Prospective Screening of Cancer Syndromes in Patients with Mesenchymal Tumors

Abstract

Background: The etiology of most mesenchymal tumors is unknown, and knowledge about syndromes with an increased risk of tumors in bone or soft tissue is sparse.

Methods: We present a prospective germline analysis of 312 patients with tumors suspected of being sarcomas at a tertiary sarcoma center. Germline and tumor whole genome sequencing, tumor transcriptome, and methylome analyses were performed.

Results: Germline pathogenic or likely pathogenic variants associated with an increased risk of tumors were detected in 24 patients (8%), of which 11 (4%) harbored a detectable second hit in the tumor. Second hits were confirmed in genes with (NF1, RB1, TP53, EXT2, and SDHC) and without (ATM, CDC73, MLH1, MSH6, POLG, and KCNQ1) known association with mesenchymal tumor predisposition. Sarcomas from two Lynch syndrome patients showed mismatch repair deficiency, predicting a treatment response to immune checkpoint inhibitors (Level 1 biomarker according to the FDA (Federal Drug Administration) and ESMO (European Society for Medical Oncology)). None of the three CHEK2 carriers had a second hit in the tumor, suggesting a weak link to sarcoma.

Conclusions: We conclude that second-hit analyses can be used in standard of care to identify syndrome-related tumors. This approach can help distinguish true manifestations of tumor syndromes from unrelated germline findings and enhance the understanding of germline predisposition in soft tissue tumors. Prospective screening using germline whole genome sequencing should be considered when comprehensive somatic sequencing is introduced into clinical practice.

Keywords: ATM; CDC73; KCNQ1; MLH1; MSH6; POLG; germline; sarcoma; second hit.

Figure 1

Histopathology and genetics of an RB1 germline-related leiomyosarcoma. Patient 101 carried a germline RB1 pathogenic variant causing hereditary retinoblastoma syndrome. The tumor harbored a deletion of the RB1 locus (13q), resulting in the loss of heterozygosity and biallelic inactivation. (A–C): Microphotographs show (A) a routine hematoxylin–eosin stain of a leiomyomatous tumor with high grade atypia, and immunohistochemistry showing (B) positivity for desmin, and (C) a loss of Rb immunoreactivity (single cells with retained Rb expression are tumor-infiltrating immune cells), as discovered in the clinical workup of the tumor. All microphotographs were captured at 400x magnification. (D): DNA abundance, measured as the bias-corrected sequence depth ratio for 10kb bins along the reference genome, appears at distinct levels corresponding to the number of copies per cancer cell. The RB1-containing segment displays a low DNA abundance, typical of a deletion. (E): The SNP (single nucleotide polymorphism) allele frequency for the RB1-containing segment shows distinct allelic imbalance, also consistent with a deletion. The high allele ratio of the pathogenic germline RB1 variant confirms the retention of the alternative allele in the tumor genome. The estimated average copy number (ploidy) is about 3.6 and the cancer cell fraction is about 60%. Colored dots represent probes located in sarcoma-associated genes.

Figure 2

Histopathology and genetics of a Lynch syndrome-associated pleomorphic liposarcoma. Patient 76 was shown to have Lynch syndrome caused by a germline MLH1 inactivation. Microphotographs of the tumor histology and immunohistochemistry, as performed in the clinical workup. All microphotographs were captured at 400x magnification. (A) Routine hematoxylin–eosin stain depicting a pleomorphic liposarcoma and immunohistochemistry with loss of (B) MLH1 and (C) PMS2 expression, with retained expression of MSH2 (D) and MSH6 (E), compatible with deficient mismatch repair (dMMR).

Figure 3

Second hits in relation to phenotype. C: chemotherapy, del: deletion, NA: no tumor tissue available, or second hit detected through immunohistochemistry, R: radiotherapy, RC: radiotherapy combined with chemotherapy, SNV: single nucleotide variant, S: suggested.

Figure 4

Methylation analysis in a patient with paraganglioma and GIST. Hypermethylation of the SDHC promoter in a patient with the clinical diagnosis Carney triad, without any germline findings and no somatic gene dose abnormalities in the SDHC region. Red dots represent the beta values of methylation from the case of interest. GIST: gastrointestinal stromal tumor, TSS: transcription start site.