How Whole Genome and Transcriptome Sequencing (WGTS) Can Contribute to Unsolved Cases in Hematology That Have Undergone Extensive Standard Diagnostic Workup: A Prospective Head-to-Head Study

Background: The current WHO classification of hematologic tumors is based on 5 diagnostic pillars: cytomorphology, histology and immunohistochemistry, immunophenotyping, chromosome banding analysis (CBA) supplemented by FISH, and molecular genetics including targeted panel sequencing. However, 5-10 % of cases with unexplained blood count changes remain a challenge for hematopathologists and often require repeat examinations. We set up the prospective SIRIUS (Solving Riddles Through Sequencing, clinicaltrials.gov: NCT05046444) study to test if WGS and/or WTS can resolve such unclear cases.

Aim: To explore the potential of WGTS to solve unclear cases with suspected hematologic neoplasms in a prospective head-to-head study compared to gold standard diagnostics with respect to 1) clear-cut diagnosis and 2) turn-around time (TAT).

Methods: Since 9/2021 we enrolled 24 cases with unsolved suspected blood cancers and after informed consent. All samples were processed by gold standard diagnostics according to WHO criteria and by WGTS. Amplification-free WGS (100x, 2x151bp) and WTS (50 Mio reads, 2x101bp) were performed on NovaSeq instruments; data were analyzed with a tumor w/o normal pipeline for single nucleotide variants (SNV, Manta), structural variants (SV, Strelka2) and copy number variations (CNV, GATK) as well as fusion transcripts (Arriba, STAR-Fusion and Manta) and expression patterns.

Results: In direct comparison with gold standard diagnostics WGTS provided additional information in 8/24 (33%) patients, mostly rare SNVs not analyzed in standard panels and CNVs too small to be detected by CBA. This included SNVs with association to Diamond-Blackfan anemia and VEXAS syndrome and a variant of uncertain significance with possible association to Pelger-Huët anomaly in one case each. Moreover, WGTS further clarified SVs already detected by CBA: One patient with Waldenstroem macroglobulinemia and unusual clinical presentation showed a complex karyotype with t(14;19) usually leading to an IGH::BCL3 rearrangement. WGS identified NECTIN2 as translocation partner, which is recurrently translocated in T cell neoplasms and was found to be rearranged to IGH in DLBCL. In a CML patient with persistent thrombocytopenia, WGS revealed a CUX1 mutation. Association with shorter survival has been described for CUX1 mutations in myeloid neoplasms (Aly et al. 2019). WGTS also led to new findings: In a patient with unexplained persistent cytopenia an Xq25 deletion comprising STAG2 was detected. STAG2 mutations are well known in myeloid neoplasms, but STAG2 deletions have not been published yet. In this patient, WGS provided evidence of clonal hematopoiesis and the diagnosis CCUS had to be made. Of note, STAG2 loss in an AML cell line resulted in altered gene expression (Smith et al. 2020) and the patient's WTS data showed consistent up- and downregulation of specific genes (e.g. SFRP2, CHI3L1), compared to normal controls and to MDS/AML patients w/o STAG2 alteration. One unsolved case showed a CNV of unknown significance (dup(20q12)) resulting in a TOP1::PLCG1 fusion; genes which are known to be affected by other SVs and SNVs in myeloid and lymphoid neoplasms. Furthermore, WGTS allowed detection of a therapeutically relevant alteration: In one patient with relapsed multiple myeloma after anti-BCMA CAR T cell therapy WGTS revealed homozygous deletion of the TNFRSF17 (BCMA) gene and strongly reduced TNFRSF17 expression. Such escape from immunotherapy by biallelic inactivation of the target has been shown to be irreversible (Da Vià et al. 2021), guiding further treatment decision as other BCMA-targeted therapies have to be excluded.

Findings of WGTS were reported within a median of 21 days after sample collection. By providing diagnostically and therapeutically relevant results, WGTS can save costs on further tests or inappropriate therapies. We assume that both TAT in routine operation and costs will finally speak in favor of WGTS in the near future.

Conclusion: Initial real-world data of the ongoing study demonstrate the power of WGTS to add valuable information to gold standard diagnostics in 1/3 of unclear cases within 3 weeks. In the context of challenging unsolved cases, close cooperation of experts from various backgrounds (hematology, pathology, human genetics and bioinformatics) in tumor boards allows to achieve the best possible diagnoses for patients.

Truger:MLL Munich Leukemia Laboratory GmbH: Current Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Current Employment. Walter:MLL Munich Leukemia Laboratory: Current Employment. Hutter:MLL Munich Leukemia Laboratory: Current Employment. Baer:MLL Munich Leukemia Laboratory: Current Employment. Nadarajah:MLL Munich Leukemia Laboratory: Current Employment. Kern:MLL Munich Leukemia Laboratory: Current Employment, Other: Ownership. Haferlach:MLL Munich Leukemia Laboratory: Current Employment, Other: Ownership. Haferlach:Munich Leukemia Laboratory: Current Employment, Other: Part ownership.