Alzbeta Zavrelova

A method to identify new molecular markers for assessing minimal residual disease in acute leukemia patients

Acute leukemias (AL) comprise a heterogeneous group of hematologic malignancies, and individual patient responses to treatment can be difficult to predict. Monitoring of minimal residual disease (MRD) is thus very important and holds great potential for improving treatment strategies. Common MRD targets include recurrent cytogenetic abnormalities and mutations in important hematological genes; unfortunately well-characterized targets are lacking in many AL patients. Here we demonstrate a technical approach for the identification and mapping of novel clone-specific chromosomal abnormalities down to the nucleotide level. We used molecular cytogenetics, chromosome microdissection, amplification of the microdissected material, and next-generation sequencing to develop PCR-based MRD assays based on unique breakpoint sequences.

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.

Detection of cytomegalovirus DNA in fecal samples as a method for CMV enterocolitis diagnosis after allogeneic stem cell transplantation

BACKGROUND Cytomegalovirus enterocolitis is a rare but potentially life threatening complication after allogeneic stem cell transplantation. Its early diagnosis and treatment are essential for a successful outcome. OBJECTIVE To determine the potential benefit of fecal CMV DNA detection in the diagnosis of CMV colitis among stem cell transplant recipients. STUDY DESIGN Biopsies from the lower gastrointestinal tract, taken during 69 episodes of diarrhea, were compared with fecal samples previously examined for CMV DNA in 45 patients after allogeneic stem cell transplantation. RESULTS Six confirmed cases of CMV colitis were observed, with 16 out of 69 (23%) fecal samples proving positive for CMV DNA. Only one positive sample correlated with histologically confirmed CMV colitis, and 15 samples were evaluated as false positive. These results provide a 16.7% sensitivity and 76.2% specificity in the diagnosis of CMV enterocolitis. CONCLUSION The examination of fecal samples for the presence of CMV DNA has very low potential in the diagnosis of CMV enterocolitis after allogeneic stem cell transplantation; therefore, a biopsy of the gastrointestinal mucosa is still warranted for correct diagnosis.

Ciprofloxacin prophylaxis during autologous stem cell transplantation for multiple myeloma in patients with a high rate of fluoroquinolone-resistant gram-negative bacteria colonization

BACKGROUND Ciprofloxacin prophylaxis used to be a standard precaution during autologous stem cell transplantation. Its benefit, with a high prevalence of fluoroqinolone resistance in the population, has recently been under scrutiny. OBJECTIVE To evaluate the impact of cessation of ciprofloxacin prophylaxis during stem cell transplantation for multiple myeloma. PATIENTS AND METHODS Data from 104 patients with multiple myeloma transplanted during the period from January 2013 to April 2015 were retrospectively reviewed. 67 received standard ciprofloxacin prophylaxis (group A) and 37 received no antibacterial prophylaxis (group B). RESULTS Febrile episodes during neutropenia, bloodstream infection (BSI) and mortality in these two cohorts were evaluated. Gram negative BSI was assessed for the colonization of quinolone-resistant gram-negative pathogens. Secondary Clostridium difficile enterocolitis presence was determined in both cohorts. There were 42 (63%), 7 (10%), and 0 febrile episodes, BSI and gram-negative BSI respectively in group A, and 34 (92%), 12 (32%), and 4 (11%) respectively in group B. The differences in the number of febrile episodes (P=0.0011) and deaths (P=0.0427) were statistically significance. Mortality was 0 and 3 (8%) in group A and group B, respectively. There was no significant difference in colonization with quinolone-resistant gram negative pathogens (25 (37%) versus 11 (30%)) between groups. The occurrence of Clostridium difficile colitis was the same in both groups. CONCLUSION We resumed ciprofloxacin prophylaxis for the following reasons. There was a significant reduction in febrile episodes, and consequently a sparing effect of antibiotics used for treatment of this condition. No difference in Clostridium difficile colitis occurred and the mortality rate of 8% in group B was unacceptably high.

Plasmacytic Post-transplant Lymphoproliferative Disorder – Case Report

1. 4th Department of Internal Medicine – Haematology, University Hospital Hradec Kralove and Charles University in Prague, Faculty of Medicine in Hradec Kralove, Hradec Kralove, Czech Republic; 2. Institute of Clinical Immunology, University Hospital Hradec Kralove and Charles University in Prague, Faculty of Medicine in Hradec Kralove, Hradec Kralove, Czech Republic; 3. Institute of Clinical Biochemistry, University Hospital Hradec Kralove and Charles University in Prague, Faculty of Medicine in Hradec Kralove, Hradec Kralove, Czech Republic; 4. The Fingerland ́s Department of Pathology, University Hospital Hradec Kralove and Charles University in Prague, Faculty of Medicine in Hradec Kralove, Hradec Kralove, Czech Republic