Clonal analyses of refractory testicular germ cell tumors

Abstract

Testicular germ cell tumors (TGCTs) are unique amongst solid tumors in terms of the high cure rates using chemotherapy for metastatic disease. Nevertheless, TGCTs still kill approximately 400 men per year, at a median age of 30 years, in the United States. This young age of mortality dramatically amplifies the impact of these deaths for the patients and their often young families. Furthermore the high cure rate makes it difficult to conduct further clinical trials of non curable disease. TGCTs are characterized by a marked aneuploidy and the presence of gain of chromosomal region 12p. Genomic testing may offer the ability to identify potentially lethal TGCTs at the time of initial diagnosis. However sequencing based studies have shown a paucity of somatic mutations in TGCT genomes including those that drive refractory disease. Furthermore these studies may be limited by genetic heterogeneity in primary tumors and the evolution of sub populations during disease progression. Herein we applied a systematic approach combining DNA content flow cytometry, whole genome copy number and whole exome sequence analyses to interrogate tumor heterogeneity in primary and metastatic refractory TGCTs. We identified both known and novel somatic copy number aberrations (12p, MDM2, and RHBDD1) and mutations (XRCC2, PIK3CA, RITA1) including candidate markers for platinum resistance that were present in a primary tumor of mixed histology and that remained after tandem autologous stem cell transplant.

Fig 1. Mapping 12p amplicons in TGCT genomes.

Copy number aberrations on chromosome 12p in three distinct TGCT aneuploid populations. The 12p amplicons included the whole 12p arm (3/5 cases) in a 2.8N population detected in case #3 and two distinct amplicons in the 2.7N and the 3.2N populations present in case #1. The X and Y axes in the CGH plots represent chromosome and log₂ratios for each TGCT.

Fig 2. Mapping 4q amplicons targeting c-KIT oncogene.

Copy number aberrations targeting 4q in case #1. The red shaded areas denote ADM2 defined copy number aberrant intervals. The X and Y axes in the CGH plots represent chromosome and log₂ratios for each TGCT.

Fig 3. Clonal analysis of refractory metastatic TGCT.

A) DNA content flow sorting of aneuploid (3.0N) and diploid (2.0N) populations from primary FFPE tissues for case #5. B) Whole genome copy number plots for 3.0N population included 12q15 and 2q 36.3 amplicons targeting MDM2 (red arrow) and both IRS2 and RHBDD1 (blue arrows) in each aneuploid genome. C) Locus specific view of each amplicon. The X and Y axes in the CGH plots represent chromosome and log₂ratios. Red shaded areas denote ADM2 defined CNV interval.

Fig 4. Clonal analysis of primary and refractory metastatic TGCT.

A) DNA content flow sorting of aneuploid (3.2N, 2.7N) and diploid (2.0N) populations from primary and metastatic FFPE tissues of case #1. The histology of each biopsy is listed above the histogram. B) Copy number aberrations included 4p amplicons targeting c-KIT (red arrow) and distinct 12p amplicons (red and blue arrows) in each aneuploid genome. In addition a gain of 3q and deletion of 13q13.3-qtel were unique to the 2.7N genomes (blue arrows). C) IGV view of the clonal XRCC2^R188H mutation in each aneuploid population and the wild type sequence in patient matched normal 2.0N population.

Fig 5. Cell lineage of metastatic TGCT.

A) Genomic aberrations within primary and refractory metastatic TGCT in case #1 include; i) shared XRCC2^R188H and RITA1^T220K mutations in an inferred malignant progenitor; ii) CNVs, 12p13.31-p11.21 amplicon, gain of 7p13.1-ptel, and loss of chromosome 9, that are private to the primary 3.2N tumor seminoma population; iii) mutations RECQL5^D480G, CRISP3^A147S, WWC2^V498F, and CNVs 12p13.31-p12.3, gain of 3q, and deletions of 13q13.3-qtel, private to the 2.7N population within the non-seminomatous primary and metastatic tumor; iv) mutations CYP19A1^T201M, and IL6^D162V, that are private to 2.7N metastatic population. B) Haematoxylin and eosin (H&E) staining and visualization of distinct TGCT histologies within the primary and metastatic tumor tissues.