Germline SAMD9/9L MDS Predisposition Syndromes Are Characterized By Complex Clonal Architecture and Lineage-Specific Escape Mechanisms Including Somatic Genetic Rescue in T and B Lymphocytes

Inherited bone marrow failure (BMF) and MDS predisposition syndromes have an intrinsic defect resulting in loss and/or dysfunction of hematopoietic stem and progenitor cells (HSPC). HSPC clones can escape this process by acquiring gene- or genome-level genetic changes (compensatory mutations or uniparental disomies (UPD)) thereby increasing cellular fitness. This process, referred to as somatic genetic rescue (SGR) has been shown to occur in >70% of patients with gain-of-function mutations in SAMD9 and SAMD9L (SAMD9/9L) genes, which are a common cause of early onset BMF and pediatric MDS with monosomy 7. It is unknown how SGR arises and in which cells it originates. Furthermore, little is known about the epistatic interactions of various rescue events. We aimed to address these questions by utilizing a newly established single cell (sc)-proteogenomic platform that can simultaneously detect single nucleotide variants (SNVs) and copy number variation (CNVs) in addition to protein expression. We applied this technology to study clonal architecture and cellular origins of SGR lesions in a cohort of clinically annotated SAMD9/9L-mutated (SAMD9/9L^mut) patients. We also asked if monosomy 7, traditionally assessed by karyotyping, can be also detected in peripheral blood (PB) of affected patients.

We first performed whole exome and panel sequencing to characterize 67 germline SAMD9/9L^mut patients. Guided by this data, we designed a sc-proteogenomic panel with 69 amplicons specific to variants in SAMD9/9L and leukemia driver genes, and 250 amplicons covering high-frequency SNVs on chromosome 7q. For validation of initial findings, we developed a second panel targeting full coding regions of SAMD9/9L genes, and additional 7p SNVs allowing for a precise delineation of uniparental isodisomy 7q (UPD7q). We included oligonucleotide-conjugated antibodies specific to major hematopoietic antigens, including CD19, CD3, CD11b, CD38 and CD34. Using this panel, we analyzed 22 samples (15 bone marrow (BM) and 7 PB) of 10 patients enrolled in local biobanking protocols.

On average, 3,845 high-quality cells were genotyped per sample, and we robustly identified SNVs, monosomy 7 and UPD7q changes in single cells. Somatic rescue SAMD9/9L^mut were found only in clones with normal (diploid) chromosome 7, UPD7q (containing two wild type SAMD9/9L alleles after mitotic recombination event) was a stand-alone clone, while monosomy 7 (SAMD9/9L^mut lost) occurred alone or together with leukemia driver mutations. We also detected lesions that were previously missed using routine clinical sequencing in 3 patients (somatic SAMD9/9L^mut or UPD7q). Most patients had branching clonal architecture (i.e., independent monosomy 7, UPD7q, and somatic SAMD9/9L^mut), while linear trajectories were rare (i.e., monosomy 7 à + leukemia driver mutation). Serial assessment of 4 patients with stable hematology (3 with monosomy 7 and 1 with an additional somatic SAMD9/9L^mut) for a period of up to 4.8 years, revealed stable clonal populations without acquisition of additional driver mutations. Quantification of clones in paired BM and PB in 4 patients demonstrated equal frequencies of monosomy 7 and somatic SAMD9/9L^mut in both compartments, demonstrating the utility of this assay in surveillance. Our findings were confirmed by sorting BM cells into different lineages followed by sequencing, error-corrected sequencing, and ddPCR. Finally, analysis of protein expression data revealed that while monosomy 7 is present exclusively in cells with myeloid phenotype, the SGR events can arise in lymphocyte lineage only; 4 patients carried clones with somatic SAMD9/9L^mut in CD3+ lymphocytes, and 2 patients had UPD7q clones in either CD3+ or CD19+ lymphocytes. These findings provide clues why most of the SGR events in patients do not result in long-lasting clinical remission since the genetic rescue can be restricted only to certain hematopoietic lineages.

In conclusion, single cell proteogenomic analysis is useful for the understanding the mechanisms of clonal evolution in SAMD9/9L syndromes. This method also offers the opportunity for less invasive surveillance of karyotype abnormalities from peripheral blood in patients who are at high risk for MDS progression.