Circulating cell-free DNA improves the molecular characterisation of Ph-negative myeloproliferative neoplasms.

Genetic studies in patients with Philadelphia-negative myeloproliferative neoplasms (MPNs) are essential to establish the correct diagnosis and to optimise their management. Recently, it has been demonstrated that it is possible to detect molecular alterations analysing cell-free DNA (cfDNA) in plasma samples, which is known as liquid biopsy. We have assessed the molecular profile of a cohort of 107 MPN patients [33 polycythaemia vera (PV), 56 essential thrombocythaemia (ET), 14 primary myelofibrosis (PMF) and 4 unclassifiable MPN] by next-generation sequencing (NGS) using cfDNA and paired granulocyte DNA. A high concentration of cfDNA in plasma was observed in patients with high molecular complexity, in MPL-mutated cases, and in PMF patients. Targeted sequencing of cfDNA showed a comparable mutational profile (100% accuracy) to the one obtained in granulocytic DNA and a strong correlation was observed between the variant allele frequency (VAF) of gene mutations in both DNA sources. The median VAF detected in cfDNA (29·0%; range: 0·95-91·73%) was significantly higher than the VAF detected in granulocytes (median 25·2%; range: 0·10-95·5%), especially for MPL mutations (44·3% vs. 22·5%). In conclusion, our data support the use of cfDNA as a fast, sensitive and accurate strategy for performing molecular characterisation of MPN patients.

Cell-Free DNA as a Biomarker at Diagnosis and Follow-Up in 256 B and T-Cell Lymphomas.

BACKGROUND: Cell-free DNA (cfDNA) analysis has become a promising tool for the diagnosis, prognosis, and monitoring of lymphoma cases. Until now, research in this area has mainly focused on aggressive lymphomas, with scanty information from other lymphoma subtypes. METHODS: We selected 256 patients diagnosed with lymphomas, including a large variety of B-cell and T-cell non-Hodgkin and Hodgkin lymphomas, and quantified cfDNA from plasma at the time of diagnosis. We further selected 49 large B-cell lymphomas (LBCL) and analyzed cfDNA levels at diagnosis (pre-therapy) and after therapy. In addition, we performed NGS on cfDNA and tissue in this cohort of LBCL. RESULTS: Lymphoma patients showed a statistically significant higher cfDNA concentration than healthy controls (mean 53.0 ng/mL vs. 5.6 ng/mL, p < 0.001). The cfDNA concentration was correlated with lymphoma subtype, lactate dehydrogenase, the International Prognostic Index (IPI) score, Ann Arbor (AA), and B-symptoms. In 49 LBCL cases, the cfDNA concentration decreased after therapy in cases who achieved complete response (CR) and increased in non-responders. The median cfDNA at diagnosis of patients who achieved CR and later relapsed was higher (81.5 ng/mL) compared with levels of those who did not (38.6 ng/mL). A concordance of 84% was observed between NGS results in tumor and cfDNA samples. Higher VAF in cfDNA is correlated with advanced stage and bulky disease. CONCLUSIONS: cfDNA analysis can be easily performed in almost all lymphoma cases. The cfDNA concentration correlated with the characteristics of the aggressiveness of the lymphomas and, in LBCL, with the response achieved after therapy. These results support the utility of cfDNA analysis as a complementary tool in the management of lymphoma patients.

MultiCOVID: a multi modal deep learning approach for COVID-19 diagnosis.

The rapid spread of the severe acute respiratory syndrome coronavirus 2 led to a global overextension of healthcare. Both Chest X-rays (CXR) and blood test have been demonstrated to have predictive value on Coronavirus Disease 2019 (COVID-19) diagnosis on different prevalence scenarios. With the objective of improving and accelerating the diagnosis of COVID-19, a multi modal prediction algorithm (MultiCOVID) based on CXR and blood test was developed, to discriminate between COVID-19, Heart Failure and Non-COVID Pneumonia and healthy (Control) patients. This retrospective single-center study includes CXR and blood test obtained between January 2017 and May 2020. Multi modal prediction models were generated using opensource DL algorithms. Performance of the MultiCOVID algorithm was compared with interpretations from five experienced thoracic radiologists on 300 random test images using the McNemar-Bowker test. A total of 8578 samples from 6123 patients (mean age 66 ± 18 years of standard deviation, 3523 men) were evaluated across datasets. For the entire test set, the overall accuracy of MultiCOVID was 84%, with a mean AUC of 0.92 (0.89-0.94). For 300 random test images, overall accuracy of MultiCOVID was significantly higher (69.6%) compared with individual radiologists (range, 43.7-58.7%) and the consensus of all five radiologists (59.3%, P < .001). Overall, we have developed a multimodal deep learning algorithm, MultiCOVID, that discriminates among COVID-19, heart failure, non-COVID pneumonia and healthy patients using both CXR and blood test with a significantly better performance than experienced thoracic radiologists.

Leukemic Involvement Is a Common Feature in Waldenström Macroglobulinemia at Diagnosis.

Waldenström Macroglobulinemia (WM) is a lymphoplasmacytic lymphoma with bone marrow (BM) involvement and IgM monoclonal gammopathy. To date, no studies have focused specifically on peripheral blood (PB) involvement. In this study, 100 patients diagnosed with WM according to the World Health Organization (WHO) criteria were included based on the demonstration of MYD88mut in BM and the availability of PB multiparametric flow cytometry (MFC) analysis. Leukemic involvement by MFC was detected in 50/100 patients. A low percentage of mature small lymphocytes in PB smears was observed in only 15 cases. MYD88mut by AS-qPCR was detected in PB in 65/100 cases. In cases with leukemic expression by MFC, MYD88mut was detected in all cases, and IGH was rearranged in 44/49 cases. In 21/50 patients without PB involvement by MFC, molecular data were consistent with circulating disease (MYD88mut by AS-qPCR 3/50, IGH rearranged 6/50, both 12/50). Therefore, PB involvement by standard techniques was detected in 71/100 patients. MYD88mut was detected in PB by dPCR in 9/29 triple negative cases. Overall, 80% of the patients presented PB involvement by any technique. Our findings support the role of PB MFC in the evaluation of patients with IgM monoclonal gammopathy and provide reliable information on correlation with molecular features. The development of a feasible MFC assay may stand as an objective tool in the classification of mature B cell neoplasms presenting with IgM monoclonal gammopathy.

Outcomes and molecular profile of oligomonocytic CMML support its consideration as the first stage in the CMML continuum.

Patients with oligomonocytic chronic myelomonocytic leukemia (OM-CMML) are currently classified according to the 2017 World Health Organization myelodysplastic syndromes classification. However, recent data support considering OM-CMML as a specific subtype of chronic myelomonocytic leukemia (CMML), given their similar clinical, genomic, and immunophenotypic profiles. The main purpose of our study was to provide survival outcome data of a well-annotated series of 42 patients with OM-CMML and to compare them to 162 patients with CMML, 120 with dysplastic type (D-CMML), and 42 with proliferative type (P-CMML). OM-CMML had significantly longer overall survival (OS) and acute myeloid leukemia-free survival than did patients with CMML, considered as a whole group, and when compared with D-CMML and P-CMML. Moreover, gene mutations associated with increased proliferation (ie, ASXL1 and RAS-pathway mutations) were identified as independent adverse prognostic factors for OS in our series. We found that at a median follow-up of 53.47 months, 29.3% of our patients with OM-CMML progressed to D-CMML, and at a median follow-up of 46.03 months, 28.6% of our D-CMML group progressed to P-CMML. These data support the existence of an evolutionary continuum of OM-CMML, D-CMML, and P-CMML. In this context, we observed that harboring more than 3 mutated genes, carrying ASXL1 mutations, and a peripheral blood monocyte percentage >20% significantly predicted a shorter time of progression of OM-CMML into overt CMML. These variables were also detected as independent adverse prognostic factors for OS in OM-CMML. These data support the consideration of OM-CMML as the first evolutionary stage within the proliferative continuum of CMML.

Molecular and cytogenetic characterization of myelodysplastic syndromes in cell-free DNA.

Molecular and cytogenetic studies are essential for diagnosis and prognosis in patients with myelodysplastic syndromes (MDSs). Cell-free DNA (cfDNA) analysis has been reported to be a reliable noninvasive approach for detecting molecular abnormalities in MDS; however, there is limited information about cytogenetic alterations and monitoring in cfDNA. We assessed the molecular and cytogenetic profile of a cohort of 70 patients with MDS by next-generation sequencing (NGS) of cfDNA and compared the results to sequencing of paired bone marrow (BM) DNA. Sequencing of BM DNA and cfDNA showed a comparable mutational profile (92.1% concordance), and variant allele frequencies (VAFs) strongly correlated between both sample types. Of note, SF3B1 mutations were detected with significantly higher VAFs in cfDNA than in BM DNA. NGS and microarrays were highly concordant in detecting chromosomal alterations although with lower sensitivity than karyotype and fluorescence in situ hybridization. Nevertheless, all cytogenetic aberrations detected by NGS in BM DNA were also detected in cfDNA. In addition, we monitored molecular and cytogenetic alterations and observed an excellent correlation between the VAFs of mutations in BM DNA and cfDNA across multiple matched time points. A decrease in the cfDNA VAFs was detected in patients responding to therapy, but not in nonresponding patients. Of note, cfDNA analysis also showed cytogenetic evolution in 2 nonresponsive cases. In summary, although further studies with larger cohorts are needed, our results support the analysis of cfDNA as a promising strategy for performing molecular characterization, detection of chromosomal aberrations and monitoring of patients with MDS.

Oligomonocytic and overt chronic myelomonocytic leukemia show similar clinical, genomic, and immunophenotypic features.

Oligomonocytic chronic myelomonocytic leukemia (OM-CMML) is defined as those myelodysplastic syndromes (MDSs) or myelodysplastic/myeloproliferative neoplasms, unclassifiable with relative monocytosis (≥10% monocytes) and a monocyte count of 0.5 to <1 × 109/L. These patients show clinical and genomic features similar to those of overt chronic myelomonocytic leukemia (CMML), although most of them are currently categorized as MDS, according to the World Health Organization 2017 classification. We analyzed the clinicopathologic features of 40 patients with OM-CMML with well-annotated immunophenotypic and molecular data and compared them to those of 56 patients with overt CMML. We found similar clinical, morphological, and cytogenetic features. In addition, OM-CMML mirrored the well-known complex molecular profile of CMML, except for the presence of a lower percentage of RAS pathway mutations. In this regard, of the different genes assessed, only CBL was found to be mutated at a significantly lower frequency. Likewise, the OM-CMML immunophenotypic profile, assessed by the presence of >94% classical monocytes (MO1s) and CD56 and/or CD2 positivity in peripheral blood monocytes, was similar to overt CMML. The MO1 percentage >94% method showed high accuracy for predicting CMML diagnosis (sensitivity, 90.7%; specificity, 92.2%), even when considering OM-CMML as a subtype of CMML (sensitivity, 84.9%; specificity, 92.1%) in our series of 233 patients (39 OM-CMML, 54 CMML, 23 MDS, and 15 myeloproliferative neoplasms with monocytosis and 102 reactive monocytosis). These results support the consideration of OM-CMML as a distinctive subtype of CMML.