Treatment in AL Amyloidosis: Moving towards Individualized and Clone-Directed Therapy

Systemic amyloid light chain (AL) amyloidosis is a rare protein deposition disease caused by a clonal B cell disorder of the bone marrow. The underlying diseases can be plasma cell disorders (monoclonal gammopathy of clinical significance, smoldering or symptomatic myeloma) or B cell non-Hodgkin’s lymphoma (e.g., Waldenstrom’s disease or marginal zone lymphoma) with secretory activity. It is crucial to characterize the underlying disease very precisely as the treatment of AL amyloidosis is directed against the (often small) B cell clone. Finally, the detection of cytogenetic aberrations of the plasma cell clone will likely play an important role for choosing an effective drug in the near future.

Whole Genome Sequencing Identifies Non-KIT Mutations and Cytogenetic Aberrations in Systemic Mastocytosis but Has Limited Sensitivity for Detection of KIT D816V

Background: Systemic mastocytosis (SM) is a hematologic neoplasm characterized by the infiltration of clonal mast cells in the bone marrow or other extra-cutaneous organs. The clinical course varies between advanced and non-advanced (indolent and smoldering SM) forms of SM. The vast majority of patients harbor the activating D816V mutation in the KIT tyrosine kinase. Additional somatic mutations in other genes have been recognized as risk factors in SM. Cytogenetic aberrations are rarely found in SM but have been associated with advanced disease. Whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) have been described as an alternative to cytogenetics and targeted molecular genetic analysis in myeloid cancers. Aim: To assess the ability of WGS/WTS to detect cytogenetic aberrations and recurrent somatic mutations in SM. Methods: 120 patients (51 female, 69 male) diagnosed with SM were analyzed with WGS/WTS and results were compared with orthogonal data of KIT D816V PCR, targeted sequencing, and cytogenetics. 47 patients (39%) were diagnosed with advanced SM (1 mast cell leukemia, 3 aggressive SM, 43 SM with associated hematologic neoplasm). For WGS, 2x151bp paired-end reads were generated on NovaSeq 6000 and HiSeqX machines (Illumina, San Diego, CA). BaseSpace's Tumor/Normal app v3 was used to call variants with Strelka Somatic Variant Caller v2.4.7 and structural variants (aberrations with >50bp in size) with Manta (v0.28.0). Genomic DNA from a mixture of multiple anonymous donors (Promega, Fitchburg, WI, USA) was used as normal. For WTS, 2x101 bp paired-end reads were produced with a median of 50 mio. reads per sample, aligned with STAR v2.5.0, and variants were called using Isaac Variant Caller v2.3.13. Results: WGS/WTS detected cytogenetic aberrations in 21% of patients: 2 patients displayed a complex aberrant karyotype, 3 balanced structural aberrations, 16 copy number alterations, and 6 copy number neutral losses of heterozygosity. Aberrations detected by chromosome banding analysis were also found by WGS in all but three patients (small clones with aberrations present in ≤20% of metaphases and <10% of interphase nuclei as determined by FISH). In contrast, WGS/WTS detected additional aberrations in 16 patients. The frequency of chromosomal aberrations detected by WGS/WTS was higher in advanced compared to non-advanced SM (34% vs. 12%, p<0.05). KIT D816V was detected by PCR in 98%, by WGS in 21% and by WTS in 46% of patients. The detection rate by WGS was significantly higher in advanced (36%) compared to non-advanced SM (12%, p<0.05) while no difference was observed for WTS (45% vs. 47%). Somatic mutations outside of KIT were analyzed within a subset of 121 genes recurrently mutated in hematologic neoplasms. 46% of patients showed non-KIT mutations with a median of 2 mutations per patient. Both frequency of non-KIT mutations as well as the median number of mutations per patient was higher in advanced (83%; n=3) compared to non-advanced SM (22%, n=1, p<0.05). Finally, we analyzed the impact of genetic aberrations on survival in our SM cohort. Patients were grouped according to the presence of chromosomal aberrations and gene mutations (non-KIT) as assessed by WGS/WTS. SM patients with both types of aberrations (n=16), one type of aberration (n=47; gene mutations only n=38; chromosomal aberrations only n=8), or no aberration but KIT D816V (n=57) showed significant differences in overall survival (p<0.05, Figure 1). Con clusions: WGS/WTS has limited sensitivity for detection of KIT D816V in SM. This finding can be explained by the low KIT D816V mutation burden typically found in bone marrow aspirates of SM patients. In line, we observed a slightly higher detection rate in advanced SM and in RNA-based WTS analysis. As WGS/WTS will be applied for the diagnostic workup of myeloid malignancies in the future and SM associated with other hematologic neoplasms may be overlooked if not specifically investigated, additional PCR-based techniques are still mandatory to rule out KIT D816V as a diagnostic criterion for SM. In contrast, WGS/WTS detects both chromosomal aberrations and additional gene mutations in patients with SM and can be used as an alternative to cytogenetics and targeted sequencing for risk assessment. In particular, the absence of genetic aberrations in WGS/WTS identifies SM patients with indolent course of the disease and favorable prognosis. Figure 1 Figure 1. Disclosures Hoermann: Novartis: Honoraria. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

Impact of Allogeneic Hematopoietic Stem Cell Transplantation in First Complete Remission and Additional Cytogenetic Aberrations at Diagnosis on Prognosis in 1256 Pediatric Patients with KMT2A-Rearranged Acute Myeloid Leukemia: A Retrospective Study By the I-BFM-SG

Introduction KMT2A-rearranged (KMT2A-r) acute myeloid leukemia (AML) is a heterogeneous genetic subgroup with a frequency of about 25% in children with AML. At the 62 nd ASH annual meeting last year, we reported on the differences in outcome of various KMT2A subgroups based on translocation partner and the significance of minimal residual disease (MRD) status during and after induction as a follow-up study of Balgobind et al., Blood 2009. The impact of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR1) and the presence of additional cytogenetic aberrations (ACAs) on prognosis have not yet been described for our cohort. Methods Data on allo-HSCT in CR1 and the presence of ACAs of 1256 KMT2A-r de novo pediatric AML patients from 15 AML study groups affiliated with the I-BFM Study Group, diagnosed between 2005 and 2016, were retrospectively collected and studied. Karyotypes were reviewed and classified by two of the authors (RW&CH). Based on translocation partners, patients were classified to the KMT2A high-risk subgroup (6q27, 10p11.2, 10p12, 4q21, and 19p13.3) or non-high-risk subgroup (9p22, 19p13, 19p13.1, 1q21, Xq24, 17q21, 1p32, and 17q12). These two categories have been used to estimate a Cox model. Patients with unknown translocation partners were excluded from these analyses (n=126). Flow cytometry MRD levels at the end of induction course 1 (EOI1) and 2 (EOI2) <0.1% were considered negative, and levels ≥0.1% positive. Kaplan-Meier's methodology was used to estimate disease-free survival (DFS) and overall survival (OS). DFS was calculated from EOI1 for patients in CR to date of relapse or death/last follow-up. OS was calculated from the time of diagnosis to date of death/last follow-up. Two-sided P-values of ≤ .01 were considered statistically significant. Covariates with P-values ≤ .05 in univariate analyses were included in multivariate analyses; allo-HSCT in CR1 was included as a time-dependent covariate in the Cox model. MRD status at EOI2 was excluded from multivariate analyses as therapy could have been adjusted to the MRD status and the number of MRD positive patients was small. Results Of 1256 pediatric patients with KMT2A-r AML, data on HSCT in CR1 and ACAs were available for 1186 (94.4%) and 1204 patients (95.9%), respectively; 211 (17.8%) patients received HSCT in CR1 and ACAs were present in 601 (49.9%) patients. Compared with the KMT2A non-high-risk subgroup, patients in the KMT2A high-risk subgroup underwent HSCT in CR1 more often (23.8% vs 15.0%; P < .001). ACAs were borderline significantly more common in the KMT2A high-risk subgroup (54.1% vs 46.4%; P = .015). Univariate analysis of the probability of DFS (Table 1) showed that the KMT2A high-risk subgroup (HR 2.1; 95% CI, 1.7-2.5), age ≥10 years (HR 1.4; 95% CI, 1.2-1.7), and MRD ≥0.1 at EOI1 (HR 1.5; 95% CI, 1.1-1.9) were associated with DFS. HSCT in CR1 was a borderline significant prognostic factor (HR 0.7; 95% CI, 0.6-0.9). In a multivariate analysis for DFS (n=515) (Table 1), the KMT2A high-risk subgroup (HR 2.0; 95% CI, 1.6-2.6), MRD ≥0.1 at EOI1 (HR 1.7; 95% CI, 1.2-2.3), and HSCT in CR1 (HR 0.6; 95% CI, 0.4-0.9) were associated with DFS. Univariate analysis of the probability of OS (Table 1) showed that the KMT2A high-risk subgroup (HR 1.8; 95% CI, 1.5-2.2), age ≥10 years (HR 1.6; 95% CI 1.3-2.0), WBC ≥100 x10 9/L (HR 1.4; 95% CI, 1.1-1.7), the presence of ACAs (HR 1.4; 95% CI, 1.2-1.7), and MRD ≥0.1 at EOI1 (HR 2.1; 95% CI, 1.6-2.7) were associated with OS. HSCT in CR1 was not associated with OS. The effect of HSCT in CR1 was not significantly different between the KMT2A high-risk and non-high-risk subgroups. In a multivariate analysis for OS (n=557) (Table 1), the KMT2A high-risk subgroup (HR 1.9; 95% CI, 1.4-2.5), age ≥10 years (HR 1.5; 95% CI, 1.1-1.9), the presence of ACAs (HR 1.6; 95% CI, 1.2-2.1), and MRD positivity at EOI1 (HR 1.9; 95% CI, 1.4-2.5) were associated with OS. Conclusions In this cohort of KMT2A-r pediatric AML patients, the presence of ACAs at diagnosis was independently associated with inferior OS, but not with DFS. This may be due to the exclusion of refractory patients in DFS analysis, who were significantly more common in the group of patients with ACAs. Analysis has yet to be performed to distinguish karyotype complexity. In addition, allo-HSCT in CR1 was an independent predictor of improved DFS, but was not a prognostic factor for OS. Figure 1 Figure 1. Disclosures Abrahamsson: wedish Children´s Cancer Foundation. Research grants and 50% senior research position for clinical research on pediatric leukemia: Research Funding. Locatelli: Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Miltenyi: Speakers Bureau; Medac: Speakers Bureau; Jazz Pharamceutical: Speakers Bureau; Takeda: Speakers Bureau.

Prognostic Relevance of Cytogenetic Chromosomal Abnormalities on Outcome Among AL Amyloidosis Patients in the Netherlands

Introduction Systemic light chain (AL) amyloidosis is a clonal plasma cell neoplasm, that carries a poor prognosis. Treatment is adapted from protocols as used in MM, and include bortezomib-based (PI) regimens with or without stem cell transplantation (SCT). PI-based regimens have improved treatment responses and outcomes. Cytogenetic analysis has become an important tool in the diagnostic process of plasma cell disorders, and evidence for prognostic significance of specific genetic abnormalities in relation to therapy efficacy is recognized in systemic AL amyloidosis. Thus far, this prognostic significance has been evaluated in single center institutions. Aim This nationwide, population-based study aimed to assess the impact of cytogenetic abnormalities on hematological response and survival among patients with systemic AL amyloidosis treated with PI-based regimens. Methods We identified 349 patients ≥18 years with systemic AL amyloidosis diagnosed between 2017 and 2019 in the Netherlands Cancer Registry, with survival follow-up until February 1, 2021. Data on therapeutic strategy was known for all individual patients. The cytogenetic aberrations studied include gain(1q), hyperdiploidy, del(17p), and IGH rearrangements, i.e. t(11;14), t(4;14), t(14;16), and t(v;14q32) (non-specified IGH rearrangement). Hematological response and overall survival (OS) were evaluated in relation to cytogenetic aberrations. OS was defined as death by any cause post-diagnosis. Uni- and multivariable analysis for establishing independent predictors of OS, i.e. age, sex, cytogenetic assessment and SCT, was performed using Cox regression. Patients diagnosed at autopsy (n=4), patients who did not start first-line therapy (n=64) or received other therapies (n=37), and patients with systemic AL amyloidosis related to Waldenström Macroglobulinemia (n=10) were excluded. Results In our analytic cohort, 234 patients (median age 67 years; 62% males) were treated with PI-based regimens. Of these patients, 153 (65%) were ≤70 years at diagnosis. SCT was performed in 70 (30%) patients following PI-based regimens. For 170 (73%) patients, cytogenetic assessment was performed. IGH rearrangements were observed in 76 patients (45%), comprised of t(11;14) in 27 patients, t(4;14) in 3 patients, t(14;16) in 2 patients and non-specified IGH rearrangements in 44 patients. Furthermore, 26 patients carried a gain(1q), 4 patients a del(17p), and 33 patients were hyperdiploid. Due to the limited patient number with del(17p), response and OS was not evaluated for this subgroup. A complete remission (CR; n=53), very good partial response (VGPR; n=66), or partial remission (PR; n=55) was accomplished for 74% of the patients, ≥VGPR for 51% of the patients. The reached hematological response was irrespective of the detected cytogenetic abnormalities. In detail, ≥VGPR was 56% for patients with a t(11;14), 62% for patients with a gain(1q), 55% for patients with hyperdiploidy, and 50% for patients with an IGH rearrangement and this was not statistical significant different from patients without these cytogenetic abnormalities. The 3-year OS was 61% for patients treated with PI-based regimens. For patients with a cytogenetic assessment (n=170), there was no significant difference in 3-year OS between patients with or without a t(11;14) (69% vs. 66%, respectively; p=0.70), with or without gain(1q) (57% vs. 68%, respectively; p=0.58), with or without hyperdiploidy (72% vs. 65%, respectively; p=0.63), or with or without an IGH rearrangement (59% vs. 72%; p=0.21). Only SCT was an independent predictor for reduced risk of mortality in uni- and multivariable analyses, overall as well as for the specific cytogenetic subgroups. Conclusion In this Dutch 'real world' population of AL amyloidosis patients treated with PI-based regimens, 74% had a ≥PR and 51% had ≥VGPR. The 3-year OS was 61%. We evaluated the cytogenetic data of 170 patients, but could not confirm a relation between PI-based regimens and outcome, overall as well as in specific cytogenetic subgroups. Patient numbers of the cytogenetic subgroups were low and we could not determine the partner gene in 44 patients with an IGH rearrangement. To the best of our knowledge, this is the first population-based study to evaluate the prognostic relevance of cytogenetic abnormalities in relation to hematological response and OS among systemic AL amyloidosis patients. Disclosures Minnema: Alnylam: Consultancy; Kite/Gilead: Consultancy; Jansen-Cilag: Consultancy; BMS: Honoraria; Celgene: Other: Hospitality.

Molecular profiling of pediatric meningiomas shows tumor characteristics distinct from adult meningiomas

In contrast to adults, meningiomas are uncommon tumors in childhood and adolescence. Whether adult and pediatric meningiomas differ on a molecular level is unclear. Here we report detailed genomic analyses of 37 pediatric meningiomas by sequencing and DNA methylation profiling. Histologically, the series was dominated by meningioma subtypes with aggressive behavior, with 70% of patients suffering from WHO grade II or III meningiomas. The most frequent cytogenetic aberrations were loss of chromosomes 22 (23/37 [62%]), 1 (9/37 [24%]), 18 (7/37 [19%]), and 14 (5/37 [14%]). Tumors with NF2 alterations exhibited overall increased chromosomal instability. Unsupervised clustering of DNA methylation profiles revealed separation into three groups: designated group 1 composed of clear cell and papillary meningiomas, whereas group 2A comprised predominantly atypical meningiomas and group 2B enriched for rare high-grade subtypes (rhabdoid, chordoid). Meningiomas from NF2 patients clustered exclusively within groups 1 and 2A. When compared with a dataset of 105 adult meningiomas, the pediatric meningiomas largely grouped separately. Targeted panel DNA sequencing of 34 tumors revealed frequent NF2 alterations, while other typical alterations found in adult non-NF2 tumors were absent. These data demonstrate that pediatric meningiomas are characterized by molecular features distinct from adult tumors.

Consistent B Cell Receptor Immunoglobulin Features Between Siblings in Familial Chronic Lymphocytic Leukemia

Key processes in the onset and evolution of chronic lymphocytic leukemia (CLL) are thought to include chronic (antigenic) activation of mature B cells through the B cell receptor (BcR), signals from the microenvironment, and acquisition of genetic alterations. Here we describe three families in which two or more siblings were affected by CLL. We investigated whether there are immunogenetic similarities in the leukemia-specific immunoglobulin heavy (IGH) and light (IGL/IGK) chain gene rearrangements of the siblings in each family. Furthermore, we performed array analysis to study if similarities in CLL-associated chromosomal aberrations are present within each family and screened for somatic mutations using paired tumor/normal whole-genome sequencing (WGS). In two families a consistent IGHV gene mutational status (one IGHV-unmutated, one IGHV-mutated) was observed. Intriguingly, the third family with four affected siblings was characterized by usage of the lambda IGLV3-21 gene, with the hallmark R110 mutation of the recently described clinically aggressive IGLV3-21R110 subset. In this family, the CLL-specific rearrangements in two siblings could be assigned to either stereotyped subset #2 or the immunogenetically related subset #169, both of which belong to the broader IGLV3-21R110 subgroup. Consistent patterns of cytogenetic aberrations were encountered in all three families. Furthermore, the CLL clones carried somatic mutations previously associated with IGHV mutational status, cytogenetic aberrations and stereotyped subsets, respectively. From these findings, we conclude that similarities in immunogenetic characteristics in familial CLL, in combination with genetic aberrations acquired, point towards shared underlying mechanisms behind CLL development within each family.

A Comprehensive Review of the Genomics of Multiple Myeloma: Evolutionary Trajectories, Gene Expression Profiling, and Emerging Therapeutics

Multiple myeloma (MM) is a blood cancer characterized by the accumulation of malignant monoclonal plasma cells in the bone marrow. It develops through a series of premalignant plasma cell dyscrasia stages, most notable of which is the Monoclonal Gammopathy of Undetermined Significance (MGUS). Significant advances have been achieved in uncovering the genomic aberrancies underlying the pathogenesis of MGUS-MM. In this review, we discuss in-depth the genomic evolution of MM and focus on the prognostic implications of the accompanied molecular and cytogenetic aberrations. We also dive into the latest investigatory techniques used for the diagnoses and risk stratification of MM patients.

Clinical and Biological Characteristics of Medullary and Extramedullary Plasma Cell Dyscrasias

Background: Extramedullary plasma cell (PC) disorders may occur as extramedullary disease in multiple myeloma (MM-EMD) or as primary extramedullary plasmocytoma (pEMP)/solitary osseous plasmocytoma (SOP). In this study, we aimed to obtain insights into the molecular mechanisms of extramedullary spread of clonal PC. Methods: Clinical and biological characteristics of 87 patients with MM-EMD (n = 49), pEMP/SOP (n = 20) and classical MM (n = 18) were analyzed by using immunohistochemistry (CXCR4, CD31, CD44 and CD81 staining) and cytoplasmic immunoglobulin staining combined with fluorescence in situ hybridization (cIg-FISH). Results: High expression of CD44, a cell-surface glycoprotein involved in cell-cell interactions, was significantly enriched in MM-EMD (90%) vs. pEMP/SOP (27%) or classical MM (33%) (p < 0.001). In addition, 1q21 amplification by clonal PC occurred at a similar frequency of MM-EMD (33%), pEMP/SOP (57%) and classical MM (44%). Conversely, del(17p13), t(4;14) and t(14;16) were completely absent in pEMP/SOP. Besides this, 1q21 amplification was identified in 64% of not paraskeletal samples from MM-EMD or pEMP compared to 9% of SOP or paraskeletal MM-EMD/pEMP and 44% of classical MM samples, respectively (p = 0.02). Conclusion: Expression of molecules involved in homing and cytogenetic aberrations differ between MM with or without EMD and pEMP/SOP.