Biallelic Inactivation Of TP53 Research Articles

Hematologic malignancies in Li-Fraumeni syndrome.

10613 Background: Li-Fraumeni Syndrome (LFS) is a hereditary cancer predisposition syndrome caused by mutations in the tumor suppressor gene TP53. Since its discovery, a growing list of cancers have been described in association with LFS, predominantly solid tumors. However, hematologic malignancies (HMs) cumulatively account for 4-10% of cancer diagnoses in LFS. In this study, we present the largest series of HMs in families with LFS with detailed clinical courses. Methods: This was a two-center retrospective observational cohort study conducted at the University of Utah and the University of Wisconsin-Madison with IRB approval. Cancer genetics clinic registries were reviewed to identify all families with LFS who also had a confirmed diagnosis of a HM. To meet criteria for LFS, the patient had to have a confirmed germline pathologic or likely pathologic TP53 variant. Patients with a TP53 variant of unknown significance (VUS) were also included if they met classical LFS testing criteria. Data was gathered by manual chart review of both the families’ histories and electronic health records. Results: Among the 121 families identified with LFS, 35 (29%) families had one or more HM. Of these, 17 individuals from 16 families (13%) had confirmed germline TP53 mutations. The most frequent HMs found were acute lymphoblastic leukemia (ALL) (n=6), non-Hodgkin lymphoma (NHL) (n=5), myelodysplastic syndromes (MDS) (n=3), and chronic lymphocytic leukemia (CLL) (n=2). Acute myeloid leukemia (AML), Langerhans cell histiocytosis (LCH), and chronic myeloid leukemia (CML) were each diagnosed once. Most cases were identified in adulthood (n=12, 71%). Of the 19 total HMs, the minority (n=5, 26%) were diagnosed post-cytotoxic therapy and only six (35%) individuals had received a diagnosis of LFS prior to HM diagnosis. The MDS/AML cohort (n=4) all exhibited bi-allelic inactivation of TP53. Two of these four patients achieved long term survival after matched-unrelated donor (MUD) hematopoietic stem cell transplant (HSCT). All three patients who underwent MUD donor HSCT had immune-mediated adverse events. Conclusions: In this study we found a significantly higher incidence of HMs in LFS than previously described, with a predominance of lymphoid over myeloid malignancies. The majority of patients had excellent outcomes after standard of care therapy pointing towards more favorable outcomes for patients with LFS and HMs than previously reported. We also report several unusual immune-mediated adverse events in our patients indicating possible immunogenicity of some TP53 variants. Most patients did not have prior exposure to cytotoxic treatments and were diagnosed with the HM prior to their LFS diagnosis. We also show that 38% of individuals with an HM have multiple family members with an HM, consistent with a familial HM pattern. This underscores the importance of detailed family and personal histories in HM patients found to carry a somatic TP53 variant.

Abstract 419: Evolutionary trajectories of complex genome rearrangements in osteosarcoma

Abstract Whole-genome sequencing (WGS) studies have revealed that osteosarcoma genomes are riddled by remarkably intricate forms of structural variants (SV), collectively known as complex genomic rearrangements (CGR). Yet, most CGR detected in osteosarcoma remain unexplained, hinting at the possibility of undiscovered mechanisms that might lead to novel therapeutic strategies. To elucidate the mechanisms underpinning cancer genome complexity in osteosarcomas and the downstream consequences of CGR during tumor evolution, we uniformly processed exiting osteosarcoma WGS data sets and performed high-depth (>120x) multi-regional short- and long-read WGS for hundreds of osteosarcomas. We found that whole genome doubling and diverse types of CGR are frequent clonal events in most osteosarcomas. Through the integration of multi-regional WGS data, we discovered that in tumors with whole-genome doubling, cancer genome evolution and oncogene amplification are the consequence of multi-generational CGR triggered by whole-genome doubling and involving multiple mechanisms, especially chromothripsis and breakage-fusion-bridge cycles. By contrast, in tumors without whole-genome doubling, biallelic inactivation of TP53 and oncogene amplification frequently occur as part of a cascade of genomic instability initiated by a single double-strand break upstream of the TP53 locus. This process results in clonal diversification and rapid tumor growth suggestive of punctuated evolution. In addition, we found that subclonal CGRs are also frequent events. Indeed, clonal expansions triggered by the acquisition of subclonal CGR often colonize distant tumor regions, suggesting that CGR act as subclonal driver events. Finally, we show that some recurrent genomic alterations are prognostic of poor survival, which we validate in independent cohorts. In sum, using WGS data, we have elucidated the mechanisms underpinning the complexity and evolution of osteosarcoma genomes. Our results indicate that diverse types of CGR, such as chromothripsis, are triggered by whole genome doubling and occur throughout tumor evolution, which primes the cancer genome for ongoing genomic instability, rapid karyotype evolution and clonal diversification. These results have implications for our understanding of the molecular basis of intra-tumor heterogeneity and drug resistance in osteosarcomas and other cancers driven by genomic instability. Citation Format: Isidro Cortes Ciriano, Jose Espejo Valle-Inclán, Solange de Noon, Katherine Trevers, Hillary Elrick, Adrienne M. Flanagan. Evolutionary trajectories of complex genome rearrangements in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 419.

Genomic analysis of primary epithelial neoplasms of the seminal vesicle identifies a subset of mucinous cystic tumours driven by KRAS mutations.

Carcinomas of the seminal vesicle are exceedingly rare, with a limited number of cases described in the literature. Reported cases span a relatively wide morphological spectrum, and their genomic features remain unexplored. In this study, we interrogated five primary epithelial neoplasms of the seminal vesicle using a targeted DNA sequencing platform (OncoPanel, 447 genes). The tumours included one adenocarcinoma with intestinal phenotype presenting after external beam radiation (for prostatic adenocarcinoma), one carcinoma with Müllerian-type clear cell phenotype, two mucinous tumours resembling low-grade mucinous neoplasms of the appendix (LAMN) and one mucinous cystadenoma. The post-radiation mucinous adenocarcinoma had genomic findings consistent with bi-allelic inactivation of TP53, as well as multiple copy-number changes with regional and chromosomal arm-level copy-number losses. The Müllerian-type clear cell carcinoma exhibited a complex copy-number profile with numerous regional and arm-level copy-number changes, as well as focal amplification events, including copy-number gain of 8q and amplification of a region within 20q13. Both low-grade mucinous tumours resembling LAMN harboured hot-spot gain-of-function KRAS variants (p.G12V and p.G13D) as the only genomic alteration. No genomic alterations were detected inthe lesion diagnosed as mucinous cystadenoma. Our results suggest that primary low-grade mucinous neoplasms of the seminal vesicle may represent a distinct entity equivalent to appendiceal counterparts, driven by gain-of-function variants of RAS GTPases. The remaining tumours showed genomic features that closely resembled those of neoplasms with comparablephenotypes and/or biological characteristics arising in other sites, suggesting that they could be managed similarly, with special considerations related to their anatomical location.

A p53 score derived from TP53 CRISPR/Cas9 HMCLs predicts survival and reveals a major role of BAX in the response to BH3 mimetics

AbstractTo establish a strict p53-dependent gene-expression profile, TP53−/− clones were derived from TP53+/+ and TP53−/mut t(4;14) human myeloma cell lines (HMCLs) using CRISPR/Cas9 technology. From the 17 dysregulated genes shared between the TP53−/− clones from TP53+/+ HMCLs, we established a functional p53 score, involving 13 genes specifically downregulated upon p53 silencing. This functional score segregated clones and myeloma cell lines as well as other cancer cell lines according to their TP53 status. The score efficiently identified samples from patients with myeloma with biallelic TP53 inactivation and was predictive of overall survival in Multiple Myeloma Research Foundation–coMMpass and CASSIOPEA cohorts. At the functional level, we showed that among the 13 genes, p53-regulated BAX expression correlated with and directly affected the MCL1 BH3 mimetic S63845 sensitivity of myeloma cells by decreasing MCL1-BAX complexes. However, resistance to S63845 was overcome by combining MCL1 and BCL2 BH3 mimetics, which displayed synergistic efficacy. The combination of BH3 mimetics was effective in 97% of patient samples with or without del17p. Nevertheless, single-cell RNA sequencing analysis showed that myeloma cells surviving the combination had lower p53 score, showing that myeloma cells with higher p53 score were more sensitive to BH3 mimetics. Taken together, we established a functional p53 score that identifies myeloma cells with biallelic TP53 invalidation, demonstrated that p53-regulated BAX is critical for optimal cell response to BH3 mimetics, and showed that MCL1 and BCL2 BH3 mimetics in combination may be of greater effectiveness for patients with biallelic TP53 invalidation, for whom there is still an unmet medical need.

Data-Driven Harmonization of 2022 Who and ICC Classifications of Myelodysplastic Syndromes/Neoplasms (MDS): A Study By the International Consortium for MDS (icMDS)

Background. The inclusion of gene mutations and chromosomal abnormalities in the 2022 WHO and ICC Classifications of MDS has enhanced diagnostic precision and is expected to improve clinical decision-making process. Although these two systems share similarities, clinically relevant discrepancies still exist and potentially cause inconsistency in their adoption in a clinical setting. In this study on behalf of the International Consortium for MDS (icMDS), we adopted a data-driven approach to provide a harmonization roadmap between the 2022 WHO and ICC classification for MDS. A modified Delphi Process consensus approach is currently ongoing among icMDS experts to finalize a harmonized MDS classification scheme. Methods. We analyzed retrospective international cohorts of patients with a diagnosis of MDS (n=7017) and AML (n=1002) according to WHO 2016 criteria. Hierarchical Dirichlet Processes were applied to define clusters capturing broad dependencies among all gene mutations and cytogenetic abnormalities. To investigate the features of importance and their impact on the clustering process, we employed the SHapley Additive exPlanations approach (SHAP). This allowed to define harmonized labels for each clinical entity. The clinical relevance of the unsupervised clustering was assessed through the analysis of phenotypic features and outcomes among each group. ( Blood 2022;140: 9828-9830) Results. Patients' characteristics are summarized in Table 1. We identified 9 clusters, defined by specific genomic features. The cluster of highest hierarchical importance was characterized by biallelic inactivation of TP53 (biTP53). According to SHAP analysis, bi TP53 was defined as 2 or more TP53 mutations, or 1 mutation with copy number loss or cnLOH. Most patients assigned to bi TP53 cluster had TP53 VAF>10% (77.9%) and complex karyotype (70.1%). Assignment to bi TP53 cluster was irrespective of blast count. Patients with monoallelic TP53 mutation segregated into other clusters. Hierarchically, the second cluster included patients with del(5q). SHAP analysis highlighted 5q deletion alone, or with one other chromosomal abnormality other than -7, and absence of bi TP53, as the most relevant features. Most of these patients had blast counts <5% (88.1%). The third distinct cluster included patients with SF3B1 mutations (in the absence of concurrent del(7q), abn3q26.2, complex karyotype or RUNX1 mutation). Most patients with MDS and SF3B1 mutation had <5% blasts (94.2%). Common co-mutated variants in the SF3B1 cluster included mutant DNMT3A (25.2%) and TET2 (38.3%). Morphologically defined MDS cases (i.e., not meeting criteria for bi TP53, del(5q) or SF3B1) were preferentially assigned to the following additional clusters: SF3B1 and concurrent higher-risk mutations (e.g., RUNX1 and ASXL1); SRSF2 and concomitant TET2 mutations; U2AF1 mutations with del(20q), del(7q) or -7; SRSF2 with TET2 mutations and co-mutational patterns including RUNX1 and ASXL1; and AML-like genomic signatures. Our analyses suggest that morphologically defined MDS is characterized by a large heterogeneity in terms of mutation profiles, not entirely captured by the presence of unilineage versus multilineage dysplasia, percentage of bone marrow blasts, and presence of hypocellularity and fibrosis. To better investigate the continuum between high risk MDS (i.e., patients with ≥10% blasts) and AML, an exploratory comparison was made using a cohort of AML (defined according to WHO 2016) patients analyzed using the same statistical methods. Only a partial overlap in genetic signatures was observed between MDS with ≥10% blasts and AML. However, similarities were observed between the AML-like MDS clusters (characterized by mutant NPM1, bZIP CEBPA, and Core Binding Factor abnormalities) and AML clusters defined by the same genetic signature, thus supporting the classification of these entities as AML, irrespective of blast count. Conclusion. Our study demonstrated the utility of a data-driven approach based on advanced statistical methods to generate a harmonized classification for MDS. Table 2 shows a provisional, hierarchical classification algorithm. Further refinement of entity labels and classification criteria is the subject of the ongoing modified Delphi Process consensus approach among icMDS experts.

TP53 Mutational Status in Myelodysplastic Neoplasm and Acute Myeloid Leukemia: Impact of Reclassification By Who 2022 and ICC Criteria - a Korean Multi-Center Study

Introduction: The WHO 2022 classification (WHO 2022) introduced the category of myelodysplastic neoplasm (MDS) with biallelic TP53 inactivation (MDS-bi TP53), while the international consensus classification (ICC) included the categories of MDS, MDS/acute myeloid leukemia (AML), and AML with mutated TP53. Particularly in MDS, multi-hit TP53 mutations are clinically important, but there were differences in the criteria defining multi-hit TP53 mutations between the WHO 2022 and ICC classifications. In this study, we investigated the TP53 mutational status in Korean adult patients with MDS and AML and evaluated the impact of TP53 mutation-related criteria of WHO 2022 and ICC on the reclassification of MDS. Methods: This study included patients aged 18 years or older who were diagnosed with MDS and AML from January 2017 to December 2022 at six institutions. The results of bone marrow examination, chromosome analysis, TP53 fluorescence in situ hybridization, and targeted next-generation sequencing including TP53 were analyzed in 1264 patients with MDS and 2112 patients with AML. Multi-hit TP53 mutation was defined based on WHO 2022 and ICC criteria as having any of the following: one TP53 mutation with variant allele frequency (VAF) ≥ 50%, one TP53 mutation with VAF ≥ 10% with 17p deletion, one TP53 mutation with VAF ≥ 10% with complex karyotype (only for ICC), two TP53 mutations with VAF ≥ 10% (only for ICC), or two TP53 mutations (only for WHO 2022). Results: TP53 mutations were observed in 9.3% (118/1264) and 9.1% (192/2112) of patients with MDS and AML, respectively. Among these, 90% of patients had a single TP53 mutation and multiple TP53 mutations were observed in 10% of patients. There was no difference according to MDS or AML ( P=0.478), and 91% of patients with a single TP53 mutation had a VAF of ≥ 10%. Median VAFs of TP53 mutations were 42.7% and 44.4% in MDS and AML, respectively ( P=0.899), and complex karyotype was observed in 76.1% (236/310) of all patients with TP53 mutations without differences between MDS and AML ( P=0.534). In contrast, 17p deletion was identified in 40.3% of AML patients, significantly higher than in MDS (27.4%, P=0.02). Within MDS, 17p deletion was observed in 46.7% in MDS with 10-19% blasts (MDS-H), significantly higher than in MDS with 0-9% blasts (MDS-L, 20.7%, P=0.008), but similar to AML ( P=0.514). When each TP53 mutation-related criterion based on WHO 2022 and ICC criteria were applied to MDS, 55.7% (49/88) and 75% (66/88) of MDS-L met the criteria for multi-hit TP53 mutation and were classified as MDS-bi TP53 (WHO 2022) and MDS with mutated TP53 (ICC), respectively. Notably, more patients were classified into this category based on ICC criteria ( P<0.001), all due to the complex karyotype-related criterion included only in the ICC. Among MDS-H, 63.3% (19/30) of patients were classified as MDS-bi TP53 (WHO 2022) based on multi-hit TP53 mutation criteria, whereas 86.7% (26/30) of patients were classified as MDS/AML with mutated TP53 (ICC) based on the single TP53 mutation criterion with VAF ≥ 10%. Overall, 57.6% (68/118) and 78% (92/118) of patients with MDS and TP53 mutations were reclassified into the TP53 mutation-related MDS or MDS/AML subgroup according to WHO 2022 and ICC, respectively. Conclusions: TP53 mutation status was not different between MDS and AML, but 17p deletion was observed at approximately 40% in MDS-H and AML, much higher than in MDS-L. Multi-hit TP53 mutation based on WHO 2022 and ICC was present at a relatively high frequency at 60-80% in MDS and AML with TP53 mutations. In particular, when ICC criteria were applied to MDS, 20% more patients were reclassified into TP53 mutation-related MDS or MDS/AML subgroup than when WHO 2022 criteria were applied.

The Frequency of Clonal Hematopoiesis Prior to AML and MDS Varies Among the Different Molecularly Defined Who Subtypes

Background: AML and MDS cells accumulate mutations over years or even decades. In the current WHO 2022 classification, some mutations are listed as “defining genetic abnormalities” (WHO DGA). A long phase of clonal hematopoiesis may precede some, but not all, AML or MDS. There are multiple scenarios in which order clonal hematopoiesis, WHO DGAs, and other mutations can occur. We analyzed whole-genome sequencing data from 583 patients with AML or MDS subtypes defined by a mutation and elucidated their role in the hierarchical sequence of mutations. Aim: (1) We compared patients with AML or MDS arising from clonal hematopoiesis to those with a founder mutation in a WHO DGA gene (e.g. NPM1). (2) We analyzed which other mutations may occur before WHO DGAs. Methods and patients: We analyzed 583 patients with AML or MDS who were classified into a WHO 2022 DGA class: MDS with low blasts and SF3B1 mutation (MDS- SF3B1, n=160), MDS with biallelic TP53 inactivation (MDS-bi TP53, n=40), AML, myelodysplasia-related (AML-MR with focus on mutations, n=151), AML with NPM1 (n=171), and AML with CEBPA (n=61). Amplification-free WGS was performed (median coverage > 100x). Genes with known relevance to myeloid malignancies were analyzed (n=47). We assumed that the mutation with the highest variant allele frequency (VAF) evolved first in the malignant process (+/- 5%). VAF was corrected for copy number changes (including the presence of one X chromosome in males) and copy-neutral loss of heterozygosity (CN-LOH). The following variant callers were used: Strelka2 (mutations and VAF), Pindel (for FLT3-ITD), HadoopCNV (CN-LOH) and GATK4 (CNV). Results: We classified patients into three groups (Figure 1a): (A) 322/583 (55%) of malignancies originate from a cell with a WHO DGA; (B) 174/583 (30%) of malignancies originate from a clonal hematopoietic cell with a DNTM3A or TET2 mutation (DT origin); (C) the remaining 87/583 (15%) of AML and MDS originate from a cell with a different initial mutation (“other”). The frequency of patients with the highest VAF in a WHO DGA varied between subtypes: MDS-bi TP53: 88%, MDS- SF3B1: 72%, AML- CEPBA: 64%, AML-MR: 57% and AML- NPM1: 27%. The highest number of patients with a disease putatively arising from DNMT3A or TET2 mutated clonal hematopoiesis was found in AML- NPM1 (49%) and the lowest in MDS-bi TP53 (5%) (for details, see Figure 1b). The group of patients with the highest VAF in neither WHO DGA nor DNMT3A/ TET2 (“other”) accounted for 17-23% of AML and only 4% and 8% of MDS- SF3B1 andMDS-bi TP53 respectively. IDH1 or IDH2 had the highest VAF in 17/583 patients (3%), especially AML- NPM1 (n=8) and AML-MR (n=7). Mutations in the spliceosome genes had the highest VAF in 12/583 patients (2%), with the combination of SRSF2 in AML- NPM1 in particular (n=6). In the following, we compared DT origin and WHO DGA origin. The differences did not affect all AML and MDS subtypes to the same extent. Patients with AML- CEBPA and DT origin were a median of 19 years older than patients with an initial CEBPA mutation (75 vs. 56 years). In AML- NPM1, the difference in median age was 11 years (71 vs. 60 years). In both groups, patients with DT origin are also predominantly male (73% vs. 44% for AML- CEBPA and 54% vs. 45% for AML- NPM1). Age and sex differences were not observed in AML-MR, MDS- SF3B1 and MDS-bi TP53 with or without DT origin. Overall survival (OS) was shorter in AML patients with DT origin compared to WHO DGA (0.7 vs. 1.8 years; p=0.073), but not in MDS (7.4 vs. 6.1 years; p=0.669). However, in a multivariate Cox analysis, including DT origin and age, age remained the only significant factor for OS. Conclusion: Using VAF to model the hierarchy of mutation evolution shows that for the majority of patients, WHO-defining mutations are early events, supporting their role as defining genetic abnormalities and (potential) drivers. However, other initial mutations occur in DNMT3A and TET2, indicating AML or MDS arising from clonal hematopoiesis,but also IDH1/ IDH2 or spliceosome genes. How these diseases will be classified in the future should be actively discussed, and aspects such as targeted therapies (with IDH inhibitors) or a second hematologic neoplasm after clonal hematopoiesis should be considered.

Proposal ofmyeloid Neoplasms with MYC-Positive Double Minutes As a Distinct Entity

Background: As reported in our preliminary work (Blood (2022) 140 (Supplement 1): 9142-9143) myeloid neoplasms (MN) with MYC-positive double minutes (dmin) are mostly AML and often show a cytomorphological proximity to APL. We here now present detailed genotypical and phenotypical characteristics to address the question if MN with MYC dmin might represent a distinct hematologic entity. Aim: In-depth characterization of cytomorphological, mutational, transcriptional and clinical features of 76 MN with MYC dmin and analysis of the amplified chromosomal region and its effect on gene expression (GE). Methods: We analyzed 60 bone marrow (BM) and 16 peripheral blood (PB) samples of 76 pts with MN and MYC dmin (36 female, 40 male; median age 75 yrs, range 44-89 yrs). The diagnosis was established following WHO guidelines. Dmin and MYC amplification were assessed by chromosome banding analysis and FISH. Cytomorphologic examination included assessment of APL-like features, i.e. high number of atypical hypergranulated promyelocytes, high number of Auer rods, faggot(-like) cells and pseudo Chediak-Higashi (PCH) granules. Molecular genetic analyses comprised a targeted NGS panel (all pts, median coverage 1500x), WGS (16 pts, median coverage 100x) and WTS (40 pts, 50 Mio reads). Mut frequencies and GE levels were compared to AML cases without MYC dmin (“non dmin” AML). Survival data were available for 41 pts (median follow-up: 12 months). Results: According to WHO 2022 most of the 76 cases were AML-MR (myelodysplasia-related) (55/76, 72 %, 9 of these post MDS or post MDS/MPN). Three (4 %) cases each were classified as AML with maturation or without maturation, while one case with AML was not further classifiable due to insufficient material quality and lack of defining markers. Other diagnoses comprised MDS with biallelic TP53 inactivation (4/76, 5 %), MDS-IB1 (1/76, 1 %), CMML-2 (4/76, 5 %), CMML-1 (1/76, 1 %), MDS/MPN (3/76, 4 %) and MPN in blast phase (1/76, 1 %). BM blast count was ≥10 % in 52/60 (87 %) pts, likely underestimated in the remaining 8 samples due to lack of particles, and all 16 pts where only PB was available showed >2 % blasts. Out of 50 pts with fully assessable cytomorphology 48 (96 %) presented with a highly dysplastic granulopoiesis, often including severe dysplasia in other myeloid lineages, independent of myelodysplasia-related genetic markers. Twenty-nine of 50 (58 %) pts were APL-like (≥2 APL-like features), but with a higher degree of maturation than APL. A complex karyotype (ck, ≥3 aberrations in addition to MYC dmin) was present in 25/76 pts (33 %), whereas 21/76 (28 %) pts presented with MYC dmin as the only cytogenetic aberration. TET2 mut, which represented the most frequent mut in MYC dmin pts (55/76, 72 %), often were biallelic events (36/55 pts, 65 %). TET2 mut were strikingly overrepresentated compared to “non dmin” AML (19 %) (p<0.001) (Figure A) and often accompanied by trisomy 4, representing the most frequent chromosomal gain (12/76, 16 %). TP53 mut (24/75, 32 %) and U2AF1 mut (20/75, 27 %) were almost always mutually exclusive (except for 2 pts). While TP53 mut were strongly associated with ck (84 % vs. 6 % in non ck, p<0.001), U2AF1 mut were associated with APL-like cytomorphology (48 % vs. 5 % (1 case) in non APL-like cases, p=0.001). Both mut were highly overrepresented in MYC dmin pts compared to “non dmin” AML ( TP53: 32 % vs. 11 %, p<0.001; U2AF1: 27 % vs. 4 %, p<0.001) (Figure A). WGS analysis revealed an amplified chromosomal region with a size varying from 4.3 - 5.6 Mb and a commonly amplified region of 4.3 Mb (Chr 8:126,422,001-130,697,000). This region encompasses 6 protein coding genes ASAP1, CYRIB, GSMDC, LRATD2, POU5F1B and MYC as well as several non-coding RNAs including the long non-coding RNA PVT1. An effect of the amplified region on GE was confirmed by overexpression of MYC (p<0.001) and PVT1 (p<0.001) compared to “non dmin” AML (Figure B). The median overall survival of 41 evaluable pts was 16 months. Only TP53 mut was independently associated with inferior survival (HR: 11.0, p=0.001). Conclusion: MN with MYC dmin are often AML-MR according to WHO 2022 definition, but even if not, show consistent features of severe dysplasia. They exhibit characteristic mutational patterns and distinct GE profiles, which are affected by a commonly amplified chromosomal region. Thus, we suggest MN with MYC dmin as a distinct genetically defined entity.

TP53 and CDKN2A alterations Define a Poor Prognostic Subgroup in Patients with Nodal T-Follicular Helper Cell Lymphoma

Nodal T-follicular helper cell lymphoma (nTFHL) is a subset of peripheral T-cell lymphoma (PTCL) characterized by a distinct immunophenotype (such as CD10, BCL6, CXCL13, PD1, and ICOS) and mutations (including TET2, RHOA G17V, and IDH2 R172). However, the impact of molecular classification based on genetic profiling on prognosis in nTFHL remains largely unknown. Here we performed targeted-capture sequencing (targeted-seq) covering 235 T/NK-cell lymphoma-related genes with a mean depth of 526×. Then, we analyzed 49 genes which were reported as drivers in the previous PTCL studies to reveal the associations between clinical phenotypes and somatic alterations in a cohort of 153 patients whose clinical information was available and who were pathologically diagnosed with nTFHL according to the WHO-HAEM5. When mutation and copy number alteration (CNA) data were combined, we identified recurrent alterations in 40 among 49 driver genes. TET2 was most frequent (68%), followed by RHOA (51%), IDH2 (27%), DNMT3A (20%), PLCG1 (9%), TP53 (7%), KMT2C (6%), VAV1 (6%), CD28 (6%), KMT2D (6%), NOTCH1 (5%), and CDKN2A (5%). To create a molecular classification based on genetic alterations in nTFHL, we conducted hierarchical clustering using driver alterations and arm-level CNAs. This analysis identified four molecular subgroups named after their characteristic genetic alterations ( Fig. 1A). TR-ID (+) ( n = 52) and TR-ID (-) ( n = 60) were defined by TFH-related mutations ( TET2 and RHOA). Among them, TR-ID (+) harbored IDH2 and/or DNMT3A mutations. A53 ( n = 8) was notable for biallelic inactivation of TP53 and/or CDKN2A and associated with marked aneuploidy. NSD ( n = 33) had no subgroup-defining alterations with the fewest number of mutations. Clinically, TR-ID (+) tumors were mostly nTFHL-angioimmunoblastic-type (AI), which is consistent with the enrichment of IDH2 and/or DNMT3A mutations. On the other hand, TR-ID (-) tumors exhibited a higher proportion of nTFHL-NOS. NSD exhibited lower LDH levels and was significantly associated with younger age and localized stage than other subgroups. A53 showed the worst prognosis with a median overall survival (OS) of 15.1 months (mos) ( Fig. 1B). By contrast, the OS of NSD was significantly superior to the others (median OS not reached). The OS of TR-ID (+) and TR-ID (-) was comparable with a median OS of 40.6 and 46.4 mos, respectively. Notably, A53 remained significant in multivariate analyses adjusted for IPI or PIT score. Thus, genetic profiling enables to classify nTFHL patients into genetically and clinically distinct subgroups, including the unfavorable prognostic subgroup (A53). Next, to identify potential genes linked to the poor prognosis of the A53 subtype, we examined the prognostic impact of individual alterations. Among the genes examined, TP53 and CDKN2A, hallmarks of A53, were significantly associated with shorter OS in univariate analysis. Consequently, cases with TP53 and/or CDKN2A alterations showed a poor prognosis (median OS 11.4 vs 181.2 mos, P = 8.2×10 -7), thereby reinforcing the negative prognostic impact of A53. Then, we investigated other prognostic factors within each molecular subgroup. First, in TR-ID (+) and (-) cases, we examined the EBV status, which was known to be associated with worse prognosis in nTFHL-AI. Among them, 22 (16%) cases were EBV-positive. Despite no observed differences in genetic and clinical factors, EBV-positive cases exhibited a worse prognosis (median OS 66.5 vs 14.9 mos, P = 0.0018). Next, we assessed the influence of TCR clonality on prognosis in NSD cases. The clonal T-cell fraction assessed by targeted-seq was highest in A53 (median, 75%) but lowest in NSD (median, 16%). OS of NSD cases was further stratified by T-cell clonality (median OS not reached vs 67.7 mos for low and high clonal T-cell fraction; P = 0.042), while the presence of driver alterations failed to predict the prognosis. These findings suggest that assessment of T-cell clonality helps to identify the favorable prognostic subgroup characterized by low tumor content. In summary, we defined four molecular subgroups with different clinical features and also clarified subgroup-specific prognostic factors in nTFHL. Particularly, we identified the molecular subgroup with unfavorable prognosis (A53) for which different clinical approach should be warranted. This classification will refine the patient prognostication and stratification in nTFHL.

Predicted Functional Consequences of TP53 Alterations and Prognosis in TP53-Mutated Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) cases with TP53 mutations ( TP53mut) are associated with poor responses to chemoimmunotherapy (Eskelund Blood 2017). Across myeloid and solid tumors, predicted functional consequences and pathogenicity of specific TP53 mutations have been linked to differential patient outcomes (Pollock Breast Cancer Res Treat 2022, Yabe Cancers 2021). We aimed to characterize the impact of these features and associated molecular alterations on treatment outcome in TP53mut MCL patients treated with standard frontline regimens and on the ongoing phase 2 BOVEN trial. The BOVEN trial aims to assess the efficacy of frontline treatment with zanubrutinib, obinutuzumab, and venetoclax in TP53mut MCL (Kumar Blood 2021). Methods: The BOVEN trial enrolled 25 patients with TP53mut MCL. Retrospective chart review was performed for 22 patients with TP53mut MCL treated at frontline with standard chemoimmunotherapy regimens (Joffe Blood 2019). Tumor histology and FISH were obtained for each case. Mutational profiling (SNV/indels, CNA) was obtained prior to therapy with various CLIA-certified targeted sequencing platforms (Cheng J Mol Diagn 2015, Dias-Santagata EMBO Mol Med 2010). Mutations in TP53 were annotated using predicted phenotypes and pathogenicity from the TP53 Database (de Andrade Cell Death Differ 2022) and from phenotypic annotation of TP53 mutations (Giacomelli Nat Genet 2018). The primary outcome measures were overall and progression-free survival (OS/PFS). Results: Each patient included in the BOVEN cohort featured variants in TP53 with 26 SNV/indels across all 25 patients. There were 25 SNV/indels across all 22 patients treated with standard regimens. Within the trial cohort 14/25 cases had evidence of biallelic inactivation of TP53 with co-occurring SNV/indels in the gene and 17p loss by FISH or TP53 copy losses by SNParray; the comparator cohort featured 11/22 cases with biallelic losses. Across cohorts, most mutations were missense (>80% in both), with the majority occurring in the DNA binding domain of the p53 protein (76% and 91%, respectively), and with >30% at known hotspot codons in both cohorts. Most mutations (>70% in both cohorts) were predicted to adversely impact WT p53 function, including nonfunctional transactivation activity and dominant negative effect on the WT allele. The majority were also predicted to be highly pathogenic by bioinformatic methods (AGVGD, SIFT, PolyPhen2, PHANTM, REVEL, BayesDel). However, none of these features correlated with outcome. In the BOVEN cohort the variant allele frequency (VAF) of the TP53 SNV/indels approached significant association with poor PFS (HR 33.2, p=0.05) but not OS (HR 28.6, p=0.09). In contrast, in the comparator cohort TP53 VAF appeared to have an opposite effect direction, though this was not significant for PFS (HR 0.98, p=0.2) or OS (HR 0.97, p=0.06). We next investigated the impact of co-occurring molecular alterations on outcome. Besides TP53, the most frequently mutated genes in the trial cohort were KMT2D (24%), ATM (16%), NOTCH2 (12%), and CCND1 (12%). Alterations in these genes did not correlate with outcome. Within the comparator cohort the recurrently mutated genes were SMARCA4 (23%), ARID1A (18%), ARID1B (14%), ATM (14%), and CCND1 (14%). As described previously in this cohort (Joffe Blood 2019), alterations in SMARCA4 were associated with worse PFS (HR 4.2, p=0.02) but not OS (HR 2.2, p=0.27). Mutational burden (SNV and CNA load) was not prognostic in either 12/25 BOVEN samples sequenced with the same panel or the comparator cohort. There was no significant difference in outcome between patients with single mutations in TP53 vs. those with biallelic losses in TP53 in either cohort. Conclusion: Across patients with TP53mut MCL treated either with standard frontline chemoimmunotherapy or on the BOVEN trial, predictions of intrinsic pathogenicity and functional consequences of the TP53 alterations were not prognostic. The impact of TP53 mutation VAF remains unclear, with seemingly opposite effect directions between the two cohorts, though neither finding was significant. As previously described in the comparator cohort, alterations in SMARCA4 are associated with worse PFS in TP53mut MCL, though this was not observed in the BOVEN cohort. There were no significant differences in outcome among patients with biallelic inactivations in TP53 or with higher burdens of somatic alterations.

Perspectives on Myelodysplastic Neoplasm Diagnosis: A Comparative Analysis of the Latest World Health Organization Classifications and the International Concensus Classification

Introduction : Myelodysplastic neoplasm (MDS) is a category of clonal hematopoietic stem cell neoplasms, defined by cytopenias and morphologic dysplasia, characterized by progressively ineffective hematopoiesis and increased risk of acute myeloid leukemia (AML). The 5th edition of the World Health Organization (WHO 2022) classification grouped MDS entities into two families: those having defining genetic abnormalities and those that are morphologically defined. The former newly included biallelic TP53 inactivation (MDS-biTP53) and SF3B1 mutation, meanwhile the latter newly included hypoplastic MDS (h-MDS) and MDS with fibrosis (MDS-F). An independent proposal, the International Consensus Classification (ICC), was also published during the same period. This study aims to compare and analyze these two classifications, focusing on MDS's diagnostic criteria and entity definition. Herein, we describe the redistribution of the MDS according to the WHO 2022 classification and ICC and evaluated their significance in predicting patient's outcomes. Methods : This study included a total of 639 patients who were diagnosed with MDS based on the 4th edition of WHO classification in Seoul St. Mary's hospital from 2016 to 2022. As part of the study, all patients were reclassified using the updated WHO 2022 classification and ICC through a comprehensive review of bone marrow aspiration and biopsy, as well as the results of whole genetic studies, including cytogenetic analysis, fluorescence in situ hybridization, and targeted panel next-generation sequencing. Risk stratification was determined by using the International Prognostic Scoring System for MDS (IPSS-R, WPSS and IPSS-M). Results : Among the 639 patients, six were classified as ‘clonal cytopenia of undetermined significance’ by WHO 2022 and ICC. In the WHO 2022 classification, 22 patients were categorized as AML, whereas ICC only identified nine of them due to low blast count (<10%). Out of the newly defined MDS patients (n=611), 12.6% (n=77) were genetically defined MDS, and 87.4% (n=534) were morphologically defined MDS. Regarding the WHO 2022 categories, MDS-biTP53 and MDS-SF3B1 accounted for 5.4% (n=33) and 6.3% (n=39) of cases, respectively, whereas h-MDS and MDS-F accounted for 11.9% (n=73) and 1.8% (n=11), respectively. As for the ICC, MDS, not otherwise specified (NOS) accounted for 327 patients with 77 having single lineage dysplasia and 250 having multilineage dysplasia. The h-MDS was also included in these criteria. Of 33 MDS-biTP53 cased identified by the WHO 2022 classification, 25 were categorized as MDS-TP53 and 8 as MDS/AML-TP53 in the ICC, which included an additional four patients. Moreover, 72 patients were classified as MDS/AML by ICC, with 35 of them presenting genetic mutations and 9 exhibiting cytogenetic abnormalities. The median survival of the 611 patients were 86 months (95% confidence interval (CI): 65-111). The WHO 2022 classification demonstrated significant prognostic discrimination based on diagnosis subtype (P <0.001). MDS-biTP53 exhibited the worst outcome [hazard ratio (HR): 7.24, 95% CI: 2.87 to 18.22], followed by MDS-F (HR: 5.64, 95% CI: 1.44 to 22.08) and MDS-IB2 (HR: 2.76, 95% CI: 1.72 to 4.42), in comparison to MDS-LB. On the other hand, the ICC did not reveal significant prognostic significance. The distribution of IPSS-R, WPSS and IPSS-M was similar and also showed meaningful prognostic discrimination (P <0.001). Conclusions : The results validated the clinical relevance of updated WHO 2022 classification for MDS and indicated that the newly defined categories such as MDS-biTP53 and MDS-F are unique disease types evidenced by their particular prognostic significance.

Clinico-Genetic and Prognostic Analyses of 635 Patients with Myelodysplastic Neoplasms Based on the 2022 World Health Organization Classification

Myelodysplastic neoplasms (MDS) are a heterogeneous group of clonal myeloid neoplasms characterized by dysregulated hematopoiesis. In the past several years, major advances in molecular technologies and the development of next generation sequencing have deepened our understanding of MDS pathobiology. Revisions of the existing classification were warranted. Under the aegis of the International Agency for Research on Cancer, the 2022 World Health Organization (WHO) classification was released. The 2022 WHO proposed new categories including MDS with biallelic TP53 inactivation (MDS-bi TP53), MDS, hypoplastic (MDS-h) and MDS with fibrosis (MDS-f). In addition, the novel risk scoring system-Molecular International Prognostic Scoring System (IPSS-M) has been established. There are limited data that explore the application of the 2022 WHO classification of MDS in light of the IPSS-M. In this study, a cohort of 635 patients diagnosed as having primary MDS based on the 2022 WHO criteria was retrospectively reviewed. We aimed to elucidate the differences in genetic features and clinical outcomes among patients with different MDS subtypes, based on the 2022 WHO classification. The survival impact of allogeneic hematopoietic stem cell transplantation (allo-HSCT) was also evaluated. TruSight myeloid sequencing panel and the HiSeq platform were used to analyze the alterations of 54 myeloid neoplasm-relevant genes. Comparison of the genetic profiles of patients with different subtypes of MDS showed that patients with MDS-low blasts (LB) and those with MDS with LB and ring sideroblasts (RS) exhibited comparable mutation profiles, while patients with MDS- SF3B1 had different mutation pattern from that of these two subtypes. Among patients with MDS-increased blasts-1 (IB1), MDS-IB2 and MDS-f, patients with MDS-IB2 had higher frequencies of NRAS (9% vs. 2%, P=0.019) and mono-allelic TP53 (7% vs. 1%, P=0.031) mutations, and a trend of more TET2 (19% vs. 10%, P=0.059) mutations than those with MDS-IB1. In addition, patients with MDS-f exhibited lower incidence of TET2 (0% vs. 19%, P=0.020), and STAG2 mutations (8% vs. 27%, P=0.050) as compared to those with MDS-IB. Furthermore, patients with MDS-bi TP53 had specific concurrent genetic alterations with few co-occurring mutations which differed from those found in patients with MDS-IB or MDS-f. We performed pairwise survival comparisons among each subtypes of MDS (Figure 1). In low-risk MDS, patients with MDS-h had significantly longer median leukemia-free survival (LFS) and overall survival (OS) (185.5 months for median LFS and OS) compared with those with MDS-LB and RS [38.3 months for median LFS ( P=0.013) and OS ( P=0.010)], while had a similar outcome compared to those with MDS-LB [170.2 months for median LFS ( P=0.152) and OS ( P=0.146)], and MDS- SF3B1 [114.6 months for median LFS ( P=0.549) and OS ( P=0.528)]. For patients with increased blasts or bi TP53 mutations, those with bi TP53 inactivation had the worst outcomes (median LFS: 3.9 months and OS: 4.5 months, all P<0.001) compared to those with MDS-f (median LFS: 16.6 months and OS: 17.7 months), MDS-IB2 (median LFS: 10.0 months and OS: 17.7 months), and MDS-IB1 (median LFS: 25.6 months and OS: 31.4 months). Patients with MDS-f had similar survival to those with MDS-IB2 and IB-1 in terms of LFS (16.6 vs. 25.6 months, P=0.271) and OS (17.7 vs. 31.4 months, P=0.194) . Multivariable analysis showed that IPSS-M ( P<0.001), old age (HR for LFS: 1.023; for OS: 1.032, P<0.001), and the 2022 WHO categorization ( P<0.001) could predict LFS and OS independently and that HSCT could improve LFS (HR, 0.492, P=0.001) (Table 1). Subgroup analysis of the impact of transplantation using time-dependent Cox regression revealed that allo-HSCT could prolong both LFS and OS in patients with MDS-IB1 (HR for LFS: 0.293, for OS: 0.350, P<0.001), LFS alone (HR: 0.534, P=0.049) in patients with MDS-IB2, and a trend of LFS (HR: 0.305, P=0.078) in patients with MDS-f. However, allo-HSCT failed to remedy the dismal outcomes for patients with MDS-bi TP53. In conclusion, based on the evidence gathered from this cohort analysis, the 2022 WHO classification promote efficient segregation of this heterogeneous disease, with focus on the differences in molecular features and prognoses across different disease subtypes, leading to accurate MDS diagnosis and effective risk-adapted treatment.

Combination of genomic instability score and TP53 status for prognosis prediction in lung adenocarcinoma

The genomic instability (GI) /homologous recombination deficiency (HRD) score, calculated as the sum of the events of loss of heterozygosity (LOH), large-scale state transition (LST) and telomere allele imbalance (TAI), is used to guide the choice of treatment in several cancers, but its relationship with genomic features, clinicopathological characteristics and prognosis in lung cancer is poorly understood, which could lead to population bias in prospective studies. We retrospectively analyzed 1011 lung cancer patients whose tumor samples were successfully profiled by high-throughput sequencing panel including GI/HRD score. Alterations of many cancer suppressor genes were associated with higher GI/HRD scores, biallelic inactivation of TP53 was correlated with a high GI/HRD score. A combination of two gene alterations exhibited a higher GI/HRD scores than single gene alterations. The GI/HRD score was associated with advanced stages in lung adenocarcinoma but not in lung squamous cell carcinoma. Furthermore, patients with higher GI/HRD scores had significantly shorter overall survival and progression-free survival than patients with lower GI/HRD scores. Finally, patients with a combination of a higher GI/HRD scores and TP53 alteration exhibited an extremely poor prognosis compared with patients with a lower GI/HRD scores and wild-type TP53 (overall survival, training cohort, hazard ratio (HR) = 8.56, P < 0.001; validation cohort, HR = 6.47, P < 0.001; progression-free survival, HR = 4.76, P < 0.001). Our study revealed the prognostic value of the GI/HRD score in lung adenocarcinoma, but not for all lung cancer. Moreover, the combination of the GI/HRD score and TP53 status could be a promising strategy to predict the prognosis of patients with lung adenocarcinoma.

Identifying novel mechanisms of biallelic TP53 loss refines poor outcome for patients with multiple myeloma

Biallelic TP53 inactivation is the most important high-risk factor associated with poor survival in multiple myeloma. Classical biallelic TP53 inactivation has been defined as simultaneous mutation and copy number loss in most studies; however, numerous studies have demonstrated that other factors could lead to the inactivation of TP53. Here, we hypothesized that novel biallelic TP53 inactivated samples existed in the multiple myeloma population. A random forest regression model that exploited an expression signature of 16 differentially expressed genes between classical biallelic TP53 and TP53 wild-type samples was subsequently established and used to identify novel biallelic TP53 samples from monoallelic TP53 groups. The model reflected high accuracy and robust performance in newly diagnosed relapsed and refractory populations. Patient survival of classical and novel biallelic TP53 samples was consistently much worse than those with mono-allelic or wild-type TP53 status. We also demonstrated that some predicted biallelic TP53 samples simultaneously had copy number loss and aberrant splicing, resulting in overexpression of high-risk transcript variants, leading to biallelic inactivation. We discovered that splice site mutation and overexpression of the splicing factor MED18 were reasons for aberrant splicing. Taken together, our study unveiled the complex transcriptome of TP53, some of which might benefit future studies targeting abnormal TP53.

Genomic landscape of multiple myeloma with extramedullary disease: Results from a large patient database.

8058 Background: Extramedullary disease (EMD) is associated with poor outcomes and the biological mechanisms driving this phenotype are poorly understood. We used the CoMMpass registry data to explore the genomic characteristics of EMD. Methods: The CoMMpass study is a prospective, international registry of newly diagnosed MM. 1143 patients had clinical data. Patients were classified as EMD if they had EMD at any point. Genomic data from bone marrow (BM) samples at diagnosis (whole genome sequencing, whole exome sequencing (WES), and RNA sequencing (RNAseq)) were compared between EMD and non-EMD. A multivariable model was constructed based on univariable results. Differential expression (DEA) and gene set enrichment analysis (GSEA) were performed after normalization using Hallmark and MM transcriptomic subgroups. The top 24 most mutated genes in MM were selected. Results: The median overall survival (mOS) and progression free survival (PFS) was shorter for EMD. The presence of EMD, anemia at diagnosis, age &gt;65 years at diagnosis, ISS stage, and use of ASCT at the first line were independently associated with worse mOS and PFS. EMD was associated with worse mOS (HR 1.7, 95% CI 1.3 - 2.2 p &lt;0.01) and PFS (HR 1.5, 95% CI 1.23 - 1.8, p &lt;0.01) in multivariable analysis and was not abrogated by ASCT, triplet induction or maintenance. Results did not vary when landmark analysis was used. MYC amplification, deletion 18q and 1q amplification were enriched in EMD cases. TP53 mutations and TP53 bi-allelic inactivation (mutation and deletion) were enriched in EMD (Table). DEA identified 1552 genes with increased and 3344 with decreased expression (2.7%, 5.4% of total genes respectively) in EMD when compared to non-EMD. On GSEA, EMD cases were enriched for the CD-1, PR, and CTA myeloma gene expression signatures, in addition to the Hallmark pathways MYC target, E2F targets, G2M checkpoint and unfolded protein response, while the adhesion molecules pathway and MM gene signatures LB, MS, and MF were downregulated in EMD. Conclusions: EMD cases were enriched for TP53 mutations and bi-allelic TP53 inactivation, both of which are prognostic. In addition, MYC amplification, 18q deletion and 1q gain were enriched in EMD which are novel CNA associated with EMD. EMD is also enriched for distinct molecular subgroup, including enrichment for CD-1, PR, and CTA signatures, the latter two are associated with poor outcomes, and the Hallmark pathways MYC, E2F and G2M checkpoints consistent with MYC amplification and PR molecular subgroup. WES and RNAseq of matching BM and EMD tissue samples from our institution is currently being performed. [Table: see text]

Deterministic evolution and stringent selection during preneoplasia

The earliest events during human tumour initiation, although poorly characterized, may hold clues to malignancy detection and prevention1. Here we model occult preneoplasia by biallelic inactivation of TP53, a common early event in gastric cancer, in human gastric organoids. Causal relationships between this initiating genetic lesion and resulting phenotypes were established using experimental evolution in multiple clonally derived cultures over 2 years. TP53 loss elicited progressive aneuploidy, including copy number alterations and structural variants prevalent in gastric cancers, with evident preferred orders. Longitudinal single-cell sequencing of TP53-deficient gastric organoids similarly indicates progression towards malignant transcriptional programmes. Moreover, high-throughput lineage tracing with expressed cellular barcodes demonstrates reproducible dynamics whereby initially rare subclones with shared transcriptional programmes repeatedly attain clonal dominance. This powerful platform for experimental evolution exposes stringent selection, clonal interference and a marked degree of phenotypic convergence in premalignant epithelial organoids. These data imply predictability in the earliest stages of tumorigenesis and show evolutionary constraints and barriers to malignant transformation, with implications for earlier detection and interception of aggressive, genome-instable tumours.

P52 RISK STRATIFICATION COMBINING SKY92 GENE EXPRESSION PROFILING AND TRADITIONAL FISH IN MULTIPLE MYELOMA: THE FIRST PROSPECTIVE EVIDENCE IN THE R2-ISS ERA

High-risk (HR) multiple myeloma (MM) patients still have a very poor prognosis. However, the definition of HR MM remains controversial. Currently, FISH is the most commonly used method for risk stratification in MM. According to the Second Revised International Staging System (R2-ISS), HR cytogenetics is defined as presence of at least one of the following: del(17p), t(4;14), and 1q CNA. In recent years, gene expression profiling (GEP) approaches, e.g. SKY92, have been developed for detection of HR patients. In the R2-ISS era, however, data on MM risk stratification applying SKY92 in combination with traditional FISH is still lacking. Therefore, we performed the first prospective study evaluating the prognostic value of the combined SKY92 and FISH HR detection in newly diagnosed (ND) and relapsed/refractory (RR) MM. We prospectively collected bone marrow (BM) samples and clinical data of 147 MM patients. Cytogenetics were analyzed on purified CD138 positive MM cells by FISH, and HR cytogenetics was defined according to the R2-ISS classification. SKY92 risk status was determined with MMprofiler gene expression assay. Whole genome sequencing (WGS) was performed to compare the both risk stratification systems SKY92 and FISH. We included 147 patients in our study (NDMM: n=51, RRMM: n=96). SKY92 classification was available for 121 (82.3%) patients. HR SKY92 was significantly enriched in RRMM (40/76) compared with NDMM (6/45) (P<0.0001). RRMM patients with HR SKY92 showed significantly shorter progression free survival (PFS) (P<0.0001) and overall survival (OS) (P=0.0004) than standard-risk (SR). In NDMM, HR SKY92 also indicated a significantly inferior PFS (P=0.001) in comparison with SR. Of note, samples of 26 (17.7%) patients, who showed significantly lower bone marrow infiltration than the remaining patients (median: 25% vs 55%, P=0.038), did not meet the SKY92 quality control criteria. We then combined the SKY92 classification with cytogenetic analyses by FISH, which were available in 99 patients (NDMM: n=33; RRMM: n=66). We noticed a discrepancy between both risk stratification systems, with 44 (44.4%) and 58 (58.6%) patients being classified as HR by SKY92 and FISH, respectively. In total, 28 (42.4%) RRMM patients and 6 (18.2%) NDMM patients displayed HR in both SKY92 and FISH (double-HR). Regarding survival outcome in RRMM, double-HR patients showed the worst PFS (P<0.0001) and OS (P=0.0007). In NDMM, double-HR presented a negative prognostic factor for PFS (P=0.01). To compare the FISH and SKY92 classifications, we performed WGS in 16 patients who exhibited either only HR SKY92 (n=7) or only HR FISH (n=9). Interestingly, 1 patient with bi-allelic TP53 inactivation (deletion + mutation) and 6 patients harbouring 1q CNA were determined as SR by SKY92 but as HR by FISH. The median PFS was not reached after a median follow up of 371 days in these 9 patients. Moreover, 4 out of 7 patients with only HR SKY92 but SR FISH displayed 1q CNA, which was detected only by WGS, and del1p32 was found in 1 patients. Interestingly, we found CRBN mutation in 3 out of 7 patients with only HR SKY92 but SR FISH. The remaining 2 patients did not show any known HR genomic alterations, suggesting that HR MM may be associated with other factors, e.g. epigenetic modifications. Here, we provide the first prospective evidence in the R2-ISS era that advanced risk stratification combining SKY92 and FISH may help to identify the highest-risk MM.

A case of vasculogenic mesenchymal tumor in the mediastinum: whole-exome sequencing reveals origin from pre-existing germ cell tumor

Vasculogenic mesenchymal tumor (VMT), a primitive mesenchymal neoplasm enriched by various-sized atypical vessels, is a new entity that develops in mediastinal germ cell tumors (GCTs) with yolk sac tumor (YST) components after chemotherapy. Notably, patients with VMT in the residual GCT have increased risk of developing sarcomas or hematopoietic malignancies. Here, we report a late-teenage male patient with residual teratoma and high-grade VMT after chemotherapy for a mediastinal mixed GCT, including YST. Whole-exome sequencing revealed biallelic inactivation of TP53 and extensive copy number alterations that suggested whole-genome doubling. The biopsy tissue of the mixed GCT before chemotherapy exhibited overlapping genetic alterations to those in the VMT. Immunohistochemical analyses of the VMT showed that the abnormal vessels were positive for cytokeratin, glypican 3, EZH2, and IMP3. The findings that VMT inherits the genetic alterations of pre-existing mixed GCT and exhibits a partly YST-like immunophenotype might contribute to its clinical aggressiveness.

Cytogenetic Abnormalities in Multiple Myeloma: Incidence, Prognostic Significance, and Geographic Heterogeneity in Indian and Western Populations

Multiple myeloma (MM) is a genetically complex and heterogeneous neoplasm in which cytogenetics is a major factor playing an important role in the risk stratification of disease. High-risk MM based upon cytogenetic classification includes primary IGH translocations t(4;14), t(14;16), t(14;20), and secondary progressive aberrations such as gain/amp(1q), 1p deletion, del(17p), and hypodiploidy. Several studies have proved that interphase FISH can detect primary as well as secondary cryptic aberrations very efficiently in lowest 5–10% abnormal plasma cell population. The present large-scale study was undertaken to evaluate the incidence of cytogenetic abnormalities, to analyse the correlation of conventional karyotyping with FISH, and to seek the geographic heterogeneity in the incidence of primary as well as secondary aberrations in our Indian versus Western populations. We conducted prospective studies of 1,104 patients consecutively referred from the primary, secondary, and tertiary oncology centres from all over India. Interphase FISH was performed on isolated plasma cells. Karyotype analysis was done as per ISCN 2016 and 2020. FISH could detect cytogenetic abnormalities in 67.6% of the cases with an incidence of 59% non-hyperdiploidy. The incidence of IGH translocation was 26% versus literature frequency of 40–50% which was mainly due to a low incidence (6%) of t(11;14) in contrast to 15–20% in other series. Additionally, the association of secondary progressive aberrations in the hyperdiploid group rather than the non-hyperdiploid group in our patients is not a common finding. A biallelic inactivation of TP53 as an ultra-high risk factor was detected in old-aged patients. These observations disclose the novel findings and strongly indicate the racial disparity which leads to geographic heterogeneity. In contrast to FISH, conventional karyotyping could detect MM-related aberrations in 50% of cases, of which 44% revealed highly complex karyotypes with common aberrations of chromosome 1q. Overall, FISH was found to be a novel, easy approach with high success rate and capability of detection of all cytogenetic abnormalities that add valid information for the risk stratification of disease. This, in future, in combination with mutation profile and gene expression profile will help in further refinement of disease and identification of actionable targets.

Primary cutaneous SMARCA4-deficient undifferentiated malignant neoplasm: first two cases with clinicopathologic and molecular comparison to eight visceral counterparts

SMARCA4-deficient undifferentiated malignant neoplasms (SD-UMN) comprise a group of aggressive tumors with epithelioid morphology that are characterized by loss of function of SMARCA4, a component of the SWI/SNF chromatin remodeling complex. SD-UMN was first recognized in the thoracic cavity but is now appreciated to occur at multiple anatomic sites. A notable exception has been skin. Here we report the first two cases of primary cutaneous SD-UMN and compare their features to a cohort of eight visceral cases arising in lung, gastrointestinal tract, and gallbladder. Evidence for a bona fide cutaneous origin included extensive clinical, radiologic, and serologic analyses that failed to identify a metastatic source as well as the molecular identification of a UV-associated mutational pattern. The cutaneous cases showed strikingly similar morphologic, immunohistochemical, and molecular features to the visceral cases, strongly suggesting that they belong to this family of tumors. In addition to biallelic inactivation of SMARCA4, both cutaneous tumors also showed biallelic inactivation of TP53 and CDKN2A, findings which also appear common in visceral cases. One patient died of disease at 18 months after diagnosis, consistent with the aggressive nature of this tumor. Our results expand the anatomic spectrum of SD-UMN, adding this entity to an already challenging differential diagnosis that includes melanoma, squamous cell carcinoma, Merkel cell carcinoma, epithelioid sarcoma, and others. Given the potentially aggressive nature of SD-UMN, the timely and accurate diagnosis of this entity may have implications for prognosis and therapy.