The effect of HLA allele and haplotype polymorphisms on donor matching in hematopoietic stem cell transplantation – Croatian experience

The knowledge of HLA characteristics of a patient’s population helps to predict the probability of finding a MUD.The study included 170 transplanted patients for whom a search for a MUD in BMDW was performed and a sample of 4000 volunteer unrelated donors from the Croatian Bone Marrow Donor Registry (CBMDR). Patients and their MUDs were typed for HLA-A, -B, -C, -DRB1, and -DQB1 loci using PCR-SSO and PCR-SSP methods while donors were typed for HLA-A, -B, -C, and -DRB1 loci using the PCR-SSO method.A comparison of allele frequencies at tested HLA loci between patients and donors from CBMDR did not reveal significant differences. The majority of patients (117, 68.8%) had a 10/10 MUD, 45 (26.5%) patients had a 9/10 MUD and eight (4.7%) patients had an 8/10 MUD. The highest number of mismatches (MM) was present at HLA-DRB1 (19; 31.1%). The presence of DRB1*11 and DRB1*04 allelic groups among patients caused allelic MMs at HLA-DRB1 in most cases. The presence of an infrequent HLA-B∼C haplotype resulted in the HLA-C MM at antigen level in the majority of cases.The present study clarified HLA factors that cause difficulties in searching for a 10/10 MUD for Croatian patients.     

Suppression of HLA expression by lentivirus-mediated gene transfer of siRNA cassettes and in vivo chemoselection to enhance hematopoietic stem cell transplantation

Current approaches for hematopoietic stem cell (HSC) and organ transplantation are limited by donor and host-mediated immune responses to allo-antigens. Application of these therapies is limited by the toxicity of preparative and post-transplant immunosuppressive regimens and a shortage of appropriate HLA-matched donors. We have been exploring two complementary approaches for genetically modifying donor cells that achieve long-term suppression of cellular proteins that elicit host immune responses to mismatched donor antigens, and provide a selective advantage to genetically engineered donor cells after transplantation. The first approach is based on recent advances that make feasible targeted down-regulation of HLA expression. Suppression of HLA expression could help to overcome limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors. Accordingly, we have recently investigated whether knockdown of HLA by RNA interference (RNAi) enables allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors that integrate into genomic DNA, thereby permanently modifying transduced donor cells. Lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA achieved efficient and dose-dependent reduction in surface expression of HLA in human cells, and enhanced resistance to allo-reactive T lymphocyte-mediated cytotoxicity, while avoiding non-MHC restricted killing. Complementary strategies for genetic engineering of HSC that would provide a selective advantage for transplanted donor cells and enable successful engraftment with less toxic preparative and immunosuppressive regimens would increase the numbers of individuals to whom HLA suppression therapy could be offered. Our second strategy is to provide a mechanism for in vivo selection of genetically modified HSC and other donor cells. We have uniquely combined transplantation during the neonatal period, when tolerance may be more readily achieved, with a positive selection strategy for in vivo amplification of drug-resistant donor HSC. This model system enables the evaluation of mechanisms of tolerance induction to neo-antigens, and allogeneic stem cells during immune ontogeny. HSC are transduced ex vivo by lentivirus-mediated gene transfer of P140K-O⁶-methylguanine-methyltransferase (MGMT^P140K). The MGMT^P140K DNA repair enzyme confers resistance to benzylguanine, an inhibitor of endogenous MGMT, and to chloroethylating agents such as BCNU. In vivo chemoselection enables enrichment of donor cells at the stem cell level. Using complementary approaches of in vivo chemoselection and RNAi-induced silencing of HLA expression may enable the generation of histocompatibility-enhanced, and eventually, perhaps “universally” compatible cellular grafts.     

Expression of cancer stem cell markers in basal and penta-negative breast carcinomas – A study of a series of triple-negative tumors

Purpose

Breast cancer is a heterogeneous disease. Immunohistochemistry has given rise to triple-negative carcinoma (TNC). Concomitantly, biological origins of neoplasia and its heterogeneity has been strongly debated in cancer stem cells (CSC) theme. This study investigates the prevalence of basal (BCC) and penta-negative carcinomas (5NC) in TNC and establishes associations with CSC (CD44CD24).

Materials and methods

94 TNC were tested for CK5/6, HER1, CD44 and CD24, evaluated by a simple immunohistochemistry score and correlated with clinicopathological and survival data.

Results

BCC had higher tumor grades than 5NC (p = 0.004). CD44 negativity (p = 0.007) and CD44⁻CD24⁺ phenotype (p = 0.013) were associated with less vascular invasion amongst TNC. CD44 expression was associated with BCC (p = 0.007). CD44⁻CD24^−/low phenotype was associated with 5NC. None of the variables were associated with clinical outcome.

Conclusion

BCC and 5NC are closely related tumor subtypes. CD44⁻CD24^−/low phenotype was associated with 5NC and CD44⁻CD24⁺ phenotype was associated with vascular invasion. These results require histogenetic confirmation in larger studies.     

Characterization of the CDR3 structure of the Vβ21 T cell clone in patients with P210(BCR-ABL)-positive chronic myeloid leukemia and B-cell acute lymphoblastic leukemia

The clonally expanded T cells identified in most cancer patients that respond to tumor-associated antigen such as P210(BCR-ABL) protein have definite, specific antitumor cytotoxicity. T cell receptor (TCR) Vβ CDR3 repertoire diversity was analyzed in patients with chronic myeloid leukemia (CML) and BCR-ABL(+) B-cell acute lymphoblastic leukemia (B-ALL) by GeneScan. A high frequency of oligoclonal expansion of the TCR Vβ21 subfamily was observed in the peripheral blood of CML and B-ALL patients. These clonally expanded Vβ21 T cells were correlated with the pathophysiologic process of CML. A conserved amino acid motif (SLxxV) was observed within the CDR3 region in only 3 patients with CML. Preferential usage of the Jβ segments was also observed in a minority of patients. The 3-dimensional structures of the CDR3 region containing the same motif or using the same Jβ segment displayed low similarity; on the contrary, the conformation of the CDR3 region containing no conserved motif in some T cell clones was highly similar. In conclusion, our findings indicate a high frequency of TCR Vβ21 subfamily expansion in p210(BCR-ABL)-positive CML and B-ALL patients. The characterization of the CDR3 structure was complex. Regrettably, at this time it was not possible to confirm that the Vβ21 T cell clones were derived from the stimulation of p210(BCR-ABL) protein.