Inference of genomic lesions from single-cell RNAseq in myeloma improves functional intra- and inter-clonal analysis.

Smoldering Multiple Myeloma (SMM) is an asymptomatic plasma cell (PC) neoplasm that may evolve with variable frequency into multiple myeloma (MM). SMM is initiated by chromosomal translocations involving the IgH locus or by hyperdiploidy and evolves through acquisition of additional genetic lesions. In this scenario, we aimed at establishing a reliable analysis pipeline to infer genomic lesions from transcriptomic analysis, by combining single-cell RNA sequencing (scRNA-seq) with B-cell receptor sequencing and copy- number abnormality (CNA) analysis to identify clonal PCs at the genetic level along their specific transcriptional landscape. We profiled 20,465 bone marrow (BM) PCs derived from five SMM/MM patients and unbiasedly identified clonal and polyclonal plasma cells. Hyperdiploidy, t(11;14) and t(6;14) were identified at the scRNA level by analysis of chimeric reads. Subclone functional analysis was improved by combining transcriptome with CNA analysis. As examples, we illustrate the different functional properties of a light chain escape subclone in SMM, and of different B-cell and PC subclones in a patient affected by Wäldenstrom Macroglobulinemia and SMM. Overall, our data provide a proof of principle for inference of clinically relevant genotypic data from scRNAseq, which in turn will refine functional annotation of the clonal architecture of PC dyscrasias.

Clonal Hematopoiesis of Indeterminate Potential in Patients with Immunoglobulin Light Chain AL Amyloidosis.

Immunoglobulin light chain (AL) amyloidosis is characterized by the deposition of misfolded monoclonal free light chains, with cardiac complications accounting for patients' mortality. Clonal hematopoiesis of indeterminate potential (CHIP) has been associated with worse cardiovascular outcomes in the general population. Its significance in AL amyloidosis remains unclear. We collected clinical information and outcome data on 76 patients with a diagnosis of AL amyloidosis who underwent deep-targeted sequencing for myeloid neoplasia-associated mutations between April 2018 and August 2023. Variant allele fraction was set at 2% to call CHIP-associated mutations. CHIP mutations were present in AL amyloidosis patients at a higher frequency than age-matched control individuals. Sixteen patients (21%) had at least 1 CHIP mutation. DNMT3A was the most frequent mutation (7/16, 44%). Compared to patients without CHIP, patients with CHIP were enriched for the presence of t(11;14) (69% vs 25%, respectively, p = 0.004) and, for patients with renal involvement, a lower Palladini renal stage (p = 0.001). At a median follow-up of 32.5 months, the presence of CHIP was not associated with worse overall survival or major organ dysfunction progression-free survival. Larger studies and longer follow-up are needed to better define the impact of CHIP in patients with AL amyloidosis.

Impact of clonal hematopoiesis of indeterminate potential on hepatocellular carcinoma in individuals with steatotic liver disease.

BACKGROUND AND AIMS: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global epidemic and is the most rapidly rising cause of HCC. Clonal hematopoiesis of indeterminate potential (CHIP) contributes to neoplastic and cardiometabolic disorders and is considered a harbinger of tissue inflammation. CHIP was recently associated with increased risk of liver disease. The aim of this study was to examine whether CHIP is associated with HCC development in patients with SLD. APPROACH AND RESULTS: We considered individuals with MASLD-HCC (n=208) and controls with (n =414) and without (n =259) advanced fibrosis who underwent whole exome sequencing. CHIP was diagnosed when ≥2 variant callers identified a known myeloid mutation with variant allele frequency ≥2%. CHIP was observed in 116 participants (13.1%), most frequently in DNMT3A, TET2, TP53 , and ASXL1 , and correlated with age ( p <0.0001) and advanced liver fibrosis (p=0.001). Higher aspartate aminotransferase levels predicted non- DNMT3A -CHIP, in particular with variant allele frequency ≥10% (OR: 1.14, 1.03 -1.28 and OR: 1.30, 1.12 -1.49, respectively, p <0.05). After adjustment for sex, diabetes, and a polygenic risk, a score of inherited MASLD predisposition CHIP was associated with cirrhosis (2.00, 1.30 -3.15, p =0.02), and with HCC even after further adjustment for cirrhosis (OR: 1.81, 1.11 -2.00, 1.30 -3.15, p =0.002). Despite the strong collinearity among aging and development of CHIP and HCC, non- DNTM3A -CHIP, and TET2 lesions remained associated with HCC after full correction for clinical/genetics covariates and age (OR: 2.45, 1.35 -4.53; OR: 4.8, 1.60 -17.0, p =0.02). CONCLUSIONS: We observed an independent association between CHIP, particularly related to non- DNTM3A and TET2 genetic lesions and MASLD-HCC.

Pathological and genomic features of myeloproliferative neoplasms associated with splanchnic vein thrombosis in a single-center cohort.

Here, we reviewed clinical-morphological data and investigated mutational profiles by NGS in a single-center series of 58 consecutive MPN-SVT patients admitted to our hospital between January 1979 and November 2021. We identified 15.5% of PV, 13.8% of ET, 34.5% of PMF, 8.6% of SMF and 27.6% of MPN-U. Most cases (84.5%) carried JAK2V617F mutation, while seven patients were characterized by other molecular markers, namely MPL in four and CALR mutations in three cases. NGS was performed in 54 (93.1%) cases: the most frequent additional mutations were found in TET2 (27.8%) and DNMT3A (16.7%) genes, whereas 25 (46.3%) patients had no additional mutation. Cases with JAK2V617F homozygosity had a higher median number of additional mutations than those with low allele burden. More importantly, all cases of leukemic evolution were characterized by a higher median number of co-mutations, and a co-mutational pattern of high-risk lesions, such as truncating mutations of ASXL1, bi-allelic TP53 loss, and CSMD1 mutations. Nevertheless, no difference was found between cases with and without additional somatic mutations regarding fibrotic progression, SVT recurrence, other thrombo-hemorrhagic complications, or death. After a median follow-up of 7.1 years, ten deaths were recorded; fibrotic progression/leukemic evolution was ascertained in one (1.7%) and six (10.3%) patients, respectively, while 22 (37.9%) patients suffered from recurrent thrombosis. In conclusion, our data underline the importance of using NGS analysis in the management of MPN-related SVT as it can support the MPN diagnosis, particularly in "triple-negative" cases, and provide additional information with potential consequences on prognosis and therapeutic strategies.

Biodegradation of potential diesel oxygenate additives: dibutyl maleate (DBM), and tripropylene glycol methyl ether (TGME).

The addition of oxygen-bearing compounds to diesel fuel considerably reduces particulate emissions. TGME and DBM have been identified as possible diesel additives based on their physicochemical characteristics and performance in engine tests. Although these compounds will reduce particulate emissions, their potential environmental impacts are unknown. As a means of characterizing their persistence in environmental media such as soil and groundwater, we conducted a series of biodegradation tests of DBM and TGME. Benzene and methyl tertiary butyl ether (MTBE) were also tested as reference compounds. Primary degradation of DBM fully occurred within 3 days, while TGME presented a lag phase of approximately 8 days and was not completely degraded by day 28. Benzene primary degradation occurred completely by day 3 and MTBE did not degrade at all. The total mineralized fractions of DBM and TGME achieved constant values as a function of time of approximately 65% and approximately 40%, respectively. Transport predictions show that, released to the environment, DBM and TGME would concentrate mostly in soils and waters with minimal impact to air. From an environmental standpoint, these results combined with the transport predictions indicate that DBM is a better choice than TGME as a diesel additive.