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. 2024 Nov 4;22(1):994.
doi: 10.1186/s12967-024-05736-0.

High level of aneuploidy and recurrent loss of chromosome 11 as relevant features of somatotroph pituitary tumors

Julia Rymuza  1 , Paulina Kober  1 , Maria Maksymowicz  2 , Aleksandra Nyc  2 , Beata J Mossakowska  1 , Renata Woroniecka  3 , Natalia Maławska  3 , Beata Grygalewicz  3 , Szymon Baluszek  1 , Grzegorz Zieliński  4 , Jacek Kunicki  5 , Mateusz Bujko  6
Affiliations

High level of aneuploidy and recurrent loss of chromosome 11 as relevant features of somatotroph pituitary tumors

Julia Rymuza et al. J Transl Med. .

Abstract

Background: Somatotroph neuroendocrine pituitary tumors (sPitNET) are a subtype of pituitary tumors that commonly cause acromegaly. Our study aimed to determine the spectrum of DNA copy number abnormalities (CNAs) in sPitNETs and their relevance.

Methods: A landscape of CNAs in sPitNETs was determined using combined whole-genome approaches involving low-pass whole genome sequencing and SNP microarrays. Fluorescent in situ hybridization (FISH) was used for microscopic validation of CNAs. The tumors were also subjected to transcriptome and DNA methylation analyses with RNAseq and microarrays, respectively.

Results: We observed a wide spectrum of cytogenetic changes ranging from multiple deletions, recurrent chromosome 11 loss, stable genomes, to duplication of the majority of the chromosomes. The identified CNAs were confirmed with FISH. sPitNETs with multiple duplications were characterized by intratumoral heterogeneity in chromosome number variation in individual tumor cells, as determined with FISH. These tumors were separate CNA-related sPitNET subtype in clustering analyses with CNA signature specific for whole genome doubling-related etiology. This subtype encompassed GNAS-wild type, mostly densely granulated tumors with favorable expression level of known prognosis-related genes, notably enriched with POUF1/NR5A1-double positive PitNETs. Chromosomal deletions in sPitNETs are functionally relevant. They occurred in gene-dense DNA regions and were related to genes downregulation and increased DNA methylation in the CpG island and promoter regions in the affected regions. Recurrent loss of chromosome 11 was reflected by lowered MEN1 and AIP. No such unequivocal relevance was found for chromosomal gains. Comparisons of transcriptomes of selected most cytogenetically stable sPitNETs with tumors with recurrent loss of chromosome 11 showed upregulation of processes related to gene dosage compensation mechanism in tumors with deletion. Comparison of stable tumors with those with multiple duplications showed upregulation of processes related to mitotic spindle, DNA repair, and chromatin organization. Both comparisons showed upregulation of the processes related to immune infiltration in cytogenetically stable tumors and deconvolution of DNA methylation data indicated a higher content of specified immune cells and lower tumor purity in these tumors.

Conclusions: sPitNETs fall into three relevant cytogenetic groups: highly aneuploid tumors characterized by known prognostically favorable features and low aneuploidy tumors including specific subtype with chromosome 11 loss.

Keywords: Acromegaly; Cytogenetic abnormalities; DNA copy number variations; Gene expression regulation, neoplastic; Growth hormone-secreting pituitary adenoma; Pituitary tumors.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Genome-wide DNA copy number profile in somatotroph pituitary tumors. A) Representative examples of virtual karyotypes with copy number patterns observed in the study, based on low pass WGS coverage (WGS RD line), SNP biallelic frequency (CystoSNP BAF line), and microarray probe signal (CystoSNP LRR line); B) Landscape of copy number alteration (CNAs) in sPitNETs with a resolution of single chromosomal bands. The dendrograms based on similarity in the CNAs pattern indicate two main cytogenetic clusters of the tumors; C) Comparison of the number of chromosomal deletions and duplications in two major cytogenetic clusters of tumors. D) Correlation between frequency of incidence of the duplications and deletions at particular chromosomal regions (bands) and gene density; E) CNA signatures for each of the three somatotroph tumor clusters and their comparison to known CNA signatures found in human cancers [30] by measuring Euclidiean similarity; F) The comparison of CNAs profile in sPitNEts and human cancers included in TCGA project
Fig. 2
Fig. 2
The results of fluorescent in situ hybridization. A) The example of chromosome 11 loss; B and C) The example of the sample with multiple deletions with confirmed chromosome 1p loss (A) and 15q22 loss (B); D) The example of chromosome 20 duplication in sample with multiple chromosomal duplications; E) The example of chromosomes 5q15, 5q31 and 8q24 duplication in sample with multiple chromosomal duplications
Fig. 3
Fig. 3
The relationship between CNAs profile and clinico-demographical features. A) Summary of the molecular, histological and clinical characteristics. The dendrogram shows the similarity in CNAs pattern; B) Comparison of the number of CNAs in densely granulated (DG) and sparsely granulated (SG) sPitNETs, ***- indicate p = 0.0001 to 0.001; C) Comparison of CNAs number in tumors classified into one three transcriptomic subtypes according to previous profiling [20] Subtype 1 are double positive PIT1/SF1 positive DG tumors, Subtype 2 are PIT1 positive, mostly DG tumors with common GNAS mutations, Subtype 3 are mostly SG tumors; D) The expression of known prognosis-related genes in sPitNETs stratified according to CNAs based hierarchical clustering into: high aneuploidy, low aneuploidy and low aneuploidy/ chromosome 11 loss tumors, p-value from Kruskal-Wallis test; D) The correlation between aneuploidy level (CNAs number) in somatotroph tumor and the expression of known prognosis-related genes; E) Results of the analysis of independent validation set, sPitNETs from previous study [8]
Fig. 4
Fig. 4
Relationship between CNAs and genes expression or DNA methylation profiles. A) Genes expression in tumor samples clustered according to cytogenetic pattern (reflecting Fig. 1B). The heat map shows the mean expression of genes encoded by each chromosome band. The boxes indicate sites of chromosomal loss. The dendrogram shows similarity in the pattern of CNAs; B) Correlation between the expression and copy number value of each gene, Spearman R > |0.5| was used as a cut-off point for moderate/high correlation. Each dot represents a particular gene; C) Correlation between CNAs count and median DNA methylation (median β-value) at specified category of CpG sites; D) Normalized, scaled DNA methylation level in individual sPitNETs showing difference in DNA methylation at CpG island and genes promoters at deleted chromosomal regions from the methylation level from the other chromosomal regions. Boxes indicate sites of chromosomal loss. The dendrograms show similarity in pattern of CNAs; E) Correlation between expression and copy number value of each gene, Pearson R > |0.5| was used as a cut-off point for moderate/high correlation. Each dot represents a chromosomal band
Fig. 5
Fig. 5
Comparison of gene expression between sPitNETs with chromosome 11 deletion and cytogenetically stable ones. A) Results of the dimensionality reduction analysis; B) Clustering and the expression of the most variably expressed genes; C) Differentially expressed genes plotted according to chromosomal location. Each dot represents a particular gene. D) Top 10 most enriched upregulated and downregulated GO biological processes; E) All significantly enriched GO biological processes classified according to a common physiological role; F) Comparison of the expression of known cancer-related genes encoded on chromosome 11 identified as differentially expressed. **- indicate p = 0.001 to 0.01, ***- indicate p = 0.0001 to 0.001
Fig. 6
Fig. 6
Comparison of gene expression in sPitNETs with a high level of aneuploidy (multiple chromosomal duplications) and cytogenetically stable tumors. A) Results of the dimensionality reduction analysis results; B) Whole transcriptome-based clustering and the expression of top the most variably expressed genes; C) Differentially expressed genes plotted according to chromosomal location. Each dot represents a particular gene. D) Top 10 most enriched upregulated and downregulated GO biological processes; E) All significantly enriched GO biological processes classified according to a common physiological role; F) Comparison of the expression of mitotic spindle-, chromosome segregation-, DNA damage response-related genes that were identified as downregulated in tumors with high aneuploidy (multiple chromosomal duplications). *- indicates p = 0.01 to 0.05, **- indicates p = 0.001 to 0.01, ***- indicates p = 0.0001 to 0.001, ****- indicates p > 0.0001
Fig. 7
Fig. 7
The content of immune cells and tumor purity based on deconvolution of DNA methylation data (EPIC human methylation arrays, Illumina). A) Normalized immune cell content in the entire group of sPitNETs. Dendrogram shows similarity in the pattern of CNAs; B) Comparison of estimated tumor purity, as well as T-cells and B-cell content, in sPitNETs with high aneuploidy (multiple chromosomal duplications), chr. 11 loss, and cytogenetically stable tumors. * indicate p = 0.01 to 0.05
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