TCRgamma gene rearrangement analysis in skin samples and peripheral blood of mycosis fungoides patients.

BACKGROUND: Diagnosing mycosis fungoides (MF) can be challenging in the early stage of the disease because histopathological features may simulate a variety of benign inflammatory skin diseases. Assessment of T-cell clonality was found to be useful in diagnosis and follow-up of patients. OBJECTIVE: In this study, PCR-based TCRgamma gene rearrangement analysis was performed in skin and peripheral blood samples of patients with MF treated at the two largest referral centers in Serbia, and the results obtained were correlated with clinical and follow-up data. METHODS: Skin and peripheral blood samples were obtained with informed consent from 37 patients treated at the Department of Dermatology of the Military Medical Academy and the Medical Center of Serbia from 2001 to 2006. The median time of follow-up was 4 years. Multiplex PCR was used for TCRgamma gene rearrangement analysis in skin and peripheral blood samples. Clonality results were correlated with the clinical data and disease course data. RESULTS: Monoclonality was detected in skin samples of 30/37 patients (81%), in 2/5 patients with large-plaque parapsoriasis (LPP), in 28/32 (88%) patients with histologically proven MF, and in 1/16 (6%) patients with benign inflammatory dermatoses. A monoclonal pattern in both skin and peripheral blood was detected in 7/16 (44%) patients in the late stage of the disease, and in 1/7 (14%) patients in the early stage of the disease. A dominant clone was found in both skin and peripheral blood in 1/4 patients in remission, 2/5 with a stable disease, and 4/9 (44%) with disease progression. CONCLUSION: TCR-gamma gene rearrangement analysis can be regarded as a useful adjunct to diagnosis of epidermotropic lymphoproliferative disorders. The presence of a dominant clone in both the skin and peripheral blood was more frequently detected in late stages and in patients with disease progression, confirming the usefulness of clonality detection by TCR-gamma gene rearrangement analysis in follow-up of patients with primary cutaneous T-cell lymphomas.

Detection of t(14;18), P53 and RAS gene mutations and quantification of residual disease in patients with B-cell non-Hodgkin's lymphoma.

UNLABELLED: PCR analysis has been demonstrated as a valuable tool for detection of minimal residual disease (MRD) in lymphoid malignancies. However, the finding that patients with evidence of MRD sometimes remain in long-lasting remission directs further investigations toward biology of residual disease and/or quantification of MRD level. The study included 40 B-NHL patients--13/40 patients with high- (HG) and 27/40 with low-grade (LG) lymphoma. Seven patients achieved partial clinical remission (PR) and 33 patients achieved complete clinical remission (CCR) after chemotherapy. Peripheral blood samples were analyzed for MRD at up to ten follow-up points while samples of MRD+ patients and patients who achieved partial clinical response after therapy were further analyzed for the presence of t(14;18) and P53 and RAS genes mutations. The level of MRD was quantified in eight patients by PCR-limiting dilution method. RESULTS: MRD was found in 13/33 patients (12 LG and 1 HG) who achieved CCR. The incidence of relapse was significantly higher in MRD+ vs. MRD- B-NHL patients (Fisher's exact test, p = 0.0083). In the LG group the incidence of relapse between MRD+ and MRD-patients was not significantly different. In the HG group MRD was detected in only one patient who subsequently relapsed. Significant difference in DFI between MRD+ and MRD- patients was not observed. Concerning MRD+ patients in CCR and patients who achieved PR, t(14;18) was found in six patients (4 relapsed). In the same group of patients P53, K- and N-RAS mutations were not found. H-RAS mutations were found in six patients--3 relapsed and 3 remain in CCR. The calculated number of IGH copies ranged from 4800 to 44,000. Our results demonstrated positive correlation between MRD-positivity and incidence of relapse in B-NHL patients, but could not indicate significance of P53 and RAS mutations for evaluation of residual clone malignancy. The study implies that MRD level, measured at one follow-up point, does not correlate with clinical outcome. The measurement of MRD level sequentially, at different follow-up points, seems to be a better parameter for the prediction of disease course.

Analysis of T-cell clonality pattern in tumor samples of breast cancer patients.

PURPOSE AND METHODS: A large body of experimental evidence has confirmed that different tumors, including breast carcinomas, can stimulate specific T-cell-mediated immune responses. In this study we have analyzed patterns of T-cell clonality in tumor samples of 54 breast cancer patients classified as lymph node negative, N0 (n=16), or lymph node positive, N+ (n=38). The clonality of T-cells was analyzed by the PCR-PAGE method. RESULTS: Monoclonal/oligoclonal (M/O) T-cell populations were found in 15 breast cancer patients, nine N+ and six N0. In all analyzed groups (N+ + N0, N+, N0) the incidence of relapse was not significantly different between patients with M/O and patients with polyclonal T-cells. Comparison of disease-free interval (DFI) between patients divided according to the presence of TCRgamma monoclonality/oligoclonality showed a marginally significant difference only in the group of N+ patients within the first 24 months of follow-up. Patients with a M/O T-cell population had a shorter DFI than patients with a polyclonal T-cell population. This difference was not observed when the complete follow-up period was considered in the same group of patients. Furthermore, there was no significant difference in overall survival (OS) between patients with M/O and patients with polyclonal T-cells. CONCLUSION: Our results imply that tumor infiltrating T-cells are usually polyclonal. The pattern of T-cell clonality does not correlate with the incidence of relapse and the duration of DFI and OS in the analyzed groups of breast cancer patients, excluding N+ patients with M/O T-cells who had a shorter DFI in the first 24 months of follow-up. This observation suggests that polyclonal T-cell populations may provide a broader spectrum of T-cell-mediated antitumor response.

Analysis of T-cell clonality pattern in tumor samples of breast cancer patients.

PURPOSE AND METHODS: A large body of experimental evidence has confirmed that different tumors, including breast carcinomas, can stimulate specific T-cell-mediated immune responses. In this study we have analyzed patterns of T-cell clonality in tumor samples of 54 breast cancer patients classified as lymph node negative, N0 (n=16), or lymph node positive, N+ (n=38). The clonality of T-cells was analyzed by the PCR-PAGE method. RESULTS: Monoclonal/oligoclonal (M/O) T-cell populations were found in 15 breast cancer patients, nine N+ and six N0. In all analyzed groups (N+ + N0, N+, N0) the incidence of relapse was not significantly different between patients with M/O and patients with polyclonal T-cells. Comparison of disease-free interval (DFI) between patients divided according to the presence of TCRg monoclonality/oligoclonality showed a marginally significant difference only in the group of N+ patients within the first 24 months of follow-up. Patients with a M/O T-cell population had a shorter DFI than patients with a polyclonal T-cell population. This difference was not observed when the complete follow-up period was considered in the same group of patients. Furthermore, there was no significant difference in overall survival (OS) between patients with M/O and patients with polyclonal T-cells. CONCLUSION: Our results imply that tumor infiltrating T-cells are usually polyclonal. The pattern of T-cell clonality does not correlate with the incidence of relapse and the duration of DFI and OS in the analyzed groups of breast cancer patients, excluding N+ patients with M/O T-cells who had a shorter DFI in the first 24 months of follow-up. This observation suggests that polyclonal T-cell populations may provide a broader spectrum of T-cell-mediated antitumor response. (Int J Biol Markers 2005; 20: 177-83).

[Detection of B-lymphocyte clonality in samples of salivary gland tissue in patients with primary Sjogren syndrome]. / Detekcija klonalnosti B limfocita u uzorcima tkiva pljuvacnih zlezda bolesnika sa primarnim Sjegrenovim sindromom.

Intensive lymphoplasmocytic infiltration with atrophy of glandular tissue structures is the dominant patohistological feature found in exocrine glands of patients with Sjögren syndrome (SS). The infiltrates consist of T and B lymphocyte clusters that make the structures resembling germinal centers, and numerous plasmocytes that are secreting imunoglobulines locally, including autoantibodies. By applying the polymerase chain reaction (PCR) in our study we have shown the existence of dominant B cell clone in salivary glands samples of 4 out of 6 patients with SS, in the absence of clinical, routine laboratory, and patohistological signs of the lymphoma. B lymphocyte clones were detected upon the amplification of gene segment that encoded variable heavy chain immunoglobulin CDR3 region. Finding of single, dominant B lymphocyte clone could be of predictive significance, because these patients are predisposed to non-Hodgkin lymphoma (NHL) for which there is an assumption that it originates out of salivary glands from one of the clusters of proliferating B lymphocytes.

[Detection of K-ras and p-53 oncogene mutations in a patient with acute lymphoblastic leukemia before and after bone marrow transplantation]. / Detekcija mutacija u K-ras i p-53 onkogenima kod obolelog od akutne limfoblastne leukemije pre i posle transplanticije kostine srzi.

We present a case of 22 years old male patient, who was submitted to singenic transplantation of hematopoietic cells originating from the bone marrow in the remission phase of the diagnosed acute lymphoblastic leukemia (ALL). The bone marrow sample was donated by his healthy twin brother. The pretransplantation and transplantation phases were regular. We analyzed the presence of K-ras and p-53 point mutations in our patient with ALL and for the first time we had the opportunity to analyze the samples from two monozygotic twins. DNA was isolated from the peripheral blood mononuclear cells (PBMNC) by the standard procedure, of the patient with ALL before and after bone marrow (BM) transplantation and of his clinically healthy twin brother. Samples were subjected to PCR amplification of K-ras exons 1 and 2 and p-53 exons 5, 6, 7 and 8. In PBMNC of the patient with ALL before BM transplantation, mutations were observed in exon 1 of K-ras and exon 8 of p-53 gene. These mutations were found neither in PBMNC sample of his twin brother, nor in PBMNC of the patient with ALL after BM transplantation. In the p-53 exons 5, 6 and 7 and exon 2 of K-ras, there were no mutations in any analyzed samples. Detected mutations in K-ras and p-53 genes could be a part of larger genetic abnormalities and the obtained results had shown the possibility of using DNA mutational changes in the follow-up of the success of BM transplantation. The molecular disease marker that was found by this method was also significant for the detection of minimal residual disease at the molecular level.