46. Integrative cytogenetic and molecular studies unmasks `chromosomal mimicry' in hematologic malignancies

Detection or exclusion of structural variants (SVs) represents a critical component in the diagnostic evaluation, and treatment, of many hematologic malignancies. Across a large cohort (n=383) of unselected myeloid (334/383, 87%) and lymphoid (49/383,13%) tumors profiled within a calendar year, we evaluated the added clinical utility of anchored multiplex PCR (AMP) targeted RNA-fusion sequencing, to chromosome banding studies. SVs detected by either assay were categorized using a four-tier system: Tier 1 (established clinical significance), Tier 2 (possible clinical significance), Tier 3 (unknown significance) and Tier 4 (known germline variants). Of 111 tumors with Tier 1 or 2 SVs, 42 were detected by AMP studies alone, highlighting the improved detection of clinically relevant SVs in T-ALL (35%; 5/14), AML (15%, 22/150) and B-ALL (11%; 4/35). In these instances, chromosome banding failed due to cytogenetically cryptic SVs, complex cytogenetic findings, or by the outgrowth of non-neoplastic tissue. Most notably, in 2.7% (3/111) of tumors with clinically significant SVs, cytogenetic studies detected a Tier 1 structural variant (two instances of t(9;22)(q34;q11.2)/BCR::ABL1, and 1 t(8;21)(q22;q22)/RUNX1::RUNX1T1) with confirmatory FISH results, whose findings could not be recapitulated by AMP studies. Subsequent evaluation by Optical Genome Mapping and nanopore-sequencing confirmed breakpoints on the expected chromosomes, but not affecting the anticipated genes of interest. Herein, we describe the frequency and phenomenon of `Chromosomal Mimicry' in which chromosome morphology mimics a clinically relevant SV, thereby providing a false positive result, and highlighting the need for gene-level resolution in the diagnostic evaluation and clinical management of hematologic malignancies. Detection or exclusion of structural variants (SVs) represents a critical component in the diagnostic evaluation, and treatment, of many hematologic malignancies. Across a large cohort (n=383) of unselected myeloid (334/383, 87%) and lymphoid (49/383,13%) tumors profiled within a calendar year, we evaluated the added clinical utility of anchored multiplex PCR (AMP) targeted RNA-fusion sequencing, to chromosome banding studies. SVs detected by either assay were categorized using a four-tier system: Tier 1 (established clinical significance), Tier 2 (possible clinical significance), Tier 3 (unknown significance) and Tier 4 (known germline variants). Of 111 tumors with Tier 1 or 2 SVs, 42 were detected by AMP studies alone, highlighting the improved detection of clinically relevant SVs in T-ALL (35%; 5/14), AML (15%, 22/150) and B-ALL (11%; 4/35). In these instances, chromosome banding failed due to cytogenetically cryptic SVs, complex cytogenetic findings, or by the outgrowth of non-neoplastic tissue. Most notably, in 2.7% (3/111) of tumors with clinically significant SVs, cytogenetic studies detected a Tier 1 structural variant (two instances of t(9;22)(q34;q11.2)/BCR::ABL1, and 1 t(8;21)(q22;q22)/RUNX1::RUNX1T1) with confirmatory FISH results, whose findings could not be recapitulated by AMP studies. Subsequent evaluation by Optical Genome Mapping and nanopore-sequencing confirmed breakpoints on the expected chromosomes, but not affecting the anticipated genes of interest. Herein, we describe the frequency and phenomenon of `Chromosomal Mimicry' in which chromosome morphology mimics a clinically relevant SV, thereby providing a false positive result, and highlighting the need for gene-level resolution in the diagnostic evaluation and clinical management of hematologic malignancies.