Genetic Diversity within Leukemia-Associated Immunophenotype-Defined Subclones in AML

Acute myeloid leukemia (AML) is a heterogeneous clonal disease and in different patients, different combinations of mutations may be found that drive the disease. Also within patients, various leukemic clones with a different combination of mutations may co-exist, reflecting various evolutionary stages of the disease. At present, flowcytometry and mutational analyses are used to keep track of the malignant cells during and after treatment. Using panels of antibodies, abnormal cells can be identified when lineage markers are over- or underexpressed, or when abnormal differentiation leads to the co-expression of markers that normally are not expressed together on hematopoietic cells. These patient-specific leukemia-associated immunophenptypes (LAIPs) can be followed during and after treatment, and enable the early detection of resistant and recurring disease. Often, several LAIPs are present simultaneously within a patient, but their clonal relationship is not always clear. In addition, next-generation sequencing and mutation-specific PCR can be used to follow the leukemic cells. As the clinical consequences of MRD-positivity may consist of high-dose re-induction chemotherapy or stem cell transplantation, accurate results are of the highest importance. Although it has already been shown that flowcytometry and mutational analysis may have added value, in-depth information on the genetic make-up of immunophenotypically defined normal and leukemic subclones and their clonal relationship in AML is still scarce. To study the genetic similarities and differences between various normal and leukemic subclones defined by immunophenotyping, we performed an in-depth analysis of the genetic makeup of FACS-sorted immunophenotypically normal and aberrant subclones in 10 cases of AML. In total, 86 LAIPs and normal cell fractions were sorted and fully genotyped using error-corrected sequencing with a panel genes that are recurrently mutated in AML. In some cases, different aberrant populations from the same patient carried identical mutations, indicating that the leukemia was highly homogeneous, and that the phenotypically different LAIPs all were part of the same genetic clone. In these cases, monitoring the disease by following one of the present LAIPs would be sufficient, or, alternatively, following disease dynamics by molecularly monitoring one of the mutations would accurately show the dynamics of the disease. In other cases, different subclones harbored a different set of mutations. In addition, immunophenotypically normal populations frequently carried mutations, or, inversely, no mutations were found in immunophenotypically clearly aberrant clones. In these cases, following the disease by LAIPs or molecular markers would carry the risk that several subclones would be missed. In case these clones would be relevant for resistance or relapse, using either technique would be at risk to fail to detect MRD positivity. This work underscores that currently available, state-of-the-art immunophotyping and gene panels have added value and should be used simultaneously. We conclude that currently, MRD detection by molecular and immunophenotypic techniques are complementary, and should be used in parallel in order to obtain the most complete view on resistant disease and early detection of relapse. At the same time, both techniques can be further optimized with more monoclonal antibodies and genes that are screened for mutations, in order to increase their resolution. Disclosures No relevant conflicts of interest to declare.

Clonal Relationship within Two Oral Actinomyces Populations Collected from Plaque of Periodontitis Patients

Actinomyces naeslundii and A. oris are dental plaque formers involved in the pathogenesis of periodontitis. The aim of the study was to investigate the clonal relationship within two oral Actinomyces populations collected from plaque of patients with chronic periodontitis. The 223 clinical strains of A. naeslundii and A. oris were isolated from biofilm samples collected supra and subgingivally from teeth with shallow (probing depth (PD) = 3-4 mm), deep (PD = 5-6 mm) and very deep (PD ≥7 mm) pockets from 20 chronic periodontitis patients. All strains were submitted to repetitive sequence-based PCR typing using DiversiLab (BioMerieux,Marcy l´Étoile, France). Seven patients harboured only unrelated (<95% similarity) multiple isolates, while 13 harboured both similar (>95% similarity) and unrelated isolates at different sites. Identical (>98% similarity) strains were found to be present in the subgingival shallow depths more often than in the other subgingival depths. The number of clones in individual patients varied from 2 to 17 different rep-PCR genotypes. The clonal relationship within the oral populations of A. naeslundii and A. oris in an individual was unpredictable, ranging from the presence of multiple genotypes with no clonal similarity to only two different clones supra or subgingivally at different sites.

Spread of Carbapenemase Producing Klebsiella pneumoniae Isolates in Our Hospital: Investigation of Molecular Typing and Clonal Relationship

Objective: Carbapenem resistance has been reported with increasing frequency among members of Enterobacterales, especially in the last 10 years. Screening and detection of carbapenemase-producing isolates is important in terms of both directing the treatment and preventing its spread. In our study, it was aimed to determine the carbapenemase types and molecular epidemiological relationships of carbapenem resistant Klebsiella pneumoniae isolates, which were isolated sequentially from the samples sent to microbiology laboratory of our hospital. Method: A total of 32 carbapenem-resistant K. pneumoniae isolates of the samples sent to microbiology laboratory between July and September 2014, were included in the study. In addition to classical methods, identification of isolates at species level was made with BD Phoenix ID/AST automated system. Carbapenemase types (blaOXA-48, blaNDM, blaIMP, blaKPC, blaVIM and blaGES) of the isolates were investigated by PCR. The clonal relationship between the isolates was assessed with PFGE. Results: It was noted that 18 isolates were obtained from intensive care units, 9 from inpatient and 5 from outpatient departments. The blaOXA48 gene was found in all isolates while the other carbapenemase genes were not found. It was determined that strains were isolated from 32 patients in our hospital had 12 different PFGE pulsotypes, named as A-L. Among these, the most common ones were B (n=18) and closely related B1 pattern (n=2). The remaining isolates were represented by 11 different types. It was observed that the first isolate with B pulsotype was responsible for the spread of the outbreak from General Intensive Care Unit. Conclusion: It has been thought that the spread of carbapenem- resistant K. pneumoniae isolates in the hospital was probably occurred through the transfer of isolates from patients with gastrointestinal colonization to other patients through hospital staff. Therefore, the spread of the isolates in hospitals can be limited by detecting colonization with active surveillance programs and by applying contact isolation and effective infection control measures.