Germ Cell Tumor Molecular Heterogeneity Revealed Through Analysis of Primary and Metastasis Pairs

Abstract

Purpose: Although primary germ cell tumors (GCTs) have been extensively characterized, molecular analysis of metastatic sites has been limited. We performed whole-exome sequencing and targeted next-generation sequencing on paired primary and metastatic GCT samples in a patient cohort enriched for cisplatin-resistant disease.

Patients and methods: Tissue sequencing was performed on 100 tumor specimens from 50 patients with metastatic GCT, and sequencing of plasma cell-free DNA was performed for a subset of patients.

Results: The mutational landscape of primary and metastatic pairs from GCT patients was highly discordant (68% of all somatic mutations were discordant). Whereas genome duplication was common and highly concordant between primary and metastatic samples, only 25% of primary-metastasis pairs had ≥ 50% concordance at the level of DNA copy number alterations (CNAs). Evolutionary-based analyses revealed that most mutations arose after CNAs at the respective loci in both primary and metastatic samples, with oncogenic mutations enriched in the set of early-occurring mutations versus variants of unknown significance (VUSs). TP53 pathway alterations were identified in nine cisplatin-resistant patients and had the highest degree of concordance in primary and metastatic specimens, consistent with their association with this treatment-resistant phenotype.

Conclusion: Analysis of paired primary and metastatic GCT specimens revealed significant molecular heterogeneity for both CNAs and somatic mutations. Among loci demonstrating serial genetic evolution, most somatic mutations arose after CNAs, but oncogenic mutations were enriched in the set of early-occurring mutations as compared with VUSs. Alterations in TP53 were clonal when present and shared among primary-metastasis pairs.

FIG 1.

Mutational heterogeneity of paired primary and metastatic germ cell tumor (GCT) specimens analyzed by whole-exome sequencing. (A) Primary GCT samples had a low mutation rate (median, 0.42 mutations per megabase [mut/Mb]; range, 0.1-1.52 mut/Mb), whereas paired metastasis samples from the same patients demonstrated a significantly higher tumor mutational burden (median, 1.55 mut/Mb; range, 0.16-4.79 mut/Mb; Wilcoxon signed rank, P = .03). Individual mutations were designated as occurring in the primary sample (orange) and/or the metastasis sample (green) and identified as oncogenic (red) or variants of unknown significance (VUSs; blue). (B) The median percentage of concordant mutations was 32.4% (range, 0%-70.3%), with six of 10 patients having a majority of discordant mutations. Oncogenic mutations were more likely to be concordant across primary-metastasis pairs (72.7%; eight of 11 concordant) compared with variants of unknown significance (19.1%; 111 of 581 concordant; Fisher’s exact test, P = .0002).

FIG 2.

Copy number analysis and timing of mutations during tumor evolution. (A) Continuous losses and gains, as partly reflected in the discordance of whole-genome duplication and reciprocal loss of heterozygosity rates across 40 evaluable primary-metastasis pairs (sequenced by whole-exome sequencing [WES] and/or Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets—MSK-IMPACT—were reflected in the total percentage of the genome with the same major and minor integer copy number (as measured in base pairs sequenced in both samples). Ten (25%) of 40 pairs demonstrated ≥ 50% identical integer copy number (median, 38.9%; range, 16.5%-78.9%). (B) For 10 primary-metastasis pairs analyzed by WES (each represented by a different colored dot), the majority of mutations arose after the observed copy number alteration at the respective locus: (1) before: median, 2.5 mutations, range (1-10); (2) after: median, 25.5 mutations, range (8-66); Wilcoxon signed rank test, P = .006). (C) For primary-metastasis pairs analyzed by WES, oncogenic mutations were enriched in the set of early-occurring mutations compared with variants of unknown significance (VUSs). Five (62.5%) of eight oncogenic mutations occurred early compared with 30 (10.5%) of 287 VUSs in the WES samples (Fisher’s exact test, P = .001). (**) Statistical significance for the illustrated comparisons.

FIG 3.

Discordance of MDM2 amplification across primary-metastasis pairs in two patients. (A) In the first patient, the MDM2 amplification was identified only in the metastatic sample collected after chemotherapy. (B) The same finding is demonstrated in a second patient.

FIG 4.

Analysis of plasma cell-free DNA (cfDNA) samples from 11 patients using Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets—MSK-IMPACT, which enabled comparison of primary-metastasis-cfDNA trios. (A) TP53 status was concordant across primary, metastasis, and plasma cfDNA specimens in two patients whose tumors were TP53 mutant. Additional mutations in the trio samples from these two patients are also shown. (B) Across all 11 trios, a total of 19 mutations were detected (median, two mutations, range (0-5) in cfDNA, including nine oncogenic mutations. Plasma cfDNA analysis did not reveal any additional mutations undetected in tumor tissue. Nineteen mutations identified in the tumor samples were absent in the matched cfDNA sample, including seven oncogenic mutations. The majority of mutations (15 of 19) detected in cfDNA were concordant in the primary-metastasis pair. Four mutations identified by cfDNA were not concordant in the primary-metastasis pairs, with two each identified in primary-cfDNA and metastasis-cfDNA pairs, respectively. VUS, variant of unknown significance.

FIG A1.

Correlation of variant allele frequency between whole-exome sequencing (WES) and Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets—MSK-IMPACT—in the WES cohort. The variant allele frequency of mutations identified in genes covered by the MSK-IMPACT was highly correlated between WES and MSK-IMPACT (Spearman rank correlation, ⍴ = 0.93; P < 2.2 × 10⁻¹⁶).

FIG A2.

Mutational heterogeneity in the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) cohort. Across 30 evaluable patients who were sequenced only with MSK-IMPACT, 29 (34.5%) of 84 mutations were concordant. Only eight (26.7%) of 30 patients had a majority of concordant mutations.

FIG A3.

Timing of mutations during tumor evolution in the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets—MSK-IMPACT—cohort. (A) Significantly more mutations occurred after the observed copy number alterations (CNAs; before: median, zero mutations, range (0-3); after: median, two mutations, range (0-5); Wilcoxon signed rank test, P = 6.297 × 10⁻⁵). (B) Seven (41.2%) of 17 oncogenic mutations arose early versus four (11.1%) of 36 variants of unknown significance (VUSs; Fisher’s exact test, P = .03). (**) Statistical significance for the illustrated comparisons.