Immunogenicity of a third dose of the BNT162b2 COVID-19 vaccine in patients with CLL: effects on treatment selection.

Patients with chronic lymphocytic leukemia (CLL) show suboptimal responses to the vaccines against SARS-CoV-2; it has been shown though that a booster dose of the BNT162b2 vaccine may lead to a significant increase in the seroconversion rates of immunocompromised patients. We conducted a prospective, non-interventional study to evaluate the immunogenicity of a third dose of the BNT162b2 vaccine in adult patients with CLL. Sera were tested before the first, after the second, and before and after the third dose for anti-SARS-CoV-2 receptor binding domain (RBD) spike protein IgG (anti-RBD). Thirty-nine patients with CLL were included in the study. The seroconversion rate increased from 28.2% before the third dose to 64.1% after the third dose and was higher in treatment-naïve patients (72.7% versus 47.1% in actively treated patients, p = 0.042). All but one patient achieving a seroconversion after the second dose retained after the third, while eight patients not achieving a seroconversion after the second dose (38.1%), did so after the third. Moreover, patients actively treated with venetoclax had a higher seroconversion rate than those treated with ibrutinib (87.5% versus 14.3%, p = 0.001). This study confirms the beneficial effect of a third dose of the BNT162b2 vaccine on the seroconversion rate in patients with CLL. Our results also strongly suggest that the use of venetoclax is correlated with higher immunogenicity/seroconversion rates than that of ibrutinib, a finding that has been reported by another study. A treatment strategy change during the pandemic favoring the use of venetoclax may be suggested based on our results, although these results should be validated in larger studies.

Safety and immunogenicity of the BNT162b2 mRNA Covid-19 vaccine in patients with chronic lymphocytic leukemia: a prospective study.

Introduction: Immunization of patients with chronic lymphocytic leukemia (CLL) with vaccines against several infectious diseases has proven insufficient. Data on seroconversion of patients with CLL after vaccination against severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) are still young, but accumulating evidence shows low seroconversion rates. Methods: We conducted a prospective, noninterventional study evaluating the safety and immunogenicity of two doses of the BNT162b2 mRNA Covid-19 vaccine, administered 21 days apart in consecutive adult patients with CLL. Patients vaccinated with other vaccines against SARS-CoV-2, with a history of confirmed Coronavirus Disease 19 (COVID-19), with known human immunodeficiency virus infection, or with an inability to provide written informed consent were excluded. Sera were tested before the first and after the second dose of the vaccine for anti-SARS-CoV-2 receptor binding domain (RBD) spike protein IgG (anti-RBD), using the Abbott SARS-CoV-2 IgG II Quant assay (Abbott Laboratories, Abbott Park, IL, USA), with a cutoff value for seroconversion at 50 AU/ml. Results: Sixty-one patients (28 males/33 females) with CLL, with a median age of 61 years, were included in the study. The majority of the patients (82.0%) were lower (0-2) stage per the RAI staging system. The seroconversion rate at 14 days after the second dose was 45% and was correlated with RAI stage (0-2 versus 3-4; 51.0% versus 18.3%, p = 0.047), the treatment status (treatment naïve, previously treated, or actively treated patients; 63.0% versus 40.0% versus 26.1%, respectively, p = 0.031), the number of previous treatment lines (0-2 versus >2; 55.3% versus 8.3%, p = 0.004), and the platelet count of the patients (over or under 100 × 109/L; 52.9% versus 10.0%, p = 0.015). Moreover, there was a positive linear relationship between the antibody titers and the gamma-globulin levels (r = 0.182, p = 0.046) and platelet count (r = 0.277, p = 0.002). Finally, patients actively treated with venetoclax had higher antibody titers than those treated with ibrutinib (15.8 AU/ml versus 0.0 AU/ml, p = 0.047). No safety issues were identified while the emergence of adverse events was not correlated with immunogenicity. Discussion: This study confirms results from previous studies on the low seroconversion rates in patients with CLL vaccinated with the BNT162b2 mRNA Covid-19 vaccine and on the detrimental effect of advanced disease and multiple treatment lines on seroconversion, while it is suggested that treatment with venetoclax may offer a chance for higher antibody titers, suggesting a treatment strategy change during the pandemic provided that this result is confirmed by larger studies specifically designed to address this issue.

The prognostic significance of chromosome 17 abnormalities in patients with myelodysplastic syndrome treated with 5-azacytidine: Results from the Hellenic 5-azacytidine registry.

In patients with myelodysplastic syndrome (MDS), the prognostic significance of chromosome 17 abnormalities has not yet been fully elucidated, except for isochromosome 17q that has been characterized as an intermediate risk abnormality in the Revised International Prognostic Scoring System (IPSS-R). To further characterize the prognostic significance of chromosome 17 abnormalities we analyzed the hematologic and prognostic characteristics of 548 adult patients with MDS treated with 5-azacytidine through the Hellenic 5-azacytidine registry and found 32 patients with a chromosome 17 abnormality (6 with i[17q], 15 with -17, 3 with add[17p] and the rest with other rarer abnormalities, mostly translocations). The presence of a chromosome 17 abnormality was correlated with poor prognostic features (high IPSS, IPSS-R, and WPSS scores) and a low overall survival rate (15.7 vs 36.4 months for patients without chromosome 17 abnormalities, Kaplan-Meier, Log Rank P < 0.00001), but these results were confounded by the fact that most (92.3%) of the cases with a chromosome 17 abnormality (with the exception of i(17q) that was found in all cases as an isolated abnormality) were found in the context of a complex karyotype. Nevertheless, one should not ignore the contribution of chromosome 17 abnormalities to the prognostic significance of a complex karyotype since 33.8% of complex karyotypes encompassed a chromosome 17 abnormality.

Detection of CALR Mutations Using High Resolution Melting Curve Analysis (HRM-A); Application on a Large Cohort of Greek ET and MF Patients.

BACKGROUND AND OBJECTIVES: Somatic mutations in the calreticulin gene (CALR) are detected in approximately 70% of patients with essential thrombocythemia (ET) and primary or secondary myelofibrosis (MF), lacking the JAK2 and MPL mutations. To determine the prevalence of CALR frameshift mutations in a population of MPN patients of Greek origin, we developed a rapid low-budget PCR-based assay and screened samples from 5 tertiary Haematology units. This is a first of its kind report of the Greek patient population that also disclosed novel CALR mutants. METHODS: MPN patient samples were collected from different clinical units and screened for JAK2 and MPL mutations after informed consent was obtained. Negative samples were analyzed for the presence of CALR mutations. To this end, we developed a modified post Real Time PCR High-Resolution Melting Curve analysis (HRM-A) protocol. Samples were subsequently confirmed by Sanger sequencing. RESULTS: Using this protocol we screened 173 MPN, JAK2 and MPL mutation negative, patients of Greek origin, of whom 117 (67.63%) displayed a CALR exon nine mutation. More specifically, mutations were detected in 90 out of 130 (69.23%) essential thrombocythaemia cases (ET), in 18 out of 33 (54.55%) primary myelofibrosis patients (pMF) and in 9 out of 10 (90%) cases of myelofibrosis secondary to ET (post-ET sMF). False positive results were not detected. The limit of detection (LoD) of our protocol was 2%. Furthermore, our study revealed six rare novel mutations which are to be added in the COSMIC database. CONCLUSIONS: Overall, our method could rapidly and cost-effectively detect the mutation status in a representative cohort of Greek patients; the mutation make-up in our group was not different from what has been published for other national groups.

Isolated skeletal muscle recurrence of an originally nodal diffuse large B cell lymphoma: A case report and review of the literature.

RATIONALE: Diffuse large B cell lymphoma (DLBCL) is a malignancy of the B cells with extranodal primary involvement being estimated at 30% to 40% of cases. Primary skeletal muscle presentation of DLBCL is extremely rare, with an estimated incidence of about 0.5% of extranodal lymphomas, presenting mostly in the lower extremities. The possible mechanisms of muscle involvement of DLBCL include primary extranodal disease, extension from adjacent organs (such as lymph nodes) or disseminated disease. PATIENT CONCERNS: We report a case of a 70-year-old woman with an advanced initially nodal DLBCL, treated with R-CHOP, that presented with an enlargement of her left thigh and restricted mobility 3 months after completion of chemotherapy. Imaging studies were performed, which showed possible infiltration of the muscles of the left thigh, without any nodal disease present. DIAGNOSES: Muscle biopsy documented the recurrence of the lymphoma at the left thigh. INTERVENTIONS: The patient started second-line treatment with gemcitabine and vinorelbine. OUTCOMES: A partial response was achieved after the first cycle. LESSONS: The remarkable element lies in the reappearance of the lymphoma at the left thigh muscles, with no radiographic or clinical evidence of involvement of lymph nodes, despite the extensive lymph node disease at initial presentation. The further management of such recurrences remains to be clarified, as the odd biological behavior of the malignant cells dictates a special handling of the disease.

Hypereosinophilic Syndrome as a Rare Cause of Reversible Biventricular Heart Failure.

Hypereosinophilic syndrome is a rare entity that can develop secondary to overproduction of eosinophilopoietic cytokines or as idiopathic disease. Cardiac involvement, which occurs often, is divided into 3 stages, the latter of which is nonreversible and leads to severe heart failure. Early detection and treatment of the syndrome is essential. For this reason, genetic testing for the FIP1L1-PDGFRA fusion gene has recently been added to the diagnostic algorithm. Patients with this mutation are at increased risk for the development of cardiac involvement and typically respond to treatment with the tyrosine kinase inhibitor imatinib mesylate.

Prognostic Implication of the Absolute Lymphocyte to Absolute Monocyte Count Ratio in Patients With Classical Hodgkin Lymphoma Treated With Doxorubicin, Bleomycin, Vinblastine, and Dacarbazine or Equivalent Regimens.

Low absolute lymphocyte count (ALC) to absolute monocyte count (AMC) ratio (ALC/AMC) is an independent prognostic factor in Hodgkin lymphoma (HL), but different cutoffs (1.1, 1.5, and 2.9) have been applied. We aimed to validate the prognostic significance of ALC/AMC in 537 homogenously treated (doxorubicin, bleomycin, vinblastine, and dacarbazine or equivalents ± radiotherapy) classical HL patients at various cutoffs. The median ALC/AMC was 2.24 (0.44-20.50). The median AMC was 0.653 × 10(9)/L (0.050-2.070). Lower ALC/AMC was associated with established markers of adverse prognosis. In total, 477 (89%), 418 (78%), and 189 (35%) patients had an ALC/AMC ratio of ≥1.1, ≥1.5, and ≥2.9; respectively; 20% had monocytosis (≥0.9 × 10(9)/L). Ten-year time to progression (TTP) was 77% versus 55% for patients with ALC/AMC ≥1.1 and <1.1 (p = .0002), 76% versus 68% for ALC/AMC ≥1.5 and <1.5 (p = .049), 77% versus 73% for ALC/AMC ≥2.9 and <2.9 (p = .35), and 79% versus 70% for ALC/AMC ≥2.24 and <2.24 (p = .08), respectively. In stages ΙΑ/ΙΙΑ and in patients ≥60 years old, ALC/AMC had no significant effect on TTP. In advanced stages, ALC/AMC was significant only at the cutoff of 1.1 (10-year TTP 67% vs. 48%; p = .016). In younger, advanced-stage patients, the differences were more pronounced. In multivariate analysis of TTP, ALC/AMC < 1.1 (p = .007) and stage IV (p < .001) were independent prognostic factors; ALC/AMC was independent of International Prognostic Score in another model. ALC/AMC was more predictive of overall survival than TTP. At the cutoff of 1.1, ALC/AMC had independent prognostic value in multivariate analysis. However, the prognostically inferior group comprised only 11% of patients. Further research is needed prior to the widespread use of this promising marker.

Expression, prognostic significance and mutational analysis of protein tyrosine phosphatase SHP-1 in chronic myeloid leukemia.

The protein tyrosine phosphatase SHP-1 dephosphorylates BCR-ABL1, thereby serving as a potential control mechanism of BCR-ABL1 kinase activity. Pathways regulating SHP-1 expression, which could be exploited in the therapeutics of TKI-resistant chronic myeloid leukemia (CML), remain unknown. Moreover, the questions of whether there is any kind of SHP-1 deregulation in CML, contributing to disease initiation or evolution, as well as the question of prognostic significance of SHP-1, have not been definitively answered. This study shows moderately lower SHP-1 mRNA expression in chronic phase CML patients in comparison to healthy individuals and no change in SHP-1 mRNA levels after successful TKI treatment. Mutational analysis of the aminoterminal and phosphatase domains of SHP-1 in patients did not reveal genetic lesions. This study also found no correlation of SHP-1 expression at diagnosis with response to treatment, although a trend for lower SHP-1 expression was noted in the very small non-responders' group of the 3-month therapeutic milestone.

5-Azacytidine in the Treatment of Intermediate-2 and High-risk Myelodysplastic Syndromes and Acute Myeloid Leukemia. A Five-year Experience with 44 Consecutive Patients.

BACKGROUND/AIM: The hypomethylating agent 5-azacytidine has been the standard-of-care for patients with higher-risk myelodysplastic syndrome (MDS) during the past few years. Its efficacy has been proven in large clinical trials, and its safety has been shown to be superior to that of conventional treatments. PATIENTS AND METHODS: We conducted a retrospective study on the efficacy and safety of 5-azacytidine in 44 consecutive patients with MDS and acute myeloid leukemia treated with 5-azacytidine during a 63-month period. We recorded the clinical and laboratory characteristics of the patients and we analyzed the response to treatment, overall survival and adverse events during treatment. RESULTS: The median overall survival was 13 months, while serious adverse events consisted mostly of neutropenic infections. CONCLUSION: We reached two possibly valuable conclusions: Younger patients (<73 years), as well as patients receiving treatment at longer than 28-day intervals had a significantly higher overall survival.

An adult patient with systemic mastocytosis and B-acute lymphoblastic leukemia.

Mastocytosis is a myeloproliferative neoplasm characterized by clonal expansion of abnormal mast cells, ranging from the cutaneous forms of the disease to mast cell leukemia. In a significant proportion of patients, systemic mastocytosis (SM) coexists with another hematologic malignancy, termed systemic mastocytosis with an associated hematologic nonmast cell lineage disorder (SM-AHNMD). Despite the pronounced predominance of concomitant myeloid neoplasms, the much more unusual coexistence of lymphoproliferative diseases has also been reported. Imatinib mesylate (IM) has a role in the treatment of SM in the absence of the KITD816V mutation. In the setting of SM-AHNMD, eradicating the nonmast cell malignant clone greatly affects prognosis. We report a case of an adult patient with SM associated with B-lineage acute lymphoblastic leukemia (B-ALL). Three cases of concurrent adult ALL and mastocytosis have been reported in the literature, one concerning SM and two concerning cutaneous mastocytosis (CM), as well as six cases of concomitant CM and ALL in children.

Survivin messenger RNA levels in Epstein-Barr virus-positive patients with leukemic low-grade B-cell lymphomas expressing the latent membrane protein 1: evidence of apoptotic function?

BACKGROUND: Epstein-Barr virus (EBV) is a ubiquitous pathogen that chronically infects B lymphocytes and is implicated in the pathogenesis of lymphoproliferative diseases. Latent membrane protein 1 (LMP1), the major oncoprotein of the virus, has been shown to inhibit apoptosis and trigger survivin expression in malignant cell lines. LMP1 expression has been detected in patients with chronic lymphocytic leukemia, but its properties have not been studied in patients with low-grade B-cell lymphomas. Recent data show that LMP1 can simultaneously induce and inhibit apoptosis in B cells. We detected LMP1 messenger RNA (mRNA) in patients with leukemic low-grade B-cell lymphoma and correlated the expression of the antiapoptotic molecule survivin to that of LMP1 in this group of patients. PATIENTS AND METHODS: Peripheral whole blood from 64 patients with low-grade B-cell lymphoma was tested by quantitative reverse transcriptase-polymerase chain reaction (PCR) for the presence of the BXLF-1 gene of EBV, and positive samples were tested by conventional PCR for LMP1 expression. Accordingly, survivin mRNA levels were measured by quantitative reverse transcriptase PCR in all samples and compared between LMP1-positive (LMP1(+)) and LMP1(-) patients. RESULTS: The BXLF-1 gene was detected in 27 of 64 patients (42%). LMP1 was expressed in 22 of 27 (81%) EBV(+) patients. Survivin expression was found to be 6.36 times higher in LMP1(-) patients than in LMP1(+) patients (P = .008). CONCLUSION: Our results imply that in patients with non-EBV-related leukemic low-grade B-cell lymphoma, LMP1 expression is possibly correlated to apoptosis, as indicated by the lower survivin mRNA levels in LMP1(+) patients.

Systemic mastocytosis accompanied by a non-secretory plasma cell dyscrasia and nephrotic syndrome-level proteinuria in a 76-year-old patient.

We report here the interesting case of a 76-year-old man with severe proteinuria who was diagnosed with systemic mastocytosis accompanied by a clonal non-mast-cell lineage haematological disorder (a non-secretory plasma cell dyscrasia). This is a unique report of systemic mastocytosis with a non-secretory plasma cell dyscrasia and nephrotic syndrome. The pathophysiological relevance between these entities along with the probability of occult amyloidosis is discussed.

Evidence for regulation of oxidative stress by latent membrane protein 1 oncoprotein in patients with low-grade leukemic B cell lymphoma with latent Epstein-Barr virus infection.

The role of latent Epstein-Barr virus (EBV) infection in the pathogenesis of low-grade B cell non-Hodgkin lymphoma (B-NHL) has not been studied. We therefore investigated the incidence of latent EBV infection in a group of patients with leukemic low-grade B-NHL, as well as the incidence of viral latent membrane protein 1 (LMP1) oncoprotein expression in the same patient group. Furthermore, in an attempt to elucidate the role of this viral oncoprotein in non-EBV-related lymphomas, we correlated the expression of LMP1 with the level of oxidative stress, a parameter related to apoptosis. In the present study we detected lower levels of oxidative stress in the sera of LMP1-positive patients. This possibly implies an anti-apoptotic role of this viral oncoprotein in low-grade B cell lymphomas. However, LMP1 expression status did not affect expression of the major anti-apoptotic gene BCL-2.

Routine application of a novel MLPA-based first-line screening test uncovers clinically relevant copy number aberrations in haematological malignancies undetectable by conventional cytogenetics.

OBJECTIVE: The presence of numerical and/or structural chromosomal abnormalities is a frequent finding in clonal hematopoietic malignant disease, typically diagnosed through routine karyotyping and/or fluorescent in situ hybridization (FISH) analysis. Recently, the application of array comparative genomic hybridization (aCGH) has uncovered many new cryptic genomic copy number imbalances, most of which are now recognized as clinically useful markers of haematological malignancies. In view of the limitations of both FISH and aCGH techniques, in terms of their routine application as a first line screening test, we designed a new multiple ligation-dependent probe amplification (MLPA) probemix for use in addition to classic karyotype analysis. METHODS: A novel MLPA probemix was developed to interrogate copy number changes involving chromosomal regions: 2p23-24 (MYCN, ALK), 5q32-34 (MIR145A, EBF1, MIR146A), 6q21-27, 7p12.2 (IKZF1), 7q21-36, 8q24.21 (MYC), 9p24 (JAK2 V617F point mutation), 9p21.3 (CDKN2A/2B), 9p13.2 (PAX5), 10q23 (PTEN), 11q22.3 (ATM), 12p13.2 (ETV6), 13q14 (RB1, MIR15A, DLEU2, DLEU1), 17p13.1 (TP53), and 21q22.1 (RUNX1/AML1) and was applied to DNA extracted from 313 consecutive bone marrow patient samples, referred for routine karyotype analysis. RESULTS: More than half of the samples originated from newly investigated patients. We discovered clinically relevant genomic aberrations, involving a total of 24 patients (8%) all with a normal karyotype, which would have remained undiagnosed. DISCUSSION: Our data clearly indicate that routine application of this MLPA screening panel, as an adjunct to karyotype analysis, provides a sensitive, robust, rapid and low-cost approach for uncovering clinically important genomic abnormalities, which would have otherwise remained undetected.

Cessation of tyrosine kinase inhibitors in patients with chronic-phase chronic myelogenous leukemia following durable complete molecular response: a single center facing the dilemma.

Tyrosine kinase inhibitors (TKIs), namely imatinib mesylate (IM) and recently approved second-generation TKIs dasatinib and nilotinib, are currently considered the treatment of choice for newly-diagnosed chronic phase chronic myelogenous leukemia (CP-CML). Although treatment with TKIs has not yet been proven curative, it certainly accomplishes a sustained control of the disease in the vast majority of patients. More than a decade after the successful launching of IM in first-line treatment of CP-CML and the subsequent introduction of second-generation TKIs in this setting, the question of the possibility of TKI cessation in a specific subset of patients has emerged. Side-effects of TKIs, along with some patients' wish to abandon the drugs and the rising financial burden upon healthcare systems, have led to the dilemma whether IM can be safely withdrawn after achieving deep molecular remissions and which patients are suitable for this discontinuation. We examined the data of our patients with CML in search of potential canditates for cessation of TKI therapy and identified their characteristics. We also performed a thorough review of the relevant literature. Eight out of fifty patients were discriminated on grounds of sustained complete molecular response (CMR) exceeding 12 months, most of them with a low or intermediate Sokal score at diagnosis. The median interval from IM initiation to CMR was almost 2 years and the median duration of detected CMR reached 6.5 years. Based on the promising results of prospective clinical trials reporting successful cessation of treatment with TKIs on selected subgroups of patients, we decided to proceed to interruption of therapy in the specific subset of our patients and closely monitor their response.

Correlation of Fc-γ RIIA polymorphisms with latent Epstein-Barr virus infection and latent membrane protein 1 expression in patients with low grade B-cell lymphomas.

Fc-γ RIIA (CD32), a member of the family of Fc-γ receptors, participates in the phagocytosis of bound to antibody antigens. The effectiveness of this function varies for its several haplotypes, and it participates in the pathogenesis of viral infections, according to recent studies. The genetic locus of Fc-γ RIIA consists of two allelic genes: 131-Arg (R131) and 131-His (H131). Our aim was to correlate Fc-γ RIIA polymorphisms, by studying the prevalence of each allele using PCR-RFLPs (polymerase chain reaction-restriction fragment length polymorphisms), with latent Epstein-Barr virus (EBV) infection and the expression of latent membrane protein 1 (LMP1) in 40 patients with leukemic low grade B-cell lymphomas. R131 was found in 84.2% of EBV-positive patients, but only in 28.5% of EBV-negative patients (p = 0.001). A similar correlation was found for R131 and LMP1 expression (84.6% vs. 28.5%) (p = 0.002). Our results support the hypothesis that Fc-γ RIIA polymorphisms are a genetic risk factor for latent EBV infection and the expression of its oncogenic latency proteins.

Red cells with paroxysmal nocturnal hemoglobinuria-phenotype in patients with acute leukemia.

CD55 and CD59 are complement regulatory proteins that are linked to the cell membrane via a glycosyl-phosphatidylinositol anchor. They are reduced mainly in paroxysmal nocturnal hemoglobinuria (PNH) and in other hematological disorders. However, there are very few reports in the literature concerning their expression in patients with acute leukemias (AL). We studied the CD55 and CD59 expression in 88 newly diagnosed patients with AL [65 with acute non-lymphoblastic leukemia (ANLL) and 23 with acute lymphoblastic leukemia (ALL)] using the sephacryl gel test, the Ham and sucrose lysis tests and we compared the results with patients' clinical data and disease course. Eight patients with PNH were also studied as controls. Red cell populations deficient in both CD55 and CD59 were detected in 23% of ANLL patients (especially of M(0), M(2) and M(6) FAB subtypes), 13% of ALL and in all PNH patients. CD55-deficient erythrocytes were found in 6 ANLL patients while the expression of CD59 was decreased in only 3 patients with ANLL. No ALL patient had an isolated deficiency of these antigens. There was no correlation between the existence of CD55 and/or CD59 deficiency and the percentage of bone marrow infiltration, karyotype or response to treatment. However no patient with M(3), M(5), M(7) subtype of ANLL and mature B- or T-cell ALL showed a reduced expression of both antigens. The deficient populations showed no alteration after chemotherapy treatment or during disease course. This study provides evidence about the lower expression of CD55 and CD59 in some AL patients and the correlation with their clinical data. The possible mechanisms and the significance of this phenotype are discussed.