Rituximab, Cyclophosphamide and Dexamethasone (RCD) Chemoimmunotherapy for Relapsed Chronic Lymphocytic Leukaemia.

High doses of corticosteroids in combination with rituximab remain an alternative in the treatment in relapsed or refractory chronic lymphocytic leukaemia (CLL) in the current era of targeted therapies. This study retrospectively evaluates the efficacy of an RCD (rituximab, cyclophosphamide and dexamethasone) regimen in the treatment of 51 patients with relapsed CLL (median age, 72 years). Unfavourable prognostic features, such as Rai stage III/IV, unmutated IGHV, del11q, TP53 mutation/deletion, complex karyotype and bulky lymphadenopathy, were frequent. The overall response or complete remission was of 57% and 7%, respectively, and the median progression-free survival (PFS) was of 12.3 months, median time to next treatment 23.1 months and median overall survival 39.2 months. Significant independent predictors of shorter PFS were TP53 deletion/mutation, advanced Rai stage and ≥2 previous lines of treatment. The incidence of neutropenia grade ≥ 3 was of 13%. Serious (CTCAE grade 3-5) infections were found in 20% of patients. Steroid-induced diabetes or diabetes decompensation occurred in 20% patients. Treatment-related adverse events resulted in RCD dose reduction in 35% of patients. In comparison with a historical R-Dex patient group, the treatment response and/or toxicity in our group was largely similar. However, the substantial differences in the baseline clinical characteristics of the groups may affect this comparison. In conclusion, the RCD regimen is an active, time-limited therapeutic strategy for elderly patients with relapsed CLL. Further, the results of our analysis indicate that the addition of cyclophosphamide to the R-Dex regimen maintains a similar efficacy, even after 50% reduction in the dexamethasone dose.

Real-world data on efficacy and safety of obinutuzumab plus chlorambucil, rituximab plus chlorambucil, and rituximab plus bendamustine in the frontline treatment of chronic lymphocytic leukemia: The GO-CLLEAR Study by the Czech CLL Study Group.

Until recently, a combination of anti-CD20 antibody plus less intensive chemotherapy was a standard of care in elderly population with previously untreated chronic lymphocytic leukemia (CLL). The aim of this observational study was to retrospectively assess efficacy and safety of obinutuzumab + chlorambucil (G-Clb), rituximab + chlorambucil (R-Clb), and bendamustine + rituximab (BR) given as the frontline therapy within routine practice. The final analyzed dataset included 398 consecutive CLL patients from 10 hematology centers cooperating within the Czech CLL Study Group: 63 treated with G-Clb, 78 with R-Clb, and 257 with BR. There were no significant differences in prognostic and predictive markers among the groups. On the contrary, median age at the start of therapy and cumulative illness rating scale (CIRS) score was significantly higher in R-Clb group. Obinutuzumab plus chlorambucil regimen was preferably offered to elderly patients (compared to BR) with less severe comorbidities and lower CIRS score (compared to R-Clb). A time period when a treatment was indicated had also a strong impact on the choice of the regimen. The overall response rate reached 76% (30% complete remissions, CRs) in G-Clb, 75% (22% CRs) in R-Clb, and 85% (47% CRs) in BR group. Median event-free survival was 49.0 months for G-Clb, 20.3 months for R-Clb, and 37.0 months for BR group. Neutropenia grade ≥ 3 developed in 43% of G-Clb, 31% of R-Clb and in 49% of BR patients, grade ≥ 3 infections were recorded in 17% of G-Clb, 6.4% of R-Clb, and 17% of BR patients. In conclusion, real-world therapeutic activity of G-Clb appears to be at least comparable to prospective clinical trial data. R-Clb yields relatively good results in very old and severely comorbid patients.

Identification of a four-gene methylation biomarker panel in high-grade serous ovarian carcinoma.

Background The lack of effective biomarkers for the screening and early detection of ovarian cancer (OC) is one of the most pressing problems in oncogynecology. Because epigenetic alterations occur early in the cancer development, they provide great potential to serve as such biomarkers. In our study, we investigated a potential of a four-gene methylation panel (including CDH13, HNF1B, PCDH17 and GATA4 genes) for the early detection of high-grade serous ovarian carcinoma (HGSOC). Methods For methylation detection we used methylation sensitive high-resolution melting analysis and real-time methylation specific analysis. We also investigated the relation between gene hypermethylation and gene relative expression using the 2-ΔΔCt method. Results The sensitivity of the examined panel reached 88.5%. We were able to detect methylation in 85.7% (12/14) of early stage tumors and in 89.4% (42/47) of late stage tumors. The total efficiency of the panel was 94.4% and negative predictive value reached 90.0%. The specificity and positive predictive value achieved 100% rates. Our results showed lower gene expression in the tumor samples in comparison to control samples. The more pronounced downregulation was measured in the group of samples with detected methylation. Conclusions In our study we designed the four-gene panel for HGSOC detection in ovarian tissue with 100% specificity and sensitivity of 88.5%. The next challenge is translation of the findings to the less invasive source for biomarker examination, such as plasma. Our results indicate that combination of examined genes deserve consideration for further testing in clinical molecular diagnosis of HGSOC.

Improved laboratory diagnostics of Streptococcus pneumoniae in respiratory tract samples through qPCR.

The aim of this study was to test the detection performance of the cpsA, lytA and ply genes through qPCR in the identification of Streptococcus pneumoniae in respiratory tract samples. Specificity was tested on a panel of 128 streptococci and other bacteria DNA samples. The qPCR assay was tested on a total of 51 respiratory tract samples from patients with community-acquired pneumonia (CAP). The specificity of the cpsA, lytA and ply genes was 100%, 100%, and 86%, respectively. The quantitative assessment, based on lytA, determined a cutoff value of ~2x104, 4x102 and 4x102 DNA copies per 1 mL of valid sputum, tracheal aspirate and bronchial aspirate samples, respectively. The results from the present study suggest that qPCR detection of all three genes would be optimal in the accurate detection of Streptococcus pneumoniae.

Sysmex-derived COVID-19 prognostic score as an early prognostic marker for severity of the COVID-19 disease.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a life-threatening disease with a heterogeneous course. Even some young patients are at increased risk of severe course or death, as they can face severe complications. It would be very useful to have a cheap and easily available marker to predict COVID-19 course in the early stages of the disease. The COVID-19 prognostic score could be a very useful clinical indicator available at the time of primary contact with the patient. METHODS: The COVID-19 prognostic score and the clinical condition together with selected laboratory parameters were evaluated in patients with respiratory tract infection and a positive PCR test for the SARS-CoV-2 during the first contact with the patient. Prognostic significance was evaluated using receiver operating characteristic curves (ROC) and area under the curve (AUC). Selected parameters of the blood count and hemostasis, as well as selected biochemical indicators, were examined too. RESULTS: Thirty-seven of 164 patients developed serious symptoms. The COVID-19 score had one of the highest AUC values (0.855) of all markers. The highest combination of sensitivity (91.9%) and specificity (71.7%) for identifying patients with a subsequent moderate and severe course of the disease was achieved at the threshold 1.5. The predictive value of a negative test is beneficial too (0.968). CONCLUSIONS: The COVID-19 prognostic score is a promising indicator stratifying patients with COVID-19 into prognostic groups at the time of the first contact, thus allowing the timely provision of increased care in patients at high risk of severe development.

Intensive Care Infection Score (ICIS) is elevated in patients with moderate and severe COVID-19 in the early stages of disease.

BACKGROUND: Coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus is still a very dangerous and life-threatening disease with an extremely heterogeneous course. Older patients and those with comorbidities are at increased risk of death from the disease but young patients can develop potentially lethal complications too. For those reasons, numerous recent studies focus on the analysis of markers associated with early assessment of COVID-19 prognosis. Previous publications provided evidence for the Intensive Care Infection Score (ICIS) as an easy to use tool to assess the risk for bacterial infection in ICU patients based on a combination of haematologic parameters. This study evaluated the performance of ICIS as a prognostic marker of stages of disease in COVID-19 patients. METHODS: A total of 205 COVID-19 patients admitted to the University Hospital Hradec Kralove, Czech Republic, with symptoms of respiratory tract infection and a positive RT-PCR test for SARS-CoV-2 virus were enrolled in this study. Forty-nine patients developed mild COVID-19 symptoms (no oxygen therapy needed), 156 patients developed moderate or severe symptoms (supplemental oxygen therapy or death). RESULTS: ICIS predicted the mild or moderate/severe course with the highest AUC (0.773). The cut-off value (ICIS = 3.5) was selected as the value with the highest Youden index (0.423). The cut-off value could predict a mild or moderate/severe course of the disease with the highest specificity (77.6%) and positive predictive value (90.2%) of all markers used in this study. Sensitivity was 64.7%. CONCLUSION: ICIS is a reliable, cheap, fast and simply interpretable score for the early identification of moderate/severe course of COVID-19 in an early stage of the disease. ICIS> 3 predicts a severe course of the disease with high specificity and positive predictive value.

Significant change in ZAP-70 expression during the course of chronic lymphocytic leukemia.

INTRODUCTION: It is widely accepted that expression of ZAP-70 in chronic lymphocytic leukemia (CLL) remains stable in time. However, data supporting this notion are surprisingly scarce. Therefore, we assessed expression of ZAP-70 in serial samples taken during the course of the disease. PATIENTS AND METHODS: We studied 44 patients with CLL diagnosed according to NCI-WG criteria (34 men, 10 women, median age 62, range, 36-81). A total of 104 samples were examined; all patients had at least two measurements. Median interval between the first and the second sample was 13 months (range, 2-36). ZAP-70 expression was detected by flow cytometry using phycoerythrin-conjugated monoclonal antibody clone 1E7.2 and negative isotype control. Twenty percent of positive cells were considered as the threshold of positivity. RESULTS: Significant change in ZAP-70 expression (i.e. from positivity to negativity and vice versa) was detected in 15/44 patients (34%). Interestingly, 7/8 patients whose ZAP-70 expression converted to positivity had unmutated IgVH genes. In addition, the conversion was accompanied by clinical progression or relapse in all but one patient. On the other hand, 5/7 patients with loss of ZAP-70 had stable clinical course. One patient became ZAP-70-negative during treatment with prednisone for autoimmune hemolytic anemia. CONCLUSIONS: In contrast to commonly accepted opinion, significant change in ZAP-70 expression in time was detected in a substantial proportion of our patients with CLL. While the conversion to ZAP-70 negativity was found predominantly in patients with stable disease, change to positivity was typical in patients with unmutated IgVH genes at the time of progression or relapse. Based on our pilot results, repeated assessment of ZAP-70 expression might be especially useful at the time of progression or relapse in patients who were initially ZAP-70-negative.

Biological prognostic markers in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most frequent leukemic disease of adults in the Western world. It is remarkable by an extraordinary heterogeneity of clinical course with overall survival ranging from several months to more than 15 years. Classical staging sytems by Rai and Binet, while readily available and useful for initial assessment of prognosis, are not able to determine individual patient's ongoing clinical course of CLL at the time of diagnosis, especially in early stages. Therefore, newer biological prognostic parameters are currently being clinically evaluated. Mutational status of variable region of immunoglobulin heavy chain genes (IgVH), cytogenetic aberrations, and both intracellular ZAP-70 and surface CD38 expression are recognized as parameters with established prognostic value. Molecules regulating the process of angiogenesis are also considered as promising markers. The purpose of this review is to summarize in detail the specific role of these prognostic factors in chronic lymphocytic leukemia.

Herpes simplex virus resistant to acyclovir: A single-centre experience from the Czech Republic.

INTRODUCTION AND AIM: Infections caused by herpes simplex viruses (HSV) are frequent in the human population. Because of the widespread use of long-term treatment or prophylaxis by anti-herpetic antivirals in various specific medical contexts (immunosuppression, recurrent infections), the level of antiviral resistance is increasing. According to previous studies, there is a low resistance level in immunocompetent populations but a relatively high level in populations with immunodeficiency. However, there has been no study from the Czech Republic. This study presents results of a single-centre retrospective study from the Czech Republic. MATERIALS AND METHODS: Deep frozen DNA from patients with suspected clinical antiviral failure over a long time period (2009-2016) - a total of 15 isolates of HSV1 and seven of HSV2 - were examined for the presence of mutations associated with antiviral resistance. Sequence analysis was performed using an ABI PRISM 3500xL Genetic Analyzer (Applied Biosystems®). RESULTS: There were no mutations associated with resistance to antivirals inside the UL23 gene in HSV1 isolates. However, resistant mutation D672N (nucleotide change G2014A) was found inside the UL30 gene in seven of the isolates. One mutation associated with resistance to acyclovir (M183stop) was found inside the UL23 gene in one HSV2 isolate. Resistant mutation E678G (nucleotide change A2033G) was identified inside the UL30 gene in six of the HSV2 isolates. CONCLUSIONS: This study confirmed the presence of resistance mutations within the Czech population, but it will be necessary to examine a higher number of isolates for further conclusions.

First-line imatinib in elderly patients with chronic myeloid leukaemia from the CAMELIA registry: Age and dose still matter.

We retrospectively evaluated the role of age and dosage in 372 CML patients (170 women, 202 men) treated with first-line imatinib (IMA) from the records of the CAMELIA registry. The median follow-up of the patients was 82.3 (18.0-177.3) months. The treatment results of 80 elderly patients aged over 65 years at diagnosis were compared in analysis "A" with those of 292 younger patients and in analysis "B" with those of 90 patients younger than 40 and 202 patients aged 40-64. The elderly patients had statistically adverse values of the Sokal, ELTS, and ECOG scores and Charlson comorbidity index in both analyses (p from = 0.012 to ≤ 0.001). Despite a more frequent use of a daily dose lower than 400 mg - in 31 elderly patients (38.8%) than in 45 younger ones (15.4%) (p < 0.001), there were no statistically significant differences in the achievement of optimal haematological, cytogenetic, and molecular responses according to the ELN criteria in both the analyses, A and B. The comparisons of overall survival with CML-related death (OSCML) and event-free survival (EFS) were insignificant inanalysis A (p = 0.07 and 0.396, respectively) but progression-free survival (PFS) differed significantly (p = 0.007). In analysis B OSCML and PFS differed significantly (p = 0.027 and 0.003) but EFS was similar (p = 0.351). Elderly patients with a sustained dose of IMA of 400 mg/day have insignificantly better OS, PFS, and EFS compared to patients treated with a lower dosage of IMA. The results in the treatment of the elderly CML patients were comparable with those of the younger ones in terms of the probabilities of the achievement of optimal ELN responses. However, the results for the survival probabilities were influenced by age and the IMA dosage.

IDH1 and IDH2 mutations in patients with acute myeloid leukemia: Suitable targets for minimal residual disease monitoring?

OBJECTIVES: Molecular screening plays a major role in prognostic categorization and subsequent definition of treatment strategies for acute myeloid leukemia. The possibility of using IDH1/2 mutations as a marker for the monitoring of minimal residual disease (MRD) is still under investigation and remains unclear. METHODS: In this retrospective study, we evaluated 90 patients with de novo AML using Sanger sequencing (exon 4, IDH1 and IDH2). For subsequent MRD monitoring were used both methods, massive parallel sequencing and droplet digital PCR (ddPCR). RESULTS: We identified 22 patients (24%) who harboured mutations in IDH1 or IDH2 genes. Fourteen (64%) of them had other commonly used MRD markers (insertion in NPM1 and partial tandem duplication of MLL, MLL-PTD). Eight of the 22 patients had IDH1 mutations, 13 had IDH2 mutations and 1 had both IDH1 and IDH2 mutations. In our cohort, this IDH1/2 marker responded to the treatment in all of the patients and reflected the onset of the relapse very well. NPM1 mutation based MRD monitoring was more sensitive and predicted relapse earlier but IDH1/2 based monitoring was more sensitive than a method based on MLL-PTD. Both massive parallel sequencing and ddPCR were competent to monitor MRD using IDH1/2. Nevertheless, ddPCR was able to achieve a higher sensitivity in some cases and moreover this method can analyse a single sample without significant price increases. CONCLUSION: Given these data, we conclude that IDH1/2 mutations can be used as a reliable and cost-effective marker for MRD monitoring.

The significance of enzyme and transporter polymorphisms for imatinib plasma levels and achieving an optimal response in chronic myeloid leukemia patients.

INTRODUCTION: Imatinib mesylate is the drug of choice for patients with chronic myeloid leukemia (CML). Imatinib pharmacokinetics is affected by a number of transport proteins and enzymes. MATERIAL AND METHODS: In the present study we evaluated the association of eight polymorphisms in the seven genes CYP3A5*3 (rs776746), CYP3A4*1 (rs2740574), CYP2C9*3 (rs1057910), SLC22A1 (rs683369), ABCB1 (rs1045642, rs1128503), ABCG2 (rs2231142) and ABCC2 (rs717620) with imatinib plasma level and achieving an optimal clinical response in 112 CML patients (53 men and 59 women). RESULTS: No association was found between the examined polymorphisms in rs776746, rs2740574, rs1057910, rs683369, rs1045642, rs1128503, rs2231142, rs717620 and the achieved imatinib plasma level. The influence of rs776746 (CYP3A5*3) on the achievement of a complete cytogenetic response (CCyR) at 6 months was borderline non-significant (p = 0.06). Furthermore, no association was demonstrated between rs776746 polymorphisms and the achievement of a major molecular response (MMR) at 12 or 18 months. Polymorphisms rs776746, rs2740574, rs1057910, rs683369, rs1045642, rs1128503, rs2231142, rs717620 showed no impact on the optimal therapeutic response. CONCLUSIONS: Despite the results of some other studies, no other polymorphism we analyzed was associated with imatinib plasma level or clinical response. The treatment outcomes cannot be predicted using the candidate gene approach and treatment decisions cannot be made according to the polymorphisms investigated in this study.

COBLL1, LPL and ZAP70 expression defines prognostic subgroups of chronic lymphocytic leukemia patients with high accuracy and correlates with IGHV mutational status.

The clinical course of chronic lymphocytic leukemia (CLL) is highly variable. Patients with unmutated IGHV (U-CLL) usually progress rapidly, whereas patients with mutated IGHV (M-CLL) have a more indolent disease. The expression of several genes correlates closely with the IGHV mutational status and could be used to assess prognosis in CLL. We analyzed the prognostic relevance of COBLL1, LPL, and ZAP70 gene expression, which correlated with IGHV mutational status (p < 0.0001), in 117 CLL patients and established a prognostic parameter dividing the tested cohort according to the disease aggressiveness. Our prognostic parameter was validated on an independent cohort of 161 CLL patients and achieved a high accuracy (94%). Patients divided according to the prognostic parameter differ in overall survival and time to first treatment (p < 0.0001, HR = 2.300/5.970, 95% CI: 1.587-3.450/4.621-15.86). Our approach provides a reliable alternative method to prognosis assessment via IGHV mutational status analysis.

Genotypes of SLC22A4 and SLC22A5 regulatory loci are predictive of the response of chronic myeloid leukemia patients to imatinib treatment.

BACKGROUND: Through high-throughput next-generation sequencing of promoters of solute carrier and ATP-binding cassette genes, which encode drug transporters, we aimed to identify SNPs associated with the response to imatinib administered for first-line treatment of patients with chronic myeloid leukemia. METHODS: In silico analysis using publicly available databases was done to select the SLC and ABC genes and their promoters for the next-generation sequencing. SNPs associated with the imatinib response were identified using Fisher's exact probability tests and subjected to the linkage disequilibrium analyses with regulatory loci of concerned genes. We analyzed cumulative achievement of major molecular response and probability of event free survival in relation to identified SNP genotypes in 129 CML patients and performed multivariate analysis for determination of genotypes as independent predictors of outcome. Gene expression analysis of eight cell lines naturally carrying different genotypes was performed to outline an impact of genotypes on the gene expression. RESULTS: We observed significant differences in the frequencies of the rs460089-GC and rs460089-GG (SLC22A4) genotypes among rs2631365-TC (SLC22A5) genotype carriers that were associated with optimal and non-optimal responses, respectively. Loci rs460089 and rs2631365 were in highly significant linkage disequilibrium with 12 regulatory loci in introns of SLC22A4 and SLC22A5 encoding imatinib transporters. Genotype association analysis with the response to imatinib indicated that rs460089-GC carriers had a significantly higher probability of achieving a stable major molecular response (BCR-ABL1 transcript level below or equal to 0.1% in the international scale). In contrast, the rs460089-GG represented a risk factor for imatinib failure, which was significantly higher in rs460089-GG_rs2631365-TC carriers. CONCLUSIONS: This exploratory study depicted potentially important genetic markers predicting outcome of imatinib treatment, which may be helpful for tailoring therapy in clinical practice.

Five years of experience with rituximab plus high-dose dexamethasone for relapsed/refractory chronic lymphocytic leukemia.

INTRODUCTION: High-dose methylprednisolone (HDMP) in combination with rituximab is active in the treatment of relapsed/refractory chronic lymphocytic leukemia (CLL), but serious infections are frequent. Recently published data suggested that high-dose dexamethasone might be equally effective as HDMP despite a lower cumulative dose. MATERIAL AND METHODS: We performed retrospective analysis of 60 patients with relapsed/refractory CLL (median age: 66 years; range: 37-86) treated with rituximab plus dexamethasone (R-dex) at a single tertiary center between September 2008 and October 2012. The schedule of R-dex consisted of rituximab 500 mg/m(2) i.v. day 1 (375 mg/m(2) in cycle 1) and dexamethasone 40 mg orally on days 1-4 and 10-13 repeated every 3 weeks for a maximum of 8 cycles. Unfavorable prognostic features were frequent (Rai stages III/IV in 67%, unmutated IgVH 82%, del 11q 43%, TP53 mutation/deletion 23%, bulky lymphadenopathy 58% of patients). RESULTS: Overall response (OR)/complete remission (CR) was achieved in 75/3%. At the median follow-up of 21 months, median progression-free survival (PFS) was 8 months, median time to next treatment 12.9 months and median overall survival 25.5 months. Refractoriness to fludarabine (p = 0.04) and age ≥ 65 years (p = 0.03) were significant predictors of shorter PFS. R-dex was successfully used for debulking before allogenic stem cell transplantation in 7 patients (12%). Serious (CTCAE grade III/IV) infections occurred in 27% of patients; 20% of patients developed steroid diabetes requiring temporary short-acting insulin. CONCLUSIONS: Our results show that R-dex is an active and well-tolerated regimen for patients with relapsed/refractory CLL; however, major infections remain frequent despite combined antimicrobial prophylaxis.

Cervical microbiota in women with preterm prelabor rupture of membranes.

OBJECTIVE: To analyze the cervical microbiota in women with preterm prelabor rupture of membranes (PPROM) by pyrosequencing and to document associations between cervical microbiota, cervical inflammatory response, microbial invasion of the amniotic cavity (MIAC), histological chorioamnionitis, and intraamniotic infection (IAI). STUDY DESIGN: Sixty-one women with singleton pregnancies complicated by PPROM were included in the study. Specimens of cervical and amniotic fluid were collected on admission. The cervical microbiota was assessed by 16S rRNA gene sequencing by pyrosequencing. Interleukin (IL)-6 concentration in the cervical fluid and amniotic fluid was measured by ELISA and lateral flow immunoassay, respectively. RESULTS: Four bacterial community state types [CST I (Lactobacillus crispatus dominated), CST III (Lactobacillus iners dominated), CST IV-A (non-Lactobacillus bacteria dominated), and CST IV-B (Gardnerella vaginalis and Sneathia sanguinegens dominated)] were observed in the cervical microbiota of women with PPROM. Cervical fluid IL-6 concentrations differed between CSTs (CST I = 145 pg/mL, CST III = 166 pg/mL, CST IV-A = 420 pg/mL, and CST IV-B = 322 pg/mL; p = 0.004). There were also differences in the rates of MIAC, of both MIAC and histological chorioamnionitis, and of IAI among CSTs. No difference in the rate of histological chorioamnionitis was found among CSTs. CONCLUSIONS: The cervical microbiota in PPROM women in this study was characterized by four CSTs. The presence of non-Lactobacillus CSTs was associated with a strong cervical inflammatory response and higher rates of MIAC, both MIAC and histological chorioamnionitis, and IAI representing a PPROM subtype with pronounced inflammation. CST I represents the dominant type of PPROM with a low rate of MIAC, IAI, and the combination of MIAC and histological chorioamnionitis.