Feasibility and clinical utility of comprehensive genomic profiling of hematological malignancies

Abstract Identification of genetic alterations through next‐generation sequencing (NGS) can guide treatment decision‐making by providing information on diagnosis, therapy selection, and prognostic stratification in patients with hematological malignancies. Although the utility of NGS‐based genomic profiling assays was investigated in hematological malignancies, no assays sufficiently cover driver mutations, including recently discovered ones, as well as fusions and/or pathogenic germline variants. To address these issues, here we have devised an integrated DNA/RNA profiling assay to detect various types of somatic alterations and germline variants at once. Particularly, our assay can successfully identify copy number alterations and structural variations, including immunoglobulin heavy chain translocations, IKZF1 intragenic deletions, and rare fusions. Using this assay, we conducted a prospective study to investigate the feasibility and clinical usefulness of comprehensive genomic profiling for 452 recurrently altered genes in hematological malignancies. In total, 176 patients (with 188 specimens) were analyzed, in which at least one alteration was detected in 171 (97%) patients, with a median number of total alterations of 7 (0–55). Among them, 145 (82%), 86 (49%), and 102 (58%) patients harbored at least one clinically relevant alteration for diagnosis, treatment, and prognosis, respectively. The proportion of patients with clinically relevant alterations was the highest in acute myeloid leukemia, whereas this assay was less informative in T/natural killer‐cell lymphoma. These results suggest the clinical utility of NGS‐based genomic profiling, particularly for their diagnosis and prognostic prediction, thereby highlighting the promise of precision medicine in hematological malignancies.


| INTRODUC TI ON
Hematological malignancies are diagnosed and classified using morphology, immunophenotype, and genetic alterations. A diverse array of genetic alterations underlies their pathogenesis, as exemplified by more than 250 recurrent alterations listed in the latest WHO classification. 5 In addition, reflecting their diversity and the presence of rare disease types, there have been many genetic studies in hematological malignancies, which have identified novel genetic alterations even recently. [6][7][8] Moreover, a non-negligible proportion of patients with myeloid neoplasms, such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), have pathogenic germline variants in leukemia-predisposing genes, such as RUNX1 and DDX41 6,9 , illuminating the potential necessity of genomic testing for germline susceptibility in hematological malignancies. Recent studies investigated the feasibility and utility of NGSbased genomic profiling in hematological malignancies, which enables simultaneous examination of many somatic alterations at once. [10][11][12] However, its potential may be underestimated because these studies did not sufficiently cover driver genes listed in the WHO classification as well as recently discovered ones. In addition, germline controls and/or RNA panel were lacking, and several disease types, including T/natural killer-cell non-Hodgkin lymphoma (T/ NK-NHL), were not well investigated in these studies. Therefore, to address these issues, here we devised an integrated DNA/RNA profiling assay that can simultaneously detect various types of somatic alterations and germline variants in 452 genes recurrently altered in various hematological malignancies. Using this assay, we have recently discovered ones, as well as fusions and/or pathogenic germline variants. To address these issues, here we have devised an integrated DNA/RNA profiling assay to detect various types of somatic alterations and germline variants at once. Particularly, our assay can successfully identify copy number alterations and structural variations, including immunoglobulin heavy chain translocations, IKZF1 intragenic deletions, and rare fusions. Using this assay, we conducted a prospective study to investigate the feasibility and clinical usefulness of comprehensive genomic profiling for 452 recurrently altered genes in hematological malignancies. In total, 176 patients (with 188 specimens) were analyzed, in which at least one alteration was detected in 171 (97%) patients, with a median number of total alterations of 7 (0-55). Among them, 145 (82%), 86 (49%), and 102 (58%) patients harbored at least one clinically relevant alteration for diagnosis, treatment, and prognosis, respectively. The proportion of patients with clinically relevant alterations was the highest in acute myeloid leukemia, whereas this assay was less informative in T/natural killer-cell lymphoma. These results suggest the clinical utility of NGS-based genomic profiling, particularly for their diagnosis and prognostic prediction, thereby highlighting the promise of precision medicine in hematological malignancies.

K E Y W O R D S
comprehensive genomic profiling, hematological malignancy, next-generation sequencing, precision medicine, somatic alteration performed a prospective hospital-based cohort study to investigate the potential of NGS-based comprehensive genomic profiling in 188 specimens of hematological malignancies.

| Workflow of clinical sequencing
We performed a prospective cohort study to evaluate the feasibility and clinical utility of integrated DNA/RNA profiling assay using tumor and germline specimens. The workflow is divided into four parts: (i) patient enrollment/specimen collection, (ii) library preparation, (iii) sequencing and bioinformatic analysis, and (iv) assessment of clinical relevance ( Figure 1A). DNA and RNA were independently processed for library preparation and hybrid selection.

| Patients and specimens
Patients aged 1 year or older who were diagnosed with untreated or relapsed/refractory hematological malignancies according to the WHO classification 5 and would intend to receive chemotherapy and/or hematopoietic stem cell transplantation (HSCT) were enrolled. The availability of tumor specimens with tumor content of 5% or more was required (≥20% was recommended). Fresh or formalinfixed paraffin-embedded (FFPE) specimens of bone marrow (BM), peripheral blood (PB), fluid aspirate, lymph node, or tumor tissues were collected. Buccal swab was obtained as a matched germline control. The study was approved by the National Cancer Center Institutional Review Board, and all patients and/or their legal guardians (when minors were enrolled) provided written informed consent for this study.

| Design of integrated DNA/RNA profiling assay
We designed a hybridization capture-based DNA/RNA profiling assay to detect SNVs, indels, SVs, CNAs, and fusions (Tables S1-S3). These include almost all driver alterations listed in the Japanese Society of Hematology (JSH) Guideline for Genomic Testing in Hematological Malignancies (2021 release) (JSH Genome Guideline; http://www.jshem.or.jp/genom gl/) (Tables S4-S6) Table S1). The RNA panel was designed to detect (i) CDSs of 44 genes targeted by recurrent fusions, (ii) CDSs and untranslated regions of 32 genes targeted by recurrent activating SVs, and (iii) IG/TCR genes (Table S2). To increase the sensitivity to detect fusions, we have extended our previous method 13 to design probes for not only fused exons and their adjacent exons but also junction sequences of 134 well-known fusions ( Figure 1B; Table S3). The performance of our assay was assessed for (i) 233 mutations in five cell lines detected at an allele frequency of 0.05 or more by amplicon sequencing with QIAseq Human Myeloid Neoplasms Panel (Qiagen), and (ii) 35 SVs/fusions and IKZF1 intragenic deletion in 31 cell lines described in previous reports. In addition, using clinical specimens, fusions were evaluated using 13 different RT-PCR tests and five different FISH tests, and rearrangements and deletion of 17p (del17p) using eight different FISH tests. (Promega), respectively. Library preparation for targeted DNA sequencing (DNA-seq) was performed using a custom SureSelect library (Agilent Technologies), as previously described. 14,15 Library preparation for targeted RNA sequencing (RNA-seq) was also performed using a custom SureSelect library with SureSelect XT RNA direct according to the manufacturer's instructions. These libraries were sequenced using the NextSeq 500 (Illumina) to generate 150 bp paired-end reads to ≥400× average depth for DNA-seq and ≥3 million uniquely mapped reads for RNA-seq either in-house or at RIKEN GENESIS. Quality control results were compared using a two-sided Welch's t-test.

| Mapping and mutation detection
For targeted DNA-seq data, sequence alignment and mutation calling were performed using the Genomon pipeline version 2.6.2 (https:// github.com/Genom on-Proje ct/), as previously described. 14,15 Sequencing reads were mapped to the custom reference genome

| SV and CNA detection
SVs and CNAs were detected using the Genomon pipeline and the CNACS algorithm, respectively, as previously described. 14,15 Putative SVs were manually curated and further filtered by removing Guideline were analyzed for SVs and focal CNAs (Table S6).

| Fusion detection
For targeted RNA-seq data, sequence alignment and fusion calling were performed using the Genomon RNA pipeline version 2.6.2, with a slight modification of those used for poly-A RNA-seq. 14,15 Candidate fusions with 10 or more supporting reads in tumor were filtered by excluding (i) endogenous IG/TCR recombination; and (ii) those detected in in-house pooled normal specimens. Finally, mapping errors were removed by visual inspection with IGV. As IGH/DUX4 and STIL-TAL1 fusions are difficult to detect, these fusions were individually reviewed.

| Definition and interpretation of clinically relevant alterations
Clinical evidence levels (A-D) were assigned to each genetic alteration in terms of diagnosis, treatment, and prognostic prediction for each disease subtype at the time of NGS-based genomic profiling according to the JSH Genome Guideline (Table S6)

| Design and validation of integrated DNA/RNA profiling assay
We designed NGS-based comprehensive genomic profiling using tumor and germline specimens. Integrated DNA/RNA analysis with elaborated panel design can provide increased breadth and improved sensitivity to detect not only fusion-generating SVs but also activating SVs, which causes aberrant expression of oncogenes ( Figure 1B, Tables S1-S3). As the excellent performance of targeted DNA-seq for detecting SNVs and CNAs has been established (Table S7), 14,15 we then evaluated the analytical accuracy for fusion-generating and activating SVs by integrated DNA/RNA analysis ( Figure 1C, Table   S8). We detected all 24 known fusions, three FLT3-internal tandem duplications (ITDs), and 1 KMT2A-partial tandem duplication (PTD), with no false positive call observed. We also detected eight of nine activating SVs, consisting of IGH translocations (including IGH/MYC, IGH/BCL2, IGH/CCND1, IGH/CRLF2, and IGH/DUX4) and GATA2/ MECOM rearrangement. In addition, we confirmed successful detection of an intragenic deletion of IKZF1 19 ( Figure 1D). These observations suggest acceptable sensitivity for various kinds of SVs and intragenic deletions.

| Quality evaluation of NGS-based genomic profiling
Between 2019 and 2021, 197 specimens from 185 patients were enrolled in our prospective cohort study ( Figure 1A,E). Nine specimens were excluded, and the remaining 188 specimens were submitted to further analysis (Table S9). The specimens consisted of 81 fresh and 107 FFPE specimens, including tumors from excisional and 0.97 (0.56-0.99) in normal tissues, respectively. Fresh specimens tended to show a higher depth than FFPE specimens, which included all six (4%) specimens with low coverage (<0.85), whereas no difference was observed across tissues and biopsy procedures ( Figure S1A,B). Except for nine specimens collected after allogeneic HSCT, genome-wide copy number was evaluable in 142 (79%) of 179 specimens, which contained all fresh specimens but only 64% of FFPE specimens (Figures 1E and S1C). As the amount of input RNA was too small in 13 (7%) specimens, the remaining 175 specimens were analyzed by targeted RNA-seq ( Figures 1E and S1D). The read number was higher in fresh than FFPE specimens, with a median of 7.1 (0.9-13.3) × 10 6 ( Figure S1E). DNA integrity number was lower in FFPE specimens stored for 1 year or longer than those stored for less than 1 year (p < 0.01) ( Figure S1F). The median turnaround time (TAT), defined as time from specimen receipt to molecular tumor board, was 50.5 (21-167) days.

| Somatic alterations detected by NGS-based genomic profiling
Among the 188 specimens, five patients submitted two specimens from different sites (10 specimens), and six patients submitted two or more specimens at different time points (13 specimens).
Therefore, a total of 176 patients with various backgrounds were analyzed ( Figure 1G, Tables Figure 3A). All SV partners of BCL2 and CCND1 were IGH ( Figure 3A, Table S11).

| Extensive heterogeneity of somatic alterations in our cohort
The entire cohort showed extensive heterogeneity of somatic alterations, while reflecting the genetic landscape of each disease type reported by previous genetic studies 20-24 ( Figure S2). As confirmed in cell lines, integrated DNA/RNA profiling successfully identified one GATA2/MECOM inversion and five KMT2A-PTDs ( Figure 3C, Table   S11). In addition to well-known disease-defining SVs and fusions, our assay detected various kinds of SVs and fusions, such as PVT1-SUPT3H fusion and DUSP22 rearrangement characteristic of blastic plasmacytoid dendritic cell neoplasm (BPDCN) and ALK-negative anaplastic large cell lymphoma, respectively. Moreover, we identified rare IG/TCR-involving SVs, including IGH/BCL3 and TRB/NOTCH1, as well as kinase fusions, such as TRAF1-ALK, TRIM24-FGFR1, and XPO1-ROS1, which can be a promising therapeutic target ( Figure 3C).

| Differences in somatic alterations across disease types
Disease-specific frequencies revealed quite different genetic profiles and associated clinical evidence across disease types (Figures 4 and   5, Table S14). These frequencies were comparable or slightly higher than those reported by the FoundationOne Heme panel, 10 demonstrating the promising performance of our assay (Tables S15-S17).

| Detection of cancer-related germline variants
Germline variants causing hereditary cancers were identified in six (3%) patients, all of which were of level A evidence (Table S18).
Among them, one AML patient harbored a DDX41 germline variant and one AML, one MDS, and three ALL/LBL patients harbored deleterious BRCA1 or BRCA2 germline variants associated with hereditary breast and ovarian cancers. All six patients were referred for genetic counseling, and three of them requested confirmatory testing and were validated to have the variants.

| Multi-site and multi-timepoint analyses
Multi-site and multi-timepoint analyses were performed for five and six patients, respectively (Table S9). In one patient with a history of essential thrombocythemia who developed myeloid sarcoma, an MPL mutation was found in both BM and sarcoma specimens, while several alterations, including RUNX1 mutation and 7q deletion, were detected only in sarcoma specimens, suggesting that these alterations contribute to the clonal evolution ( Figure S4A). In one CLL patient analyzed before and after BTK inhibitor therapy, a PLCG2 mutation, known to confer BTK inhibitor resistance (level D), was detected only after progression ( Figure S4B). These results suggest that serial analysis is helpful to uncover somatic alterations underlying therapeutic resistance.

| DISCUSS ION
Through a prospective hospital-based cohort study, we demonstrate   in CLL and CRBN mutation in MM. 35,36 Serial sampling may provide additional therapeutically relevant information, given the mutational differences between diagnosis and relapse. [37][38][39] Although the currently targetable alterations are inadequate for hematological malignancies, comprehensive genomic profiling can serve as a platform for identifying appropriate candidate individuals for clinical trials investigating molecularly targeted therapies.
Comprehensive genomic profiling can help predict patient prognosis and determine transplant candidacy, particularly in AML and ALL/LBL. In addition, such genetic information may be useful to consider the timing of therapeutic intervention in MDS, MPN, and indolent lymphomas. As clinical evidence for the utility of genetic alterations to predict prognosis is still limited, additional clinicogenetic studies are required.
In our study, TAT was approximately 50 days, which prevents the use of the profiling result for determining induction therapy for acute leukemia. Therefore, it is essential to shorten TAT and combine with conventional methods to treat rapidly progressing malignan-