Chromosomal alteration patterns in PitNETs: massive losses in aggressive tumors

Abstract

The molecular biology of pituitary neuroendocrine tumors (PitNETs) revealed few recurrent mutations and extensive chromosomal alterations, with the latter being the driving force in a subset of these lesions. Addressing the need for an easily applicable diagnostic tool, we conducted a retrospective study of 61 PitNETs operated at a tertiary care center. All cases were subtyped according to the 2022 WHO Classification of Endocrine Tumors. A genome-wide next-generation sequencing panel targeting 1500 single nucleotide polymorphisms (SNPs) was used to classify chromosomal imbalances, loss of heterozygosity, and copy number variations in DNA from formalin-fixed paraffin-embedded tissues. We identified four distinct chromosomal patterns, with varying distribution among different tumor lineages. Forty-two of 61 (69%) PitNETs showed chromosomal alterations. Gonadotroph PitNETs showed mostly quiet genomes. The majority of lactotroph PitNETs (19/20, 95%) were altered, exhibiting a gained genome and a remarkably low recurrence rate. Nine of ten (90%) corticotroph PitNETs harbored chromosomal alterations, of which two aggressive corticotroph tumors and one metastatic corticotroph PitNET showed massive chromosomal losses, leading to near-haploid/near-homozygous genomes. The comparison of the molecular profile of primary and recurrent PitNETs of five patients showed no significant accumulation of alterations over time. A simple genome-wide 1500-SNP test can be used in the identification of outspoken aggressive subsets of PitNETs by the occurrence of a near-haploid/near-homozygous genome. Furthermore, the presence of neoplastic tissue in the resected material can be potentially confirmed for non-gonadotroph PitNETs under suboptimal histological assessment conditions.

Keywords: CNV; LOH; PitNET; PitNETs; SNP analysis; chromosome alterations; cnLOH; copy number alterations; endocrine pathology; endoreduplication; genome doubling; genome haploidization; loss of heterozygosity; molecular diagnostics; near-haploid genome; near-haploidization; near-homozygous genome; pituitary tumor; prolactinoma.

Figure 1

Overview of the cohort with selected clinical characteristics of the patients, PitNET subtypes, imbalance–LOH–CNV alterations noted per chromosomal arm, and corresponding patterns. (A) Cases are sorted by lineage and further by pattern, from the least number of disrupted chromosomes to those most affected. The chromosome arms with the highest frequency of alterations in the cohort were 12q and 12p, primarily exhibiting imbalances due to chromosomal copy number gain. Following chromosome 12, the frequently altered chromosomes in descending order of frequency are 20, 19, X, 3, 5, 7, and 8, showing imbalances due to copy number gain, while chromosome 11 predominantly exhibited LOH or imbalance due to copy number loss. Chromosome 1, frequently mentioned in the literature, displayed alterations in only 14 of the 61 PitNETs. (B) A simplified overview of the distribution of the observed chromosomal alteration patterns in different lineages of PitNETs.

Figure 2

Examples of Patterns 1–4 as observed in the PitNET cohort (A–D). Scoring of informative SNPs resulted in the patterns of imbalance or loss of heterozygosity (LOH) combined with copy number variation (CNV) analysis across the entire study cohort. The upper section of such analysis shows the coverage of all 1500 SNPs used, the middle section represents the CN profiles, and the lower section depicts the allelic frequency for each SNP. Chromosome-wide genotypes were subsequently extrapolated and indicated as AB; A0 or 0B; and AA, BB, and AABB, the latter three occurring after genome doubling or endoreduplication. In some cases, locus-restricted copy-neutral LOH can be observed (also carrying genotype AA or BB). Chromosomal gains with imbalances in the SNP typing exhibit genotype AAB or ABB. (A) Example of Pattern 1: patient 51, a female patient with a primary silent gonadotroph PitNET, showing no imbalance or LOH or CNV alterations on any of the chromosomes, with heterozygous genotype of AB extrapolated. (B) Example of Pattern 2: patient 49, a male patient with a primary mammosomatotroph PitNET, exhibiting LOH of chromosomes 1p, 2, 8, 11, and 13 due to copy number loss, with an extrapolated genotype A0 or 0B. Chromosome 13 shows a difficult-to-interpret heterogeneous pattern with some imbalances with loss-like deviation and an extrapolated genotype of AA. There is only one chromosome X (male), genotypes A0 and 0B. (C) Example of Pattern 3: patient 20, a male patient with a primary sparsely granulated lactotroph PitNET, displaying extensive imbalances with copy number gain on chromosomes 3, 7, 8, 9, 12, 19, 21, and 22, with extrapolated genotypes AAB and ABB. There is again only one chromosome X (male). (D) Example of Pattern 4: patient 22, a female patient with a primary sparsely granulated lactotroph PitNET, exhibiting a complex CNV pattern with copy-neutral LOH on chromosomes 1, 2, 4, 10, 20, and 22 and imbalances with copy number gains on chromosomes 3, 7, 8, 11, 12, 14, 17, 19, and X, with extrapolated genotypes AA or BB and AAB or ABB, respectively. The copy-neutral LOH was scored as such because, in the copy number scoring in the middle panel, the height of chromosome 1 with LOH is similar to the heterozygous chromosome 6 (genotype AA vs AB, respectively). There is also a heterozygous state with whole chromosome gain on chromosomes 5 and 9 showing genotype AABB.

Figure 3

Near-haploid genome as seen with genome-wide 1500-SNP testing in the tumor of parent 52 (panel A) and with WGS in the tumor of patient 56 (panel B). (A) GW-LOH testing and imbalance–LOH–CNV analysis on tumor of patient 52: this testing shows a near-homozygous genome with multiple LOH (chromosomes 1, 2, 3, 4, part of 5q, 6, 8, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, and X), which is consistent with possible endoreduplication. These data fit with Pattern 2 of abnormalities. A previous recurrence of 4–5 years earlier shows an identical pattern of chromosomal alterations. (B) WGS result of case 56: LOH on chromosomes 1, 2, 3, 4, 6, 8, 10, 11, 13, 15, 17, 19, 20, 21, 22, and 23 can be observed. Molecular tumor cell percentage: 65%. The data point to Pattern 2 of chromosomal abnormalities. Endoreduplication occurred, leading to a near-homozygous genome (estimated ploidy 1.3).