Appearance Of New Clones Research Articles

Multiple Clonal TCR Gene Rearrangements Are Typical in Peripheral T-Cell Lymphoma Not Otherwise Specified

Introduction: Peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) is a heterogeneous subgroup of PTCL with no consistent immunophenotypic, genetic and clinical features. PTCL-NOS represents less than 29% of all T-cell lymphomas in adults and often has an aggressive course with extranodal and systemic involvement. This study is focused on the application of clonality testing methods for PTCL-NOS diagnosis and monitoring.Patients and Methods: Biopsies, blood and bone marrow samples were tested for 30 patients with PTCL-NOS: the ratio of male/female was 15/15, median age was 56 years (32-75), and 22 of 30 patients had IV stage of the disease. TCRG and TCRB gene rearrangements were evaluated for T cell clonality analysis. We used PCR-based methods (BIOMED-2 standardized clonality protocol) followed by GeneScan analysis on the ABI PRISM 3130 Genetic Analyzer (Applied Biosystems).Results: Clonal TCR gene rearrangements were found in 29 of 30 patients (97%): TCRG in 27 of 30 (90%) and TCRB in 29 of 30 (97%). Primary biopsies or bone marrow samples showed oligoclonal pattern of rearrangements (more than 3 clonal peaks in TCRG or TCRB GeneScan profiles) in 13 of 30 patients (43%). New clonal rearrangements in affected tissues (skin, tonsil, CSF, lymph nodes and etc.) during disease progression were found in 15 of 24 (63%) patients. Various representations of clonal rearrangements in different tissues indicate the presence of several tumor clones in the majority of cases (19 of 30 patients; 63%). The presence of several clones correlated with the number of tissues examined (rs = 0.6, p <0.0005). The detection rate of additional clonal rearrangements was 90% (9 of 10 patients) when 3 and more tissues were tested. The number of clonal rearrangements was not correlated with age (p = 0.43) or the stage of disease (p = 0.29). One can speculate that the presence of multiple clones in the PTCL-NOS could reflect genetic instability of this tumor.Conclusion: Oligoclonality, clonal evolution and the appearance of new clones in the progression of the disease are typical features of PTCL-NOS. Multiple clonal rearrangements complicate diagnostics and assessment of minimal residual disease in this disease. DisclosuresNo relevant conflicts of interest to declare.

P77: Clonal evolution of breast tumor during neoadjuvant chemotherapy and metastasis

Background There are numerous evidences suggesting that tumor evolution follows the laws of Darwinian evolution, whereby individual tumor cell clones have private genetic aberrations, including chromosomal abnormalities. The combined effect of genetic instability and differential selective pressures of the microenvironment and chemotherapy can result in the creation of new tumor clones (Navin et al., 2011; Ng et al., 2012). The aim of this study is to show breast tumor clonal evolution during neoadjuvant chemotherapy (NAC) using microarray analysis. Material and methods Breast cancer patients ( n = 26) with stage IIA to IIIC (T1-4N0-3M0), were treated with NAC (FAC or CAX regimens). DNA was extracted from 26 samples of tumor tissue derived before or after NAC using QIAamp DNA mini Kit (Qiagen, Germany). Copy Number Aberrations (CNA, deletions and amplifications, or Loss and Gain, respectively) and number of mutant clones were detected in pre- and post-NAC tumor samples using the high density microarray platform Affymetrix (USA) CytoScanTM HD Array. This study was approved by Tomsk Cancer Research Institute review board. Results We have revealed that 19% (5/26) of patients during the NAC showed the decrease in the number of mutant clones and CNA frequency right up to their complete elimination (genetic regression) at one case. In 7 (27%) cases chemotherapy had no any effect on number of mutant clones and the frequency of CNA in tumor. In the tumors of 10 patients the elimination of some mutant clones as well as the formation of new clones with deleted genetic material occurred under the influence of chemotherapy. 6 patients have demonstrated the appearance of new tumor clones with gene amplifications which were associated with the development of metastases in 83% of cases (5/6). All other patients ( n = 21) who has not acquired the new tumor clones with Gain function mutation after NAC did not manifest distant metastasis in 5-year follow-up (Kaplan–Meier, p = 0.00001 Log-rank test). Conclusion The first time evidence is presented that the formation of new tumor clones may occur during the NAC. Metastasis of breast cancer is associated with the appearance of new clones with DNA amplifications. Detection of these clones allow getting new prognostic factor in breast cancer. The study was supported by the Russian Foundation for Basic Research – Russia (Project 15-04-03091).

HTLV-1 clonality during chronic infection and BLV clonality during primary infection

HTLV-1 persists by driving clonal proliferation of infected T-lymphocytes. A high proviral load predisposes to the inflammatory and malignant diseases associated with HTLV-1. Yet the reasons for the remarkable variation within and between individuals in the abundance of HTLV-1-infected clones remain unknown. We demonstrate that negative selection dominates during chronic infection, favouring establishment of proviruses integrated in transcriptionally silenced DNA: this selection is significantly stronger in asymptomatic carriers. We postulated that this selection occurred mainly during the primary infection. We are testing this hypothesis in an animal model by studying the BLV clonality during the primary infection in cows. By measuring the proviral load, the anti-BLV immune response and the BLV clonality we aim to quantify the impact of the immune response on the rate of infectious spread and on the selection of proviruses inserted in a particular genomic environment. Co-infection with Strongyloides stercoralis or Staphylococcus appears to be another risk factor for the development of HTLV-1 associated diseases. We observed that HTLV-1 clonality is altered by co-infection with these pathogens with an increase of both the number and the abundance of the infected T-cell clones. The genomic characteristics of the proviral integration sites in the most abundant clones differ significantly between co-infected individuals and those with HTLV-1 alone, implying the existence of different selection forces in co-infected patients. The rate of appearance of new clones in patients co-infected with Strongyloides stercoralis is higher than in patients with HTLV-1 alone. By comparing skin lesions and blood samples from patients with Infective Dermatitis associated with HTLV-1 (IDH), we observed a significant proportion of distinct infected clones between the two compartments. The skin lesions seem to be a site for HTLV-1 infectious spread.

Fanconi Anemia: Myelodysplasia as a Predictor of Outcome

The adverse potential of the development of myelodysplastic syndrome (MDS) in Fanconi anemia (FA) was examined in a retrospective study of 41 FA patients who had bone marrow morphology and chromosomes reviewed by a single group. Thirty-three patients had adequate cytogenetic studies, and 16 (48%) had one or more abnormal studies: nine initially, and seven more on follow-up. Cytogenetic clonal variation was frequent, including disappearance of clones, clonal evolution, and appearance of new clones. The estimated five-year survival with a cytogenetic clone is 0.40, compared to 0.94 without a clone. Morphologic myelodysplasia (MDS), independent of a cytogenetic clone, was found in 13/41 patients (32%). The estimated five-year survival with MDS is 0.09, versus 0.92 without MDS. Leukemia developed in three patients whose initial cytogenetic clones prior to leukemia were t(1;18), t(5;22) and monosomy 7; the one with t(1;18) also had MDS. Our results focus on marrow morphology, and suggest that morphologic MDS may be more important than classical cytogenetics in prediction of an adverse outcome.

Evolution of an endemic methicillin-resistant Staphylococcus aureus population in an Australian hospital from 1967 to 1996.

The evolution over 30 years of a population of methicillin-resistant Staphylococcus aureus (MRSA) from a tertiary referral hospital was studied by phylogenetic analysis of SmaI-generated restriction fragment length polymorphisms (RFLPs). The results suggest that a new clone of MRSA appeared at the hospital in the early 1980s, which, although usually retaining its ancestral phage-type, developed four different RFLP pulsotypes in the next 16 years. This finding indicates that multiple RFLP patterns in MRSA do not necessarily represent multiple clones deriving from different mec gene transfer events. Such variation within a clone may be significant in the interpretation of RFLP patterns during outbreaks and emphasizes the need to use two typing methods in studies of such populations. Since the appearance of new clones of MRSA is a relatively rare event, cross-infection control is paramount in the prevention of MRSA dissemination.

The clonal antibody response to Pseudomonas aeruginosa heat shock protein is highly diverse in cystic fibrosis patients

The GroEL protein of Pseudomonas aeruginosa belongs to the bacterial 60-65 kDa heat shock protein family. A strong antibody response to GroEL has been found in cystic fibrosis (CF) patients with chronic pulmonary infection caused by P. aeruginosa. Clonotypes of IgG1 and IgG2 antibodies against GroEL were studied in 60 consecutive sera from 18 CF patients with chronic P. aeruginosa infection using isoelectric focusing in combination with affinity immunoblotting. The persistent antigenic stimulation in CF patients with chronic P. aeruginosa infection induced numerous IgG1 and particularly IgG2 antibody clones against GroEL. The appearance of new clones with time reflected the long duration of the chronic infection. A striking addition of new clonotypes during the observation period occurred when a new unrelated bacterium (Burkholderia cepacia) had become established as a cause of the pulmonary infection, or when the P. aeruginosa infection became chronic.

Chromosome banding patterns in patients with chronic myelocytic leukemia

One hundred and nine patients with Ph 1-positive chronic myelocytic leukemia were cytogenetically studied with banding methods. Seventy-eight patients were studied in the chronic phase and 39 patients in the blastic phase. The standard translocation was present in 107 cases. Two patients showed complex translocations involving chromosomes No. 6, 9, 22, 11 and No. 9, 22, 11, respectively. Ph 1-negative cells were detected in 8 cases (7%). Chromosome aberrations in addition to the Ph 1 chromosome were observed in 6 cases (8%) during the chronic phase. The karyotypic findings during the blastic phase were similar to those reported in the past [trisomy 8, iso(17q), and a second Ph 1]. The significance of Ph 1-negative cells, the geographic heterogeneity of the chromosomal aberrations, the effect of chemotherapy on the appearance of new clones, and the importance of the materials and methods used for the comparison of cytogenetic patterns at different laboratories are discussed.