Elisa Bono

Driver somatic mutations identify distinct disease entities within myeloid neoplasms with myelodysplasia

Our knowledge of the genetic basis of myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) has considerably improved. To define genotype/phenotype relationships of clinical relevance, we studied 308 patients with MDS, MDS/MPN, or acute myeloid leukemia evolving from MDS. Unsupervised statistical analysis, including the World Health Organization classification criteria and somatic mutations, showed that MDS associated with SF3B1-mutation (51 of 245 patients, 20.8%) is a distinct nosologic entity irrespective of current morphologic classification criteria. Conversely, MDS with ring sideroblasts with nonmutated SF3B1 segregated in different clusters with other MDS subtypes. Mutations of genes involved in DNA methylation, splicing factors other than SF3B1, and genes of the RAS pathway and cohesin complex were independently associated with multilineage dysplasia and identified a distinct subset (51 of 245 patients, 20.8%). No recurrent mutation pattern correlated with unilineage dysplasia without ring sideroblasts. Irrespective of driver somatic mutations, a threshold of 5% bone marrow blasts retained a significant discriminant value for identifying cases with clonal evolution. Comutation of TET2 and SRSF2 was highly predictive of a myeloid neoplasm characterized by myelodysplasia and monocytosis, including but not limited to, chronic myelomonocytic leukemia. These results serve as a proof of concept that a molecular classification of myeloid neoplasms is feasible.

Minimal morphological criteria for defining bone marrow dysplasia: a basis for clinical implementation of WHO classification of myelodysplastic syndromes.

The World Health Organization classification of myelodysplastic syndromes (MDS) is based on morphological evaluation of marrow dysplasia. We performed a systematic review of cytological and histological data from 1150 patients with peripheral blood cytopenia. We analyzed the frequency and discriminant power of single morphological abnormalities. A score to define minimal morphological criteria associated to the presence of marrow dysplasia was developed. This score showed high sensitivity/specificity (>90%), acceptable reproducibility and was independently validated. The severity of granulocytic and megakaryocytic dysplasia significantly affected survival. A close association was found between ring sideroblasts and SF3B1 mutations, and between severe granulocytic dysplasia and mutation of ASXL1, RUNX1, TP53 and SRSF2 genes. In myeloid neoplasms with fibrosis, multilineage dysplasia, hypolobulated/multinucleated megakaryocytes and increased CD34+ progenitors in the absence of JAK2, MPL and CALR gene mutations were significantly associated with a myelodysplastic phenotype. In myeloid disorders with marrow hypoplasia, granulocytic and/or megakaryocytic dysplasia, increased CD34+ progenitors and chromosomal abnormalities are consistent with a diagnosis of MDS. The proposed morphological score may be useful to evaluate the presence of dysplasia in cases without a clearly objective myelodysplastic phenotype. The integration of cytological and histological parameters improves the identification of MDS cases among myeloid disorders with fibrosis and hypocellularity.

Clinical significance of somatic mutation in unexplained blood cytopenia

Unexplained blood cytopenias, in particular anemia, are often found in older persons. The relationship between these cytopenias and myeloid neoplasms like myelodysplastic syndromes is currently poorly defined. We studied a prospective cohort of patients with unexplained cytopenia with the aim to estimate the predictive value of somatic mutations for identifying subjects with, or at risk of, developing a myeloid neoplasm. The study included a learning cohort of 683 consecutive patients investigated for unexplained cytopenia, and a validation cohort of 190 patients referred for suspected myeloid neoplasm. Using granulocyte DNA, we looked for somatic mutations in 40 genes that are recurrently mutated in myeloid malignancies. Overall, 435/683 patients carried a somatic mutation in at least 1 of these genes. Carrying a somatic mutation with a variant allele frequency ≥0.10, or carrying 2 or more mutations, had a positive predictive value for diagnosis of myeloid neoplasm equal to 0.86 and 0.88, respectively. Spliceosome gene mutations and comutation patterns involving TET2, DNMT3A, or ASXL1 had positive predictive values for myeloid neoplasm ranging from 0.86 to 1.0. Within subjects with inconclusive diagnostic findings, carrying 1 or more somatic mutations was associated with a high probability of developing a myeloid neoplasm during follow-up (hazard ratio = 13.9, P < .001). The predictive values of mutation analysis were confirmed in the independent validation cohort. The findings of this study indicate that mutation analysis on peripheral blood granulocytes may significantly improve the current diagnostic approach to unexplained cytopenia and more generally the diagnostic accuracy of myeloid neoplasms.

Inappropriately low hepcidin levels in patients with myelodysplastic syndrome carrying a somatic mutation of SF3B1

Somatic mutations of the RNA splicing machinery have been recently identified in myelodysplastic syndromes. In particular, a strong association has been found between SF3B1 mutation and refractory anemia with ring sider-oblasts, a condition characterized by ineffective erythropoiesis and parenchymal iron overload. We studied the relationship between SF3B1 mutation, erythroid activity and hepcidin levels in myelodysplastic syndrome patients. Erythroid activity was evaluated through the proportion of marrow erythroblasts, soluble transferrin receptor and serum growth differentiation factor 15. Significant relationships were found between SF3B1 mutation and marrow erythroblasts (P=0.001), soluble transferrin receptor (P=0.003) and serum growth differentiation factor 15 (P=0.033). Serum hepcidin varied considerably, and multivariable analysis showed that the hepcidin to ferritin ratio, a measure of adequacy of hepcidin levels relative to body iron stores, was inversely related to the SF3B1 mutation (P=0.013). These observations suggest that patients with SF3B1 mutation have inappropriately low hepcidin levels, which may explain their propensity to parenchymal iron loading.

Therapy-Related Late Adverse Events in Hodgkin’s Lymphoma

Hodgkins lymphoma (HL) is one of the most curable hematologic diseases with an overall response rate over 80%. However, despite this therapeutic efficacy, HL survivors show a higher morbidity and mortality than other people of the same age because of long-term therapy-related events. In the last decades, many efforts have been made to reduce these effects through the reduction of chemotherapy dose, the use of less toxic chemotherapeutic agents, and the introduction of new radiation techniques. In this paper, we will describe the main long-term effects related to chemotherapy and radiotherapy for HL, the efforts to reduce toxicity made in the last years, and the clinical aspects which have to be taken into consideration in the followup of these patients.

Independent prognostic impact of tumour-infiltrating macrophages in early-stage Hodgkin's lymphoma.

Although patients with early‐stage Hodgkins lymphoma have a high rate of cure, a portion of these are resistant to or relapse after standard treatment. Current prognostic criteria based on clinical and laboratory parameters at diagnosis do not allow to accurately identify the subset of patients with less favourable clinical outcome. An increased number of tumour‐infiltrating macrophages was found to be associated with shortened survival in patients with classic Hodgkins Lymphoma. The aim of this study was to assess the clinical significance of the proportion of CD68‐positive infiltrating macrophages in patients with early‐stage classic Hodgkins lymphoma. By using immunohistochemistry technique, we evaluated for CD68 expression diagnostic biopsies of 106 patients affected by supradiaphragmatic early‐stage classic Hodgkins lymphoma treated at our institution since 2000 to 2010. All patients were treated with adriamycin, bleomycin, vinblastine, and dacarbazine chemotherapy followed by radiotherapy in the majority. The 2‐year overall survival and progression‐free survival (PFS) in the entire cohort were 97% and 83% respectively. The 2‐year PFS was statistically different between patients with favourable and those with unfavourable prognosis according to the European Organisation for Research and Treatment of Cancer (EORTC) risk criteria (96% vs 79%, p = 0.039) and between patients having less than 25% of CD68‐positive infiltrating macrophages and those with more than 25% (85% vs 67%, p = 0.012). All patients with favourable EORTC criteria had CD68 expression lower than 25%. Within those with unfavourable EORTC criteria, patients with a CD68+ count greater than 25% had a worse 2‐year PFS than patients having values lower than 25% (64% vs 82%, p = 0.03). Moreover, in multivariate analysis, after adjusting for CD68+ macrophages count and EORTC score, only CD68+ macrophages count higher than 25% retained a prognostic effect on PFS (hazard ratio = 2.8, 95%CI: 1.1–7.6, p = 0.038). Our data show that a proportion of tumour‐infiltrating macrophages greater than 25% is associated with unfavourable clinical outcome in patients with early‐stage Hodgkins lymphoma Copyright

Risk of hepatitis B reactivation under treatment with tyrosine-kinase inhibitors for chronic myeloid leukemia.

In February 2016, the European Medicines Agency (EMA) released a document stating that, according to available evidence, the marketing authorization holders of BCR-ABL tyrosine-kinase inhibitors (TKI), namely imatinib, dasatinib, nilotinib, bosutinib, and ponatinib-containing medical products should amend the product information to provide supplementary information on the risk of hepatitis B virus (HBV) reactivation (http://www.ema.europa.eu/docs/ en_GB/document_library/PRAC_recommendation_on_signal/2016/). The EMA document recommends that patients should be tested for HBV infection before initiating treatment with TKIs as reactivation of hepatitis B in chronic carriers of this virus has occurred after these patients received BCR-ABL TKIs. Some cases resulted in acute hepatic failure or fulminant hepatitis leading to liver transplantation or a fatal outcome. In Italy, hematologists are particularly conscious of the risk of hepatitis B virus (HBV) reactivation under treatment for hematologic malignancies. In our country HBsAg prevalence is about 2%, that is a lower-intermediate endemicity level according to Schweitzer et al. [1]; on the other hand, the seroprevalence of resolved infection (HBsAg negative, HBcAb positive, HBsAb positive/negative) ranges from 8% to 36.9%, and is higher than that in other Western Europe countries.[2–4] HBV reactivation under chemo/immunochemotherapy is most common in HBsAg carriers; however, the risk is not negligible in HBsAg negative patients with resolved infection. In this setting, reactivation is related to the persistence of replication-competent HBV in the form of covalently closed circular DNA within the nuclei of hepatocytes [5] and is more likely to occur in patients receiving monoclonal antibodies and high-dose steroids. [6] To what extent BCR-ABL TKIs might represent a trigger for HBV reactivation is not known. In a recent issue of Leukemia & Lymphoma, Sor a et al. [7] have reported on 122 patients with chronic myeloid leukemia (CML) treated with BCR-ABL TKIs: none of the 11 patients with resolved infection experienced reactivation. We would like to provide additional information adding our experience on this specific issue in a larger number of patients. Based on the high national seroprevalence for HBV infection in Italy, since the ‘90s patients with hematologic malignancies have been screened for HBV at our institution before starting treatment. Taking advantage from this institutional policy, we retrospectively collected data in 187 consecutive patients with early or late chronicphase CML who started TKI treatment between January 2000 and December 2015 at our institution. We were able to assess the prevalence of HBV infection and the incidence of HBV reactivation in 157 cases; 30 patients could not be included because of incomplete data on HBV status (20) or because vaccinated (10). Clinical characteristics are detailed in Table 1. Patients HBV negative at TKI treatment start (baseline) were not systematically reassessed during follow-up, but all alive patients had HBV status completely reassessed at the time the present study was planned. Serum HBV DNA level was serially evaluated in HBsAg positive patients, while patients with resolved infection were monitored outside a predefined schedule. HBV reactivation was defined as a marked increase in HBV replication ( 2log increase from baseline levels or a new appearance of HBV DNA to a level of 100 IU/ml) in a person with previously stable or undetectable level, reverse HBsAg seroconversion or appearance of HBV DNA in serum in the absence of HBsAg.[8] HBV status was assessed using commercially available kits. Concurrently with evaluation for HBV, our patients underwent assessment for hepatitis C virus (HCV) serological status. As per clinical practice, liver enzymes were evaluated every 3–4 months under TKI therapy, along with a comprehensive biochemical panel. This retrospective study was approved by the Ethics Review Board at our Institution. At the time of present analysis, 145 patients are alive, while 12 died (causes of death: blastic progression in 6, primary second tumor in 5, preexisting HCC in 1).