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Papers by Marcelo A Fernandez-vina

Blood, 2008

Introduction/Methods: A key component of improving the success rate of allogeneic HSCT for AML in... more Introduction/Methods: A key component of improving the success rate of allogeneic HSCT for AML in CR1 is to reduce non-relapse mortality (NRM), which, if excessive, will deny the benefit of the graft-versus-leukemia effect. UD HSCT has traditionally been associated with high NRM rates. We reported previously on the significant reduction of NRM using the conditioning regimen of fludarabine 40mg/m2, IV busulfan 130 mg/m2 for 4 days and thymoglobulin. Here we analyze the outcomes of all patients (n=37) with AML in CR1 treated with this regimen and UD HSCT in our institution from January 2002 to December 2007. High-resolution allele level HLA typing was performed for all donorrecipient pairs for HLA-A, -B, -C, DRB1 and DQB1; up to one mismatch was allowed (9/10). Median follow up is 30 months (range, 9–72). Results: Median age was 48 years (range, 13–68); 30% (n=11) were older than 54 years and 51% (n=19) were male. Eleven patients (30%) had secondary AML. Prognostic cytogenetics classi...

Blood, 2006

The HLA class II DP locus encode for both subunits of DPB1 heterodimers, which have low levels of... more The HLA class II DP locus encode for both subunits of DPB1 heterodimers, which have low levels of expression on the cell surface of antigen presenting cells. We hypothesized that donor-recipient HLA-DP mismatch would lead to an increased incidence of acute (a) graft-versus-host disease (GVHD), and that 2 mismatches would likely be even more significant. Methods: We studied 84 consecutive patients (pts) with myeloid leukemias in complete remission (CR) transplanted from 01/02 to 02/06. Preparative regimens were ablative IV Busulfan-based (n=58) or Cy/TBI (n=2), and reduced intensity (Fludarabine (Flu)/Bu 130 mg/m2/2 doses plus Gleevec (n=8), and Flu/Melphalan 140 mg/m2 (n=16). Stem cell (SC) source was bone marrow (n=70) or peripheral blood (n=14). ATG was given in 78 cases. GVHD prophylaxis was tacrolimus and mini-methotrexate in all cases, with additional pentostatin in 31 pts. High-resolution typing was sequence-based for HLA-A, B, DRB1; SSP was used for DRB3/4/5, DQB1 and DPB1, a...

Human Immunology, 2015

Aim Swabs created from BLCLs transformed from peripheral blood mononuclear cells (PBMCs) or expan... more Aim Swabs created from BLCLs transformed from peripheral blood mononuclear cells (PBMCs) or expanded from existing BLCLs are used in the Be The Match Registry (BTMR) HLA QC program. The aim of this project was to document mutation as a cause of HLA typing discrepancies observed in 2 BLCLs. The first discrepancy arose in expanded BLCLs, sequenced in 2002 on a donor-derived BLCL. BLCL swabs were routinely typed multiple times using SSO and SBT methodologies as B∗49:01; however, in 2009, an SBT lab reported a potential new allele, B∗49:15, which was described in 2010 and confirmed in 2011 on the expanded BLCL by two labs. B∗49:15 differs from B∗49:01:01 by a single non-synonymous nucleotide polymorphism at NT 176 in exon 2, G/C. In 2013, a lab reported that the B49-specific amplicon showed a double peak, suggesting two alleles were present. Typing of other loci ruled out contamination. The second discrepancy arose in a BLCL transformed from patient-derived PBMC, originally typed as C∗02:09 using DNA extracted from PBMC in 2004. BLCL swabs repeatedly typed as C∗02:02 by SSO, SBT, and NGS. C∗02:09 differs from C∗02:02:01 by a synonymous nucleotide polymorphism at NT 315 in exon 2, A/G, and a non-synonymous polymorphism at NT 613 in exon 3, C/T. Methods PBMCs, fresh buccal swabs and expanded BLCL swabs were obtained for SBT and NGS of B∗49:15. Archived extracted DNA, PBMCs, failed cell culture, and transformed BLCL swabs were obtained for SBT of C∗02:09. Results Three labs confirmed B∗49:01:01 by SBT and NGS on archived PBMCs and newly obtained buccal swabs. NGS revealed the BLCL DNA contained both B∗49:15 and B∗49:01:01 in a 64/36 ratio and a 75/25 ratio in 2 samples. C∗02:09 was confirmed in archived extracted DNA used for the original allele description and in archived PBMC. SBT confirmed C∗02:02 on a failed cell culture from the PBMC to BLCL expansion and on additional transformed BLCL swabs. Conclusion It is likely these discrepancies arose as the result of mutations that occurred during the transformation of PBMC to BLCL and/or during the expansion of existing BLCLs. As a result of this work, B∗49:15 was deleted from the IMGT/HLA database in 6/2014. C∗02:09 was typed by 2 labs using DNA from the same cell, but should now be confirmed by SBT on a second subject. Purified DNA extracted from whole blood has begun to replace BLCL swabs in the BTMR QC program.

Human Immunology, 2013

Aim Natural killer cells (NKC) express many different cell surface receptors including KIR. KIR g... more Aim Natural killer cells (NKC) express many different cell surface receptors including KIR. KIR genes recognize polymorphic HLA class I including HLA-A, B & C allotypes. The KIR genes after binding specific ligands may transmit inhibitory or activating signals. NKC sense & kill target cells lacking MHC class I molecules & release various cytokines on activation. UCB is an increasingly used source of HSC for pts without matched adult donor for Tx. The goal of the study was to attain frequencies (freq) of HLA-L in UCBU aimed at providing information (info) for finding UCB with NK benefit for HSC Tx pts. Methods Total of 21,002 UCBU from Cord Bank at MD Anderson was included. HLA typing of class I & II loci were obtained using PCR/probe based methods & sequence-based typing as needed. Freq of Bw4 alleles, C1 and C2 HLA-C alleles were obtained. Results KIR3DL1 recognizes allotypes with HLA-Bw4 serological motif; KIR2DL1 recognizes HLA-C allotypes with lysine at codon 80 (group 2 - C2); & KIR2DL2/KIR2DL3 recognize C alleles with asparagine at residue 80 (group 1 - C1). Among the 21,002 UCBU, 70.7% carry Bw4 alleles. UCB bearing C1/C2 heterozygous genotype (GT) accounted for 45.6%, C1/C1 homozygous GT for 39.6%, & C2/C2 GT for 14.7% only. Conclusions Incompatibility of KIR HLA-L between pt & donor has been shown to impact graft outcome (GO) in HSC Tx. NKC from the donor greatly contribute to eradication of leukemia blasts escaping the preparative regimen & to clearance of residual host dendritic cells & T lymphocytes thus preventing graft-versus-host disease & graft rejection. This study showed high freq of Bw4-bearing GT, increased freq of C1/C2, & C1/C1 but not C2/C2 GT in UCBU. These freq info could be useful for prediction of the possibility for HSC Tx pts, especially those with Bw6 or C2/C2 GT, to find suitable UCB donor(s) with beneficial NK reactivity, thereby improving GO. Further study of diversification of KIR HLA-ligands in pts would be warranted to assess the importance of these findings.

Human Immunology, 2007

Mismatches in MICA and low expression loci (LEL) DRB3/4/5,DQB1,DPB1 and mutations (SNPs 8, 12,13)... more Mismatches in MICA and low expression loci (LEL) DRB3/4/5,DQB1,DPB1 and mutations (SNPs 8, 12,13) in NOD2/CARD15 were investigated in 134 recipient/unrelated-donor BMT pairs matched in HLA-A/B/C/ DRB1 (8/8) and in 51 pairs with one mismatch at these loci (7/8). Diagnoses were AML, MDS and CML. Univariate and multivariate analyses with the Cox regression model were used to test the effect of these variables in several outcomes including Transplant Related Mortality (TRM) and Acute Graft-versus-Host Disease (aGvHD). Only significant associations are shown. In the 8/8 group, 3 or more mismatches in MICA/ LEL associated with TRM in the multivariate (ORϭ3.87, pϽ0.01) and unviariate analysis when compared to 1-2 mismatches at these loci. One or two mismatches at MICA/LEL showed increased risk for TRM (ORϭ6.25; p not significant). In a previous study we showed that 1-2 mismatches at the class II LEL loci associated with aGvHD in patients transplanted in remission. In the 7/8 group, 3 or more mismatches in MICA or in the low expression loci (ORϭ3.87, pϽ0.04) as well as the presence for NOD2/CARD mutations in the patient (ORϭ3.95, pϽ0.02) associated with grade II-IV aGVHD in the multivariate analysis. Multiple mismatches in HLA loci and MICA appear to have a significant deleterious effect in transplant outcome and should be evaluated as a whole when selecting donors for BMT. NOD/CARD15 mutations may identify patients with more stringent matching requirements.

Human Immunology, 2002

The high degree of polymorphism of the HLA-system generates a large number of genotypes in differ... more The high degree of polymorphism of the HLA-system generates a large number of genotypes in different populations. We investigated the degree of genotype diversity that is generated by PCR SSOP IR complying with NMDP standards for HLA-A,-B and DRB loci. We used a set of 43, 62 and 31 probes for HLA-A,-B and DRB1/3/5, respectively. We have analyzed the HLA-A/-B/-DRB1/3/5 unique probe hybridization patterns (UPP) of 20357 unrelated donors representative of all ethnicities in USA. All donors were typed with the same set of reagents and current code nomenclature. We observed a large number of UPP (17379) that are only a small fraction of all possible genotypes (1.80 x 10 ϩ13). In our panel, 85% of the donors showed different unique genotypes. The first 9 frequent genotypes range from 0.39 to 0.025 % and are likely composed by the 8 best known Caucasian haplotypes. Only 15% of the individuals in the panel have a genotype that was present more than once, illustrating the large diversity of the HLA-system and reflects the difficulties to find fully matched unrelated donors. Analysis of cumulative frequencies showed that 3130 UPP are present in 30% of the donors, 7201 UPP in the 50% and 15344 in 90% of the donors studied. In spite of the high number of observed UPP, these only represent 9.5 x 10-8 % of all possible genotypes. This data suggests that it is necessary to span the unrelated donor registries in order to increase the probabilities to find allele level matched donors. Refining the resolution of certain frequent polymorphic segments in different ethnic groups increases the level of resolution and allows to discriminate the mos common alleles of a particular group. Typing of donors with these sets of reagents allows to narrow down the number of potential donors matching a given patient making the selection of higher resolution tests more cost effective and more efficient. The refinement of the HLA-typing strategy will effectively accelerate the donor search process.

Human Immunology, 2002

Molecular HLA typing has some advantages over classical CDC method. It does not require high cell... more Molecular HLA typing has some advantages over classical CDC method. It does not require high cell viability and complement. In addition, this technology provides higher level of specificity and sensitivity. High-level Class II molecular typing is used for stem cells transplantation in our laboratory. Furthermore, this technique is implicated, when Class II CDC HLA typing for solid organ transplantation is impossible because of low cell number or poor viability. Here we present the case of a rare DRB1/DQB1 association revealed in Filipino renal transplant candidate. High resolution molecular HLA-DR/DQ typing was performed using the PCR-SSP One Lambda kit. The alleles of the following genes were detected: DRB1*1502; DRB5*0101; DRB4*01xx; DQB1*0502/0503. The observed DRB1*1502-DQB1*0502 haplotype was reported to have a high frequency in the Filipino population. However, we did not observe any DRB1*04, DRB1*07 or DRB1*09 lineage associated alleles. The DRB1*04 subtyping One Lambda kit was used to investigate if any DRB1*04 alleles were present. The amplified PCR products were loaded and run on a twenty well, 1.8% agarose gel instead of One Lambda's standard minigel. The DRB1*0405 allele specific pattern of positive reactions ranging from 100 bp to 250 bp in size was observed. To confirm the presence of the DRB1*0405 allele, sequence based typing (SBT) was performed, and DRB1*1502/0405 heterozygocity was detected. The observed DRB1*0405-DQB1*0503 association had been reported in the literature as an unusual Asian/Filipino haplotype. Thus, in the case of unusual allelic associations and/or poor mini gel resolution, the use of a regular high concentration agarose gel and SBT is recommended to define the existing alleles.

Current Opinion in Hematology, 2011

Purpose of review This review summarizes the recent developments in the clinical research and the... more Purpose of review This review summarizes the recent developments in the clinical research and therapeutic applications for prevention and treatment of postallogeneic stem cell transplant (SCT) relapse. Specifically, we address various maneuvers to optimize the graft-versus-leukemia (GVL) effect while preventing graft-versus-host-disease (GVHD). Recent findings Alloreactive natural killer (NK) cells can be recruited to mediate GVL effect by careful mismatching on the killer-cell immunoglobulin-like receptors (KIRs) ligand. Donor posttransplant NK cell infusions have led to remission in poor-risk acute myeloid leukemia (AML). Donor lymphocyte infusions may also be used both prophylactically or at the time of leukemia relapse. Posttransplant hypomethylating agents have emerged as promising therapies to safeguard against relapse. Prevention of GVHD while preserving GVL effect using third-party regulatory T cells is under investigation. Adoptive T-cell transfer against specific leukemic antigens and immunotherapy exploiting the marrow microenvironment seem promising as well. Summary In the setting of allogeneic SCT, cellular and immunotherapy needs to be thoroughly investigated for its potential to fight minimal residual disease. The tight balance between GVL and GVHD needs to be harnessed in an optimal fashion to lead to long-term durable remission.

Cancer Research, 2010

Associations between HLA encoded factors with susceptibility and resistance to development of sev... more Associations between HLA encoded factors with susceptibility and resistance to development of several common autoimmune disorders has been demonstrated in studies conducted more than 30 years ago. Subsequent studies have refined the mapping to these associations. In recent years it has been shown that HLA factors contribute to clearance of viruses including hepatitis C virus, Human Herpes Virus 8 or Epstein-Barr virus (EBV) and with decreased rates of progression to AIDS. EBV may also play a significant role in the pathogenesis of the Inflammatory Breast Cancer (IBC) accounting for some of the peculiar lymphangiogenic mechanism of growth and disease progression. We compared the distribution of alleles of HLA-A, -B, -C and DRB1 loci in 66 IBC patients of European ancestry with that of 776 ethnically matched healthy unrelated volunteers. HLA typing was performed utilizing PCR based assays combined with olugonucleotide hybridization and nucleotide sequencing. We observed that the allel...

Human Immunology, 2015

ABSTRACT Compared to Sanger sequencing, next-generation sequencing offers advantages for high res... more ABSTRACT Compared to Sanger sequencing, next-generation sequencing offers advantages for high resolution HLA genotyping including increased throughput, lower cost, and reduced genotype ambiguity. Here we describe an enhancement of the Roche 454 GS GType HLA genotyping assay to provide very high resolution (VHR) typing, by the addition of 8 primer pairs to the original 14, to genotype 11 HLA loci. These additional amplicons help resolve common and well-documented alleles and exclude commonly found null alleles in genotype ambiguity strings. Simplification of workflow to reduce the initial preparation effort using early pooling of amplicons or the Fluidigm Access Array™ is also described. Performance of the VHR assay was evaluated on 28 well characterized cell lines using Conexio Assign MPS software which uses genomic, rather than cDNA, reference sequence. Concordance was 98.4%; 1.6% had no genotype assignment. Of concordant calls, 53% were unambiguous. To further assess the assay, 59 clinical samples were genotyped and results compared to unambiguous allele assignments obtained by prior sequence-based typing supplemented with SSO and/or SSP. Concordance was 98.7% with 58.2% as unambiguous calls; 1.3% could not be assigned. Our results show that the amplicon-based VHR assay is robust and can replace current Sanger methodology. Together with software enhancements, it has the potential to provide even higher resolution HLA typing. Copyright © 2015. Published by Elsevier Inc.

Frontiers in Immunology, May 7, 2021

The KIR (killer-cell immunoglobulin-like receptor) region is characterized by structural variatio... more The KIR (killer-cell immunoglobulin-like receptor) region is characterized by structural variation and high sequence similarity among genes, imposing technical difficulties for analysis. We undertook the most comprehensive study to date of KIR genetic diversity in a large population sample, applying next-generation sequencing in 2,130 United States European-descendant individuals. Data were analyzed using our custom bioinformatics pipeline specifically designed to address technical obstacles in determining KIR genotypes. Precise gene copy number determination allowed us to identify a set of uncommon gene-content KIR haplotypes accounting for 5.2% of structural variation. In this cohort, KIR2DL4 is the framework gene that most varies in copy number (6.5% of all individuals). We identified phased high-resolution alleles in large multi-locus insertions and also likely founder haplotypes from which they were deleted. Additionally, we observed 250 alleles at 5-digit resolution, of which 90 have frequencies ≥1%. We found sequence patterns that were consistent with the presence of novel alleles in 398 (18.7%) individuals and contextualized multiple orphan dbSNPs within the KIR complex. We also identified a novel KIR2DL1 variant, Pro151Arg, and demonstrated by molecular dynamics that this substitution is predicted to affect interaction with HLA-C. No previous studies have fully explored the full range of structural and sequence variation of KIR as we present here. We demonstrate that pairing high-throughput sequencing with state-of-art computational tools in a large cohort permits exploration of all aspects of KIR variation including determination of population-level haplotype diversity, improving understanding of the KIR system, and providing an important reference for future studies.

Frontiers in Immunology, May 29, 2023

Patients in need of hematopoietic stem cell transplantation often rely on unrelated stem cell don... more Patients in need of hematopoietic stem cell transplantation often rely on unrelated stem cell donors matched in certain human leukocyte antigen (HLA) genes. Donor search is complicated by the extensive allelic variability of the HLA system. Therefore, large registries of potential donors are maintained in many countries worldwide. Population-specific HLA characteristics determine the registry benefits for patients and also the need for further regional donor recruitment. In this work, we analyzed HLA allele and haplotype frequencies of donors of DKMS Chile, the first Chilean donor registry, with self-assessed "non-Indigenous" (n=92,788) and "Mapuche" (n=1,993) ancestry. We identified HLA alleles that were distinctly more abundant in the Chilean subpopulations than in worldwide reference populations, four of them particularly characteristic for the Mapuche subpopulation, namely B*39:09g, B*35:09, DRB1*04:07g, and DRB1*16:02g. Both population subsamples carried haplotypes of both Native American and European origin at high frequencies, reflecting Chile's complex history of admixture and immigration. Matching probability analysis revealed limited benefits for Chilean patients (both non-Indigenous and Mapuche) from donor registries of non-Chilean donors, thus indicating a need for ongoing significant donor recruitment efforts in Chile.

Journal of Immunology, Oct 19, 2004

Blood, 2008

Introduction/Methods: A key component of improving the success rate of allogeneic HSCT for AML in... more Introduction/Methods: A key component of improving the success rate of allogeneic HSCT for AML in CR1 is to reduce non-relapse mortality (NRM), which, if excessive, will deny the benefit of the graft-versus-leukemia effect. UD HSCT has traditionally been associated with high NRM rates. We reported previously on the significant reduction of NRM using the conditioning regimen of fludarabine 40mg/m2, IV busulfan 130 mg/m2 for 4 days and thymoglobulin. Here we analyze the outcomes of all patients (n=37) with AML in CR1 treated with this regimen and UD HSCT in our institution from January 2002 to December 2007. High-resolution allele level HLA typing was performed for all donorrecipient pairs for HLA-A, -B, -C, DRB1 and DQB1; up to one mismatch was allowed (9/10). Median follow up is 30 months (range, 9–72). Results: Median age was 48 years (range, 13–68); 30% (n=11) were older than 54 years and 51% (n=19) were male. Eleven patients (30%) had secondary AML. Prognostic cytogenetics classi...

Blood, 2006

The HLA class II DP locus encode for both subunits of DPB1 heterodimers, which have low levels of... more The HLA class II DP locus encode for both subunits of DPB1 heterodimers, which have low levels of expression on the cell surface of antigen presenting cells. We hypothesized that donor-recipient HLA-DP mismatch would lead to an increased incidence of acute (a) graft-versus-host disease (GVHD), and that 2 mismatches would likely be even more significant. Methods: We studied 84 consecutive patients (pts) with myeloid leukemias in complete remission (CR) transplanted from 01/02 to 02/06. Preparative regimens were ablative IV Busulfan-based (n=58) or Cy/TBI (n=2), and reduced intensity (Fludarabine (Flu)/Bu 130 mg/m2/2 doses plus Gleevec (n=8), and Flu/Melphalan 140 mg/m2 (n=16). Stem cell (SC) source was bone marrow (n=70) or peripheral blood (n=14). ATG was given in 78 cases. GVHD prophylaxis was tacrolimus and mini-methotrexate in all cases, with additional pentostatin in 31 pts. High-resolution typing was sequence-based for HLA-A, B, DRB1; SSP was used for DRB3/4/5, DQB1 and DPB1, a...

Human Immunology, 2015

Aim Swabs created from BLCLs transformed from peripheral blood mononuclear cells (PBMCs) or expan... more Aim Swabs created from BLCLs transformed from peripheral blood mononuclear cells (PBMCs) or expanded from existing BLCLs are used in the Be The Match Registry (BTMR) HLA QC program. The aim of this project was to document mutation as a cause of HLA typing discrepancies observed in 2 BLCLs. The first discrepancy arose in expanded BLCLs, sequenced in 2002 on a donor-derived BLCL. BLCL swabs were routinely typed multiple times using SSO and SBT methodologies as B∗49:01; however, in 2009, an SBT lab reported a potential new allele, B∗49:15, which was described in 2010 and confirmed in 2011 on the expanded BLCL by two labs. B∗49:15 differs from B∗49:01:01 by a single non-synonymous nucleotide polymorphism at NT 176 in exon 2, G/C. In 2013, a lab reported that the B49-specific amplicon showed a double peak, suggesting two alleles were present. Typing of other loci ruled out contamination. The second discrepancy arose in a BLCL transformed from patient-derived PBMC, originally typed as C∗02:09 using DNA extracted from PBMC in 2004. BLCL swabs repeatedly typed as C∗02:02 by SSO, SBT, and NGS. C∗02:09 differs from C∗02:02:01 by a synonymous nucleotide polymorphism at NT 315 in exon 2, A/G, and a non-synonymous polymorphism at NT 613 in exon 3, C/T. Methods PBMCs, fresh buccal swabs and expanded BLCL swabs were obtained for SBT and NGS of B∗49:15. Archived extracted DNA, PBMCs, failed cell culture, and transformed BLCL swabs were obtained for SBT of C∗02:09. Results Three labs confirmed B∗49:01:01 by SBT and NGS on archived PBMCs and newly obtained buccal swabs. NGS revealed the BLCL DNA contained both B∗49:15 and B∗49:01:01 in a 64/36 ratio and a 75/25 ratio in 2 samples. C∗02:09 was confirmed in archived extracted DNA used for the original allele description and in archived PBMC. SBT confirmed C∗02:02 on a failed cell culture from the PBMC to BLCL expansion and on additional transformed BLCL swabs. Conclusion It is likely these discrepancies arose as the result of mutations that occurred during the transformation of PBMC to BLCL and/or during the expansion of existing BLCLs. As a result of this work, B∗49:15 was deleted from the IMGT/HLA database in 6/2014. C∗02:09 was typed by 2 labs using DNA from the same cell, but should now be confirmed by SBT on a second subject. Purified DNA extracted from whole blood has begun to replace BLCL swabs in the BTMR QC program.

Human Immunology, 2013

Aim Natural killer cells (NKC) express many different cell surface receptors including KIR. KIR g... more Aim Natural killer cells (NKC) express many different cell surface receptors including KIR. KIR genes recognize polymorphic HLA class I including HLA-A, B & C allotypes. The KIR genes after binding specific ligands may transmit inhibitory or activating signals. NKC sense & kill target cells lacking MHC class I molecules & release various cytokines on activation. UCB is an increasingly used source of HSC for pts without matched adult donor for Tx. The goal of the study was to attain frequencies (freq) of HLA-L in UCBU aimed at providing information (info) for finding UCB with NK benefit for HSC Tx pts. Methods Total of 21,002 UCBU from Cord Bank at MD Anderson was included. HLA typing of class I & II loci were obtained using PCR/probe based methods & sequence-based typing as needed. Freq of Bw4 alleles, C1 and C2 HLA-C alleles were obtained. Results KIR3DL1 recognizes allotypes with HLA-Bw4 serological motif; KIR2DL1 recognizes HLA-C allotypes with lysine at codon 80 (group 2 - C2); & KIR2DL2/KIR2DL3 recognize C alleles with asparagine at residue 80 (group 1 - C1). Among the 21,002 UCBU, 70.7% carry Bw4 alleles. UCB bearing C1/C2 heterozygous genotype (GT) accounted for 45.6%, C1/C1 homozygous GT for 39.6%, & C2/C2 GT for 14.7% only. Conclusions Incompatibility of KIR HLA-L between pt & donor has been shown to impact graft outcome (GO) in HSC Tx. NKC from the donor greatly contribute to eradication of leukemia blasts escaping the preparative regimen & to clearance of residual host dendritic cells & T lymphocytes thus preventing graft-versus-host disease & graft rejection. This study showed high freq of Bw4-bearing GT, increased freq of C1/C2, & C1/C1 but not C2/C2 GT in UCBU. These freq info could be useful for prediction of the possibility for HSC Tx pts, especially those with Bw6 or C2/C2 GT, to find suitable UCB donor(s) with beneficial NK reactivity, thereby improving GO. Further study of diversification of KIR HLA-ligands in pts would be warranted to assess the importance of these findings.

Human Immunology, 2007

Mismatches in MICA and low expression loci (LEL) DRB3/4/5,DQB1,DPB1 and mutations (SNPs 8, 12,13)... more Mismatches in MICA and low expression loci (LEL) DRB3/4/5,DQB1,DPB1 and mutations (SNPs 8, 12,13) in NOD2/CARD15 were investigated in 134 recipient/unrelated-donor BMT pairs matched in HLA-A/B/C/ DRB1 (8/8) and in 51 pairs with one mismatch at these loci (7/8). Diagnoses were AML, MDS and CML. Univariate and multivariate analyses with the Cox regression model were used to test the effect of these variables in several outcomes including Transplant Related Mortality (TRM) and Acute Graft-versus-Host Disease (aGvHD). Only significant associations are shown. In the 8/8 group, 3 or more mismatches in MICA/ LEL associated with TRM in the multivariate (ORϭ3.87, pϽ0.01) and unviariate analysis when compared to 1-2 mismatches at these loci. One or two mismatches at MICA/LEL showed increased risk for TRM (ORϭ6.25; p not significant). In a previous study we showed that 1-2 mismatches at the class II LEL loci associated with aGvHD in patients transplanted in remission. In the 7/8 group, 3 or more mismatches in MICA or in the low expression loci (ORϭ3.87, pϽ0.04) as well as the presence for NOD2/CARD mutations in the patient (ORϭ3.95, pϽ0.02) associated with grade II-IV aGVHD in the multivariate analysis. Multiple mismatches in HLA loci and MICA appear to have a significant deleterious effect in transplant outcome and should be evaluated as a whole when selecting donors for BMT. NOD/CARD15 mutations may identify patients with more stringent matching requirements.

Human Immunology, 2002

The high degree of polymorphism of the HLA-system generates a large number of genotypes in differ... more The high degree of polymorphism of the HLA-system generates a large number of genotypes in different populations. We investigated the degree of genotype diversity that is generated by PCR SSOP IR complying with NMDP standards for HLA-A,-B and DRB loci. We used a set of 43, 62 and 31 probes for HLA-A,-B and DRB1/3/5, respectively. We have analyzed the HLA-A/-B/-DRB1/3/5 unique probe hybridization patterns (UPP) of 20357 unrelated donors representative of all ethnicities in USA. All donors were typed with the same set of reagents and current code nomenclature. We observed a large number of UPP (17379) that are only a small fraction of all possible genotypes (1.80 x 10 ϩ13). In our panel, 85% of the donors showed different unique genotypes. The first 9 frequent genotypes range from 0.39 to 0.025 % and are likely composed by the 8 best known Caucasian haplotypes. Only 15% of the individuals in the panel have a genotype that was present more than once, illustrating the large diversity of the HLA-system and reflects the difficulties to find fully matched unrelated donors. Analysis of cumulative frequencies showed that 3130 UPP are present in 30% of the donors, 7201 UPP in the 50% and 15344 in 90% of the donors studied. In spite of the high number of observed UPP, these only represent 9.5 x 10-8 % of all possible genotypes. This data suggests that it is necessary to span the unrelated donor registries in order to increase the probabilities to find allele level matched donors. Refining the resolution of certain frequent polymorphic segments in different ethnic groups increases the level of resolution and allows to discriminate the mos common alleles of a particular group. Typing of donors with these sets of reagents allows to narrow down the number of potential donors matching a given patient making the selection of higher resolution tests more cost effective and more efficient. The refinement of the HLA-typing strategy will effectively accelerate the donor search process.

Human Immunology, 2002

Molecular HLA typing has some advantages over classical CDC method. It does not require high cell... more Molecular HLA typing has some advantages over classical CDC method. It does not require high cell viability and complement. In addition, this technology provides higher level of specificity and sensitivity. High-level Class II molecular typing is used for stem cells transplantation in our laboratory. Furthermore, this technique is implicated, when Class II CDC HLA typing for solid organ transplantation is impossible because of low cell number or poor viability. Here we present the case of a rare DRB1/DQB1 association revealed in Filipino renal transplant candidate. High resolution molecular HLA-DR/DQ typing was performed using the PCR-SSP One Lambda kit. The alleles of the following genes were detected: DRB1*1502; DRB5*0101; DRB4*01xx; DQB1*0502/0503. The observed DRB1*1502-DQB1*0502 haplotype was reported to have a high frequency in the Filipino population. However, we did not observe any DRB1*04, DRB1*07 or DRB1*09 lineage associated alleles. The DRB1*04 subtyping One Lambda kit was used to investigate if any DRB1*04 alleles were present. The amplified PCR products were loaded and run on a twenty well, 1.8% agarose gel instead of One Lambda's standard minigel. The DRB1*0405 allele specific pattern of positive reactions ranging from 100 bp to 250 bp in size was observed. To confirm the presence of the DRB1*0405 allele, sequence based typing (SBT) was performed, and DRB1*1502/0405 heterozygocity was detected. The observed DRB1*0405-DQB1*0503 association had been reported in the literature as an unusual Asian/Filipino haplotype. Thus, in the case of unusual allelic associations and/or poor mini gel resolution, the use of a regular high concentration agarose gel and SBT is recommended to define the existing alleles.

Current Opinion in Hematology, 2011

Purpose of review This review summarizes the recent developments in the clinical research and the... more Purpose of review This review summarizes the recent developments in the clinical research and therapeutic applications for prevention and treatment of postallogeneic stem cell transplant (SCT) relapse. Specifically, we address various maneuvers to optimize the graft-versus-leukemia (GVL) effect while preventing graft-versus-host-disease (GVHD). Recent findings Alloreactive natural killer (NK) cells can be recruited to mediate GVL effect by careful mismatching on the killer-cell immunoglobulin-like receptors (KIRs) ligand. Donor posttransplant NK cell infusions have led to remission in poor-risk acute myeloid leukemia (AML). Donor lymphocyte infusions may also be used both prophylactically or at the time of leukemia relapse. Posttransplant hypomethylating agents have emerged as promising therapies to safeguard against relapse. Prevention of GVHD while preserving GVL effect using third-party regulatory T cells is under investigation. Adoptive T-cell transfer against specific leukemic antigens and immunotherapy exploiting the marrow microenvironment seem promising as well. Summary In the setting of allogeneic SCT, cellular and immunotherapy needs to be thoroughly investigated for its potential to fight minimal residual disease. The tight balance between GVL and GVHD needs to be harnessed in an optimal fashion to lead to long-term durable remission.

Cancer Research, 2010

Associations between HLA encoded factors with susceptibility and resistance to development of sev... more Associations between HLA encoded factors with susceptibility and resistance to development of several common autoimmune disorders has been demonstrated in studies conducted more than 30 years ago. Subsequent studies have refined the mapping to these associations. In recent years it has been shown that HLA factors contribute to clearance of viruses including hepatitis C virus, Human Herpes Virus 8 or Epstein-Barr virus (EBV) and with decreased rates of progression to AIDS. EBV may also play a significant role in the pathogenesis of the Inflammatory Breast Cancer (IBC) accounting for some of the peculiar lymphangiogenic mechanism of growth and disease progression. We compared the distribution of alleles of HLA-A, -B, -C and DRB1 loci in 66 IBC patients of European ancestry with that of 776 ethnically matched healthy unrelated volunteers. HLA typing was performed utilizing PCR based assays combined with olugonucleotide hybridization and nucleotide sequencing. We observed that the allel...

Human Immunology, 2015

ABSTRACT Compared to Sanger sequencing, next-generation sequencing offers advantages for high res... more ABSTRACT Compared to Sanger sequencing, next-generation sequencing offers advantages for high resolution HLA genotyping including increased throughput, lower cost, and reduced genotype ambiguity. Here we describe an enhancement of the Roche 454 GS GType HLA genotyping assay to provide very high resolution (VHR) typing, by the addition of 8 primer pairs to the original 14, to genotype 11 HLA loci. These additional amplicons help resolve common and well-documented alleles and exclude commonly found null alleles in genotype ambiguity strings. Simplification of workflow to reduce the initial preparation effort using early pooling of amplicons or the Fluidigm Access Array™ is also described. Performance of the VHR assay was evaluated on 28 well characterized cell lines using Conexio Assign MPS software which uses genomic, rather than cDNA, reference sequence. Concordance was 98.4%; 1.6% had no genotype assignment. Of concordant calls, 53% were unambiguous. To further assess the assay, 59 clinical samples were genotyped and results compared to unambiguous allele assignments obtained by prior sequence-based typing supplemented with SSO and/or SSP. Concordance was 98.7% with 58.2% as unambiguous calls; 1.3% could not be assigned. Our results show that the amplicon-based VHR assay is robust and can replace current Sanger methodology. Together with software enhancements, it has the potential to provide even higher resolution HLA typing. Copyright © 2015. Published by Elsevier Inc.

Frontiers in Immunology, May 7, 2021

The KIR (killer-cell immunoglobulin-like receptor) region is characterized by structural variatio... more The KIR (killer-cell immunoglobulin-like receptor) region is characterized by structural variation and high sequence similarity among genes, imposing technical difficulties for analysis. We undertook the most comprehensive study to date of KIR genetic diversity in a large population sample, applying next-generation sequencing in 2,130 United States European-descendant individuals. Data were analyzed using our custom bioinformatics pipeline specifically designed to address technical obstacles in determining KIR genotypes. Precise gene copy number determination allowed us to identify a set of uncommon gene-content KIR haplotypes accounting for 5.2% of structural variation. In this cohort, KIR2DL4 is the framework gene that most varies in copy number (6.5% of all individuals). We identified phased high-resolution alleles in large multi-locus insertions and also likely founder haplotypes from which they were deleted. Additionally, we observed 250 alleles at 5-digit resolution, of which 90 have frequencies ≥1%. We found sequence patterns that were consistent with the presence of novel alleles in 398 (18.7%) individuals and contextualized multiple orphan dbSNPs within the KIR complex. We also identified a novel KIR2DL1 variant, Pro151Arg, and demonstrated by molecular dynamics that this substitution is predicted to affect interaction with HLA-C. No previous studies have fully explored the full range of structural and sequence variation of KIR as we present here. We demonstrate that pairing high-throughput sequencing with state-of-art computational tools in a large cohort permits exploration of all aspects of KIR variation including determination of population-level haplotype diversity, improving understanding of the KIR system, and providing an important reference for future studies.

Frontiers in Immunology, May 29, 2023

Patients in need of hematopoietic stem cell transplantation often rely on unrelated stem cell don... more Patients in need of hematopoietic stem cell transplantation often rely on unrelated stem cell donors matched in certain human leukocyte antigen (HLA) genes. Donor search is complicated by the extensive allelic variability of the HLA system. Therefore, large registries of potential donors are maintained in many countries worldwide. Population-specific HLA characteristics determine the registry benefits for patients and also the need for further regional donor recruitment. In this work, we analyzed HLA allele and haplotype frequencies of donors of DKMS Chile, the first Chilean donor registry, with self-assessed "non-Indigenous" (n=92,788) and "Mapuche" (n=1,993) ancestry. We identified HLA alleles that were distinctly more abundant in the Chilean subpopulations than in worldwide reference populations, four of them particularly characteristic for the Mapuche subpopulation, namely B*39:09g, B*35:09, DRB1*04:07g, and DRB1*16:02g. Both population subsamples carried haplotypes of both Native American and European origin at high frequencies, reflecting Chile's complex history of admixture and immigration. Matching probability analysis revealed limited benefits for Chilean patients (both non-Indigenous and Mapuche) from donor registries of non-Chilean donors, thus indicating a need for ongoing significant donor recruitment efforts in Chile.

Journal of Immunology, Oct 19, 2004