond), DAXX/ATRX (red square), and other gene-level mutations (gray circles) for the copy-number model (number of ALT alleles/ploidy) that best represents the pathologist-estimated purities across the different cohorts. A fraction of 1.0 indicates a homozygous variant, and 0.5 a heterozygous variant. Any deviations from these values represent variance in the observed allelic fractions
Fig. 5
Fig. 5
Meta-analysis of the CGH datasets for the highly aneuploid PANET tumors (High-CI) against the low aneuploid PANET tumors (Low-CI). The parameters being compared are the metastasis status of the tumor type (Met+, metastasis present; Met−, no metastasis) and the functional status (F, functional; NF, non-functional)
Fig. 6
Fig. 6
Proposed molecular progression mechanism for pancreatic neuroendocrine tumors. Normal islet cells acquire a mutation in MEN1, and ATRX or DAXX which leads to perturbed deposition of H3 histone variants H3.3 and CENP-A at nucleosomes in centromeric sites. This results in premature sister chromatid separation and loss of one allele, followed by a series of genome duplications
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