Bone marrow specimens are the core of the diagnostic workup of patients with cytopenia. To explore whether next-generation sequencing (NGS) could be used to rule out malignancy without bone marrow ...specimens, we incorporated NGS in a model to predict presence of disease in the bone marrow of patients with unexplained cytopenia. We analyzed the occurrence of mutations in 508 patients with cytopenia, referred for primary workup of a suspected hematologic malignancy from 2015 to 2020. We divided patients into a discovery (n = 340) and validation (n = 168) cohort. Targeted sequencing, bone marrow biopsy, and complete blood count were performed in all patients. Mutations were identified in 267 (53%) and abnormal bone marrow morphology in 188 (37%) patients. Patients with isolated neutropenia had the lowest frequency of both mutations (21%) and abnormal bone marrow morphology (5%). The median number of mutations per patient was 2 in patients with abnormal bone marrow morphology compared with 0 in patients with a nondiagnostic bone marrow morphology (P < .001). In a multivariable logistic regression, mutations in TET2, SF3B1, U2AF1, TP53, and RUNX1 were significantly associated with abnormal bone marrow morphology. In the validation cohort, a model combining mutational status and clinical data identified 34 patients (20%) without abnormal bone marrow morphology with a sensitivity of 100% (95% confidence interval: 93%-100%). Overall, we show that NGS combined with clinical data can predict the presence of abnormal bone marrow morphology in patients with unexplained cytopenia and thus can be used to assess the need of a bone marrow biopsy.
•Twenty percent of bone marrow biopsies can be omitted by combining clinical data and NGS to assess risk of bone marrow disease before biopsy.•Disease in the bone marrow is rarely observed in patients with isolated neutropenia.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Objectives
Flow cytometry (FC) is, together with morphology, genetics, and cytogenetics, used in the diagnostic assessment of cytopenia, as its value in evaluating bone marrow dysplasia been ...highlighted by several studies. However, despite the development of algorithms and guidelines, there is still a lack of standardization of the FC assessment of bone marrow dysplasia.
Methods
By combining FC, together with morphological analysis and cytogenetic/molecular assessment in a training cohort of 209 patients, we created a novel score, ProGraME, which includes four parameters, each from a different cell lineage (Progenitor cells, Granulocytes, Monocytes, Erythroid precursors), solely based on relevant population gating. Points for ProGraME were attained for: lymphoid precursors ≤5% of all CD34+ cells (1.5 point); a granulocyte‐to‐lymphocyte side‐scatter ratio ≤6 (1 point); a monocyte CD33‐CV% ≥ 63 (2 points), and an erythroid precursor CD36‐CV% ≥ 65 (2 points).
Results
Using a cutoff of ≥2 as suggestive of dysplasia, ProGraME showed a sensitivity of 91% and a specificity of 81% in the training cohort and 95% and 75%, respectively, in an independent validation cohort of 159 patients. In addition, ProGraME had a very high negative predictive value of 97.1% and 97.8% in the training and validation cohorts, respectively, offering a useful tool for excluding bone marrow dysplasia. Finally, among the 23 CCUS patients that scored positive for dysplasia with ProGraME in the training cohort, 16 of them (69%) carried high‐risk mutations, suggesting that FC might help identify early changes of dysplasia.
Conclusions
ProGraME can potentially optimize the FC diagnosis of low‐risk myelodysplasia without minimal requirements of flow analysis other than accurate population gating.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Introduction Clonal cytopenia of undetermined significance (CCUS) is a recently recognized hematological disorder characterized by the presence of one or more cytopenias, evidence of clonal ...hematopoiesis and not fulfilling the criteria for a myeloid neoplasm. In contrast to myelodysplastic syndromes (MDS) where 95% of the patients are anemic at time of diagnosis, that is only the case for around 60% of patients with CCUS (Weeks et al. Prediction of Risk for Myeloid Malignancy in Clonal Hematopoiesis. NEJM Evid. 2023). Furthermore, as recent studies on survival in CCUS primarily included younger patients (Weeks et al. NEJM Evid. 2023) or population-based cohorts not specifically referred for diagnostic work up of unexplained cytopenia (UC) (Rossi et al. Clinical relevance of clonal hematopoiesis in persons aged ≥80 years, Blood 2021) additional survival data on patients with CCUS are warranted. In this study, we investigated survival outcomes in patients with CCUS referred for primary work up of UC. Methods We included 241 patients with CCUS and compared them to 144 patients with low-risk MDS, defined as having less than 5% blasts in the bone marrow. Bone marrow biopsies and next-generation sequencing (NGS) were performed in all patients and cytogenetic analyses using G-band karyotyping was done in 362 (94%) of cases. For NGS, we used a gene panel with the most commonly mutated genes in MDS. The patients were included at 6 different institutions in Denmark from 2013 and onwards. A cut-point of 5 years follow-up was chosen for more robust survival data, and patients were censored at this time point if followed for more than 5 years. Patients with CCUS were stratified into 3 groups dependent on the type of cytopenia present at diagnosis. CCUS patients with pancytopenia (n = 35), and CCUS patients without pancytopenia were split based on whether they were anemic (n = 143) or not (n =63). We compared overall survival between the CCUS groups and low-risk MDS in a Cox proportional hazards model adjusted for age, sex, number of variants and presence of high-risk variants. Results Of the 241 CCUS patients, 178 (74%) were anemic (hemoglobin < 12 and 13 g/dL for women and men, respectively), while this was the case for 130 (90%) of the patients with MDS. A total of 772 variants were identified in the entire cohort with a median of 2 (IQR: 1-3) per patient for both CCUS and MDS patients. The overall survival at 5 years was 45% for low-risk MDS patients and 62% for CCUS patients. For CCUS patients without anemia, CCUS patients with anemia and CCUS patients with pancytopenia overall survival at 5 years was 85%, 52% and 55%, respectively (p < 0.0001). In the adjusted Cox proportional hazards model, we found a significantly superior overall survival for CCUS patients without anemia compared to those with anemia (HR: 0.28, 95%-CI: 0.13-0.58, p = 0.001) and pancytopenia (HR: 0.22, 95%-CI: 0.09-0.53, p = 0.001) and patients with low-risk MDS (HR: 0.18, 95%-CI: 0.09-0.38, p < 0.001). There was no significant survival difference between CCUS patients with pancytopenia and low-risk MDS patients (HR: 0.67, 95%-CI: 0.46-1.50, p = 0.5). Of the remaining variables, age was the strongest prognostic factor with hazard ratio increasing with a factor 1.05 (95%-CI: 1.03-1.07, p < 0.001) per year aged. Predicted survival probabilities for a 65-year-old and 80-year-old person demonstrate a substantial difference in overall survival across all 3 categories of CCUS (figure 1). Discussion Here we show that the presence of anemia impacts overall survival among CCUS patients. We observe that CCUS patients without anemia, representing 26% of CCUS patients in our cohort, have a superior overall survival compared to those with anemia, even with a comparable mutational profile. Our findings indicate that other factors than mutational signatures impact overall survival. Moreover, the prevalence of CCUS increases with age, and our findings demonstrate a considerable difference in survival between a 65-year-old and 80-year-old person diagnosed with CCUS. This raises the concern that prediction models can overestimate the survival rates if the elderly group of CCUS patients is not well represented in these models. In conclusion, we show that age and hemoglobin levels are important factors in overall survival for CCUS patients, and that patients with CCUS without anemia have a superior overall survival and should be considered as a separate group within the CCUS category.
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IJS, IMTLJ, KILJ, NLZOH, NUK, SAZU, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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