Marrow Donor Registry Research Articles

Recognition of the three deduced probable human leukocyte antigen haplotypes in association with HLA-A∗31:30 (A∗31:30-B∗15-DRB1∗14) and HLA-B∗40:55 (A∗02:07-B∗40:55-DRB1∗04:05 and A∗26:01-B∗40:55-DRB1∗09:01) in a Taiwanese population

ObjectivesHLA-A∗31:30 and HLA-B∗40:55 are two rarely observed alleles in the HLA-A locus and HLA-B locus, respectively. The objective of this study is to report three deduced probable human leukocyte antigen (HLA) haplotypes in association with HLA-A∗31:30 and HLA-B∗40:55 in unrelated bone marrow hematopoietic stem cell donors. Materials and methodsA sequence-based typing method was used to confirm the two low-incidence alleles observed. A polymerase chain reaction was performed to amplify exons 2, 3, and 4 of the HLA-A, -B, and -C loci and exon 2 of the HLA-DRB1 locus with group-specific primer sets. Amplicons were sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction Kit in both directions according to the manufacturer's protocols. ResultsThe DNA sequence of A∗31:30 is identical to A∗31:01:02 in exons 2, 3, and 4, except for a nucleotide substitution at residue 539 (T→G) resulting in an amino acid replacement at position 156 (Leu→Trp). We deduced the probable HLA haplotype in association with A∗31:30 as A∗31:30-B∗15-DRB1∗14. The DNA sequence of B∗40:55 is identical to B∗40:01:01 in exons 2, 3, and 4 except for a nucleotide exchange at residue 814 (G→A) resulting in an amino acid substitution at position 248 (Val→Met). Two probable HLA haplotypes associated with B∗40:55 may be deduced as A∗02:07-B∗40:55-DRB1∗04:05 and A∗26:01-B∗40:55-DRB1∗09:01. ConclusionInformation about the deduced HLA haplotypes associated with the rare A∗31:30 and B∗40:55 alleles that we reported here is valuable for HLA tissue typing laboratories for reference purposes and for stem cell transplantation donor search coordinators to determine the likelihood of finding compatible donors in unrelated bone marrow donor registries for patients bearing these two uncommon HLA alleles. Because A∗31:30 and B∗40:55 have been found in Taiwanese population so far, we think the haplotypes that we reported here are most likely conserved in their population.

Volunteer unrelated donor experience after administration of filgrastim and apheresis for the collection of haemopoietic stem cells: the Australian perspective

Voluntary donations of peripheral blood stem cells after administration of filgrastim (granulocyte-colony stimulating factor, G-CSF) are undertaken throughout the world by healthy individuals, but the short-, medium- and long-term adverse events during and after donation are not fully understood. We document the experience of donors of peripheral blood stem cells mobilised by G-CSF at Australian Bone Marrow Donor Registry collection centres. When the Australian Bone Marrow Donor Registry commenced collecting mobilised peripheral blood stem cells, based on data used for registration of G-CSF, all adverse reactions in donors were documented prospectively to determine the rate and severity of events. A total of 512 consecutive first-time donors assessed between July 2001 and March 2010 were included in this study. The median age at work-up was 40 years and 71% of donors were male. A large proportion of donors (91%) experienced bone pain during administration of G-CSF, and in fewer numbers headache (61%) and fatigue (61%). Bone pain was associated with a body mass index of overweight/obese (P = 0.03). Headache (P = 0.03), muscle pain (P = 0.03) and fatigue (P = 0.001) were all significantly associated with female sex. More than a quarter (28%) of donations involved a range of complications at collection. The incidence of short- and medium-term symptoms and events observed provide support for the information provided to unrelated donors at counselling. Follow up of the consequences of unrelated voluntary donation remains important to provide accurate and relevant information to prospective donors.

136-P: THE RELEVANCE OF ALLELE LEVEL A-B-DRB1 HAPLOTYPES IN THE POPULATION STRUCTURE AND THE CLINICAL SETTING IN MEXICAN MESTIZOS

Aim Allele DNA typing has significantly increased the level of diversity revealing multiple functional variants. Some alleles are found in multiple populations with distinctive haplotype associations, suggesting convergent evolution. HLA has been under strong selection, owing to its fundamental role in the immune response. We analyzed alleles and three loci haplotypes distribution in Mexicans, because of the need to know thoroughly the HLA structure to look into disease associations and BMT donors selection. Methods A total of 87 healthy Mexicans registered at DONORMO-The Mexican Unrelated Bone Marrow Donor Registry , born in different states of Mexico were included. DNA samples were sequenced in an ABI Prism 310. The Allele and Haplotype (HF) analysis was performed with the Arlequin 3.5.1.2 software using the maximum likelihood method. Results The 10 most prevalent A∗-B∗-DRB1∗ haplotypes (HF =0.052-0.017) were ∗02:06-∗39:05-∗04:07, Hispanic; ∗02:01-∗35:12-∗08:02, Mexican Natives, Mestizos & , Hispanic ; ∗02:06 -∗14:02-∗01:02, rare ; ∗02:01-∗52:01 ∗15:02 European ; ∗11:01-∗18:01-∗ 11:04, Mediterranean ; ∗24:02-∗52:01-∗14:06, rare; ∗26:01-∗44:02 -∗04:02 Hispanic; ∗33:01-∗14:02-∗01:02 Mediterranean; ∗33:01-∗78:01-∗13:02, rare ; & ∗68:03-∗39:05 -∗04:07 Mexican, Hispanic . Conclusions Even some alleles are found in many different populations, they are grouped with distinctive haplotypic associations, suggesting distinct genetic events may have occurred. Unique or rare A∗-B∗-DRB1∗ haplotypes are present such as: ∗ 02:06- ∗ 14:02- ∗ 01:02, HF=0.029%; ∗ 24:02- ∗ 52:01- ∗ 14:06, HF=0.017%, ∗ 33:01- ∗ 78:01- ∗ 13:02 HF= 0.017% . The first is rarely found in Europe. The second one is unique to Hispanics (0.00025) and the last one is rare only in Africans (0.00065). We found 12 more haplotypes not ever detected in any other population and three more with HF=0.011. The relevance of these data in transplantation are undoubtful. The process of donor selection will be directed more efficiently for patients with these genetic profiles.

139-P: HLA-A, -B, -C, -DRB1, -DQA1, -DQB1 AND -DPB1 ALLELE FREQUENCIES IN A SAUDI POPULATION USING NEXT GENERATION SEQUENCING TECHNIQUE

Next generation sequencing is a promising technique that can reveal the entire gene sequences and to the highest possible resolution without any phase ambiguities. We have employed this technique to investigate the frequencies of HLA-A, -B, -C, -DRB3/4/5, -DRB1, -DQA1, -DQB1 and -DPB1 in a Saudi cohort of healthy individuals. We employed Next Generation Sequencing using the 454 genome sequence (GS) FLX System and Conexio Assign ATF 454 software to HLA genotype eight class I and class II loci. A total of 158 healthy Saudi adults were analyzed. The most frequent allele for HLA-A was HLA-A∗02:01:01:01 (13.6%); for HLA-B, HLA-B∗50:01:01 (15.8%); for HLA-C, HLA-C∗06:02:01:01 (18.7%); for HLA-DRB1, HLA-DRB1∗07:01:01:01 (26.6%); for HLA-DRB3/4/5, HLA-DRB4∗01:01:01:01 (44.0%); for HLA-DQA1, HLA- DQA1∗02:01(26.3%); for HLA-DQB1, HLA- DQB1∗02:01:01 (20.3%) and for HLA-DPB1, HLA- DPB1∗04:01:01 (33.2%). By studying Linkage Disequilibrium in this population the following two loci combinations were well represented; B∗50:01:01-C∗06:02:01:01; DRB1∗03:01:01:01-DQB1∗02:01:01; DRB1∗07:01:01:01-DQB1∗02:02. Two extended, conserved haplotypes were well represented in this population; A∗24:02:01:01-B∗08:01:01-C∗07:02:01:01- DRB1∗03:01:01:01-DRB3∗02:02:01-DQA1∗05:01:01- DQB1∗02:01:01-DPB1∗03:01:01 and A∗30:02:01-B∗53:01:01-C∗04:01:01:01-DRB1∗07: 01:01:01-DRB4∗01:01:01:01-DQA1∗02:01-DQB1∗03:03:02- DPB1∗04:01:01. Expected heterozygosity was significantly higher than observed. We have used a highly informative technique for HLA typing of a Saudi healthy cohort to establish allele and haplotype frequencies. This should be of great importance for population studies, disease associations and future planning of the unrelated bone marrow donor registry.

Effects of Coffee Consumption, Smoking, and Hormones on Risk for Primary Sclerosing Cholangitis

Little is known about nongenetic risk factors for primary sclerosing cholangitis (PSC), except a possible protective effect of smoking. We investigated the relationship between environmental risk factors and susceptibility to PSC. A questionnaire was distributed to patients with PSC, recruited from Oslo University Hospital Rikshospitalet in Norway through 2011, and randomly chosen individuals from the Norwegian Bone Marrow Donor Registry (control subjects). Data were analyzed from 240 patients with PSC and 245 control subjects, matched for gender and age. A lower proportion of patients with PSC were daily coffee drinkers than control subjects, bothcurrently (76% vs 86%; odds ratio [OR], 0.52; 95% confidence interval [CI], 0.32-0.82; P=.006) and at the age of 18 years (35% vs 49%; OR, 0.58; 95% CI, 0.40-0.83; P= .003). The associations were mainly attributed to differences observed in men. Twenty percent of the patients were ever (current or former) daily smokers compared with 43% of control subjects (OR, 0.33; 95% CI, 0.22-0.50; P < .001). Ever daily smoking before PSC diagnosis was associated with older age at diagnosis (42 years vs 32 years; P < .001). Ever daily smoking (P < .001) and being a coffee drinker at the age of 18 years (P= .048) were independently and negatively associated with PSC. Fewer female patients with PSC than control subjects reported ever use of hormonal contraception (51% vs 85%; P < .001). Among female patients, there was a strong correlation between increasing number of children before the diagnosis of PSC and increasing age at diagnosis (r= 0.63; P < .001). Coffee consumption and smoking might protect against development of PSC. In women, the disease might be influenced by hormonal factors.

HLA‐A, ‐B, ‐C, ‐DRB1 and ‐DQB1 allele and haplotype frequencies in Saudis using next generation sequencing technique

Next generation sequencing (NGS) is a promising technique that can reveal the entire gene sequences and to the highest possible resolution without any phase ambiguities. We have used this technique to investigate the frequencies of HLA-A, -B, -C, -DRB1 and -DQB1 in a Saudi cohort of healthy individuals. We used NGS using the 454 genome sequence (GS) FLX System and Conexio assign atf 454 software to human leukocyte antigen (HLA) genotype eight class I and class II loci. A total of 158 healthy Saudi adults were analyzed. The most frequently observed allele for HLA-A was HLA-A*02:01:01:01 (13.6%); for HLA-B, HLA-B*50:01:01 (15.8%); for HLA-C, HLA-C*06:02:01:01 (18.7%); for HLA-DRB1, HLA-DRB1*07:01:01:01 (26.6%); and for HLA-DQB1, HLA-DQB1*02:01:01 (20.3%). The most common four loci haplotypes in the Saudi population were HLA-A*24:02:01:01-B*08:01:01-C*07:02:01:01-DRB1*03:01:01:01 and HLA-A*23:01:01-B*50:01:01-C*06:02:01:01-DRB1*07:01:01:01.. We have used a highly informative technique for HLA typing of a Saudi healthy cohort to establish allele and haplotype frequencies. These results should prove useful for population studies, disease associations and future planning of the unrelated bone marrow donor registry.

A novel HLA‐DRB1 allele, HLA‐DRB1*13:116, identified by sequencing‐based typing in a member of the Czech national marrow donor registry

We describe the identification of a novel HLA-DRB1 allele, DRB1*13:116, in a member of the Czech National Marrow Donor Registry. The novel allele differs from the known DRB1*13:17 variant by a nucleotide exchange at position 227 (T/A) of the coding HLA-DRB1 sequence, which causes an amino acid substitution (Phe47Tyr) in the HLA-DR beta 1 chain.

HLA-A, HLA-B and HLA-DRB1 haplotype frequencies in Piauí’s volunteer bone marrow donors enrolled at the Brazilian registry

This study aimed to report the antigen and haplotype frequencies (HFs) of volunteer bone marrow donors (VBMDs) from the state of Piauí who were enrolled in the National Volunteer Bone Marrow Donor Registry (REDOME). The research subjects were 21,943 volunteer bone marrow donors, predominantly young adult women (53.3%). The most frequent allelic group was HLA-A2, followed by -DRB1*13, -DRB1*04, -DRB1*07, -B*15, -B∗35, -B*44, -A*24 and -A*03.Of the 2,704 haplotypes observed, the three most frequent haplotypes were A*29 B*44 DRB1*07 (1.45%), A*01 B*08 DRB1*03 (1.4%) and A*03 B*07 DRB1*15 (0.92%). These three haplotypes were in linkage disequilibrium.PCA showed that 98% of the VBMDs have HLA allele frequencies that are very similar to those from Teresina, the capital city of Piauí. According to the PCA results, these municipalities are distributed with a close proximity to Teresina, which in turn has a close genetic proximity to the Hispanic ethnicity, intermediate proximity to Caucasians and Africans and a distant kinship to Amerindians. The hierarchical proximity of the population of Piauí to the Portuguese and Hispanic populations to shows the strong influence of the latter on the former.

Two conserved HLA haplotypes (HLA-A∗11:127N-B∗54:01-DRB1∗04:05 and HLA-A∗11:01-B∗40:221-C∗03:04-DRB1∗14:54-DQB1∗05:02) observed in the Taiwanese population

ObjectiveHLA-A∗11:127N and HLA-B∗40:221 are two rarely observed alleles in the HLA-A locus and in HLA-B locus, respectively. The objective of this study is to report the two plausible deduced HLA haplotypes in association with HLA-A∗11:127N and HLA-B∗40:221 in unrelated Taiwanese bone marrow stem cell donors. Materials and MethodsA sequence-based typing method was used to confirm the low incidence alleles observed. Polymerase chain reaction was carried out to amplify exons 2, 3, and 4 of the HLA-A, -B, and -C loci with group-specific primer sets. Amplicons were sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction Kit in both directions according to the manufacturer's protocol. ResultsWe deduced the two probable HLA haplotypes in association with A∗11:127N and B∗40:221 as HLA-A∗11:127N-B∗54:01-DRB1∗04:05 and HLA-A∗11:01-B∗40:221-C∗03:04-DRB1∗14:54-DQB1∗05:02, respectively. ConclusionInformation on the two deduced HLA haplotypes associated with the rare A∗11:127N and B∗40:221 alleles that we report here is valuable for HLA tissue typing laboratories for reference purposes and for stem cell transplantation donor search coordinators to determine the likelihood of finding compatible donors in unrelated bone marrow donor registries for patients bearing these two uncommon HLA alleles. Because A∗11:127N and B∗40:221 have so far been found only in the Taiwanese population, we think the haplotypes that we report here are most likely conserved in Taiwanese.

1025-LB

Aim Next generation sequencing of HLA exonic amplicons with the 454 Life Sciences GS FLX System and Conexio Assign TM -ATF software provides high resolution, high throughput HLA genotyping for 8 class I and class II loci (Bentley et al., 2009 1 , Holcomb et al., 2011 2 ). HLA typing of potential donors for Unrelated Bone Marrow Donor Registries requires using a subset of these loci at high sample throughput and low cost per sample. To maximize the throughput, we have incorporated the Fluidigm ® Access Array TM system to simplify amplicon library preparation and allow the efficient introduction of MIDs (Multiplex Identifiers) or barcodes. Methods For this application, a streamlined amplicon sequencing workflow employing many different Multiplex Identifiers (MIDs) enables multiplexed sequencing of a large number of samples, allowing researchers to meet these cost targets. The Fluidigm ® 48 x 48 Access Array TM system and the “4 primer” approach provides an efficient way to achieve parallel semi-automated genomic PCRs with simultaneous incorporation of 48 different MIDs corresponding to 48 genomic DNA samples, and up to 48 different primer pairs, making possible 2,304 reactions in one amplification run. Minimal volumes (calculated to be about 45% less cost per run) of reagents are used in this micro- fluidics-based system. During genomic PCR, the outer set of primers containing the MIDs and the 454 adaptor sequences are incorporated into the amplicons generated by the inner set of HLA specific primers containing a complementary “universal” Fluidigm tag. Pools of the resulting amplicons are used for emulsion PCR and clonal sequencing on the 454 GS FLX System, as well as genotyping with Conexio Assign TM -ATF software. Results Using the Access Array system, we have successfully (100% concordance with known genotypes) genotyped 192 samples with 8 primer pairs (covering exons 2 and 3 in HLA-A, B, C and Exon 2 in DRB1, DRB3/4/5 and DQB1) using 96 MIDs per region in a single GS FLX run on a 2 region PicoTiterPlate™ (PTP) and 96 samples using 48 MIDs per region in a 4 region PTP. An average of 166 and 137 sequence reads per amplicon were recovered respectively. We have also genotyped, in a single run, 96 samples at high resolution (14 primer pairs covering exons 2, 3, and 4 of the class I loci and exons 2 of DRB1,3/4/5, DQA1, DQB1, DPB1, and exon 3 of DQB1) recovering an average of 175 sequence reads per amplicon at 100% concordance. The system reduces the overall time for the entire process (192 samples) from 8 days for manual processing to about 4 days with 1 FTE.

Allogeneic Hematopoietic Cell Transplantation from Unrelated Donors in Multiple Myeloma: Study from the Italian Bone Marrow Donor Registry

To evaluate trends in allografting from unrelated donors, we conducted a study on 196 consecutive myeloma patients transplanted between 2000 and 2009 in Italy. Twenty-eight percent, 37%, and 35%, respectively, received myeloablative, reduced-intensity, and nonmyeloablative conditioning. In these 3 cohorts, 1-year and 5-year transplantation-related mortalities were 28.8% and 37.0%, 20.3% and 31.3%, and 25.0% and 30.3%, respectively (P = .745). Median overall survival (OS) and event-free survival from transplantation for the 3 cohorts were 29 and 10 months, 11 and 6 months, and 32 and 13 months, respectively (P = .039 and P = .049). Overall cumulative incidences of acute and chronic graft-versus-host-disease (GVHD) were 46.1% and 51.1%. By Cox multivariate analyses, chronic GVHD was significantly associated with longer OS (hazard ratio [HR], .51; P = .009), whereas the use of peripheral blood stem cells was borderline significant (HR, .55; P = .051). Better response posttransplantation was associated with longer event-free survival (HR, 2.13 to 4.25; P < .001). Acute GVHD was associated with poorer OS (HR, 2.53; P = .001). This analysis showed a strong association of acute and chronic GVHD and depth of response posttransplantation with clinical outcomes. Long-term disease control remains challenging regardless of the conditioning. In the light of these results, prospective trials may be designed to better define the role of allografting from unrelated donors in myeloma.

Probable HLA haplotypes in association with the uncommon HLA-C*03:36, -C*03:56, and -C*03:86 alleles in a Taiwanese population

ObjectiveHLA-C*03:36, -C*03:56 and -C*03:86 are three human leukocyte antigen type C (HLA-C) locus alleles that are rarely observed in the Taiwanese population. The objective of this study is to report the three plausible deduced HLA haplotypes in association with C*03:36, C*03:56, and C*03:86 in Taiwanese unrelated bone marrow stem cell donors. Materials and MethodsThe sequence-based typing method was used to confirm the low-incidence alleles observed. Polymerase chain reaction was carried out to amplify exons 2 and 3 of the HLA-C locus with group-specific primer sets. Amplicons were sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction Kit in both directions according to the manufacturer's protocol. ResultsWe confirmed the DNA sequences of C*03:36, C*03:56, and C*03:86 and deduced the three plausible HLA haplotypes in association with C*03:36 (A*33:03-B*58:01-C*03:36-DRB1*01:01), C*03:56 (A*02:01-B*48:01-C*03:56-DRB1*12:02), and C*03:86 (A*02-B*35-C*03:86-DRB1*09:01) in unrelated Taiwanese bone marrow stem cell donors. ConclusionThe three uncommon HLA-C haplotypes that we provide here are valuable for HLA tissue-typing laboratories for reference purposes and for stem cell transplantation donor search coordinators to determine the likelihood of finding compatible donors in unrelated bone marrow donor registries for patients bearing these three uncommon HLA-C locus alleles.

Identifying belief targets to increase bone marrow registry participation among students who have never donated blood

New members on bone marrow registries worldwide are needed to allow sufficient diversity in the donor pool to meet patient needs. We used the theory of planned behaviour belief-basis and surveyed students who had not donated blood previously (i.e. non-donors) (N = 150) about the behavioural, normative, and control beliefs informing their intentions to join the Australian Bone Marrow Donor Registry. Key beliefs predicting non-donors’ intentions included: viewing bone marrow donation as an invasion of the body (β = −.35), normative support from parents (β = .40), anticipating pain/side effects from giving blood (β = −.27), and lack of knowledge about how to register (β = −.30). Few non-donors endorsed these beliefs, suggesting they are ideal targets for change in strategies encouraging bone marrow donor registration.

Recognition of HLA-A*11:01-B*51:01-C*14:02-DRB1*11:01-DQB1*03:13 and HLA-A*02-B*40-C*03:77-DRB1*14 haplotypes restricted to Taiwanese

ObjectiveIn the Tzu Chi Taiwanese Bone Marrow Donor Registry individuals carrying the human leukocyte antigen (HLA)-C*03:77 allele and -DQB1*03:13 allele were registered. Here we report the confirmed DNA sequences of C*03:77 and DQB1*03:13 and the probable HLA haplotypes deduced in association with C*03:77 (i.e., HLA-A*02-B*40-C*03:77) and DQB1*03:13 (i.e., HLA-A*11:01-B*51:01-C*14:02-DRB1*11:01-DQB1*03:13) in Taiwanese. Materials and MethodsA sequence-based typing (SBT) method was used to confirm the low incidence alleles observed. Polymerase chain reaction was carried out to amplify exons 2 and 3 of the HLA-C locus with group-specific primer sets. For HLA-DQB1 allelic typing, six group-specific primer sets were used in the SBT procedure. Amplicons were sequenced using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (versions 3.1; Applied Biosystems, Foster City, CA, USA) in both directions according to the manufacturer's protocols. ResultsWe confirmed the DNA sequence of C*03:77, which is identical to the sequence of C*03:04:01:01 in exons 2 and 3 except for the nucleotide at position 527 (A→T). The nucleotide substitution caused an amino acid replacement at residue (codon) 152 (E→V). Similarly, our SBT confirmed that the DNA sequence of DQB1*03:13 is analogous to the sequence of DQB1*03:01:01:01 in exons 2 and 3, except for the nucleotide at residue 296 (T→A). The nucleotide variation caused one amino acid exchange at residue (codon) 67 (V→D). ConclusionsThe Taiwanese ethnicity of our donors identified in this study completes the ethnicity background for C*03:77 and DQB1*03:13 alleles listed on the IMGT/HLA Database and provides a strategy for marrow donor registry search coordinators to screen for unrelated HLA matched hematopoietic stem cell donors for blood disease patients bearing C*03:77 or DQB1*03:13.

High throughput HLA genotyping using 454 sequencing and the Fluidigm Access Array™ system for simplified amplicon library preparation

The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome; distinguishing the thousands of HLA alleles is challenging. Next generation sequencing of exonic amplicons with the 454 genome sequence (GS) FLX System and Conexio Assign ATF 454 software provides high resolution, high throughput HLA genotyping for eight class I and class II loci. HLA typing of potential donors for unrelated bone marrow donor registries typically uses a subset of these loci at high sample throughput and low cost per sample. The Fluidigm Access Array System enables the incorporation of 48 different multiplex identifiers (MIDs) corresponding to 48 genomic DNA samples with up to 48 different primer pairs in a microfluidic device generating 2304 parallel polymerase chain reactions (PCRs). Minimal volumes of reagents are used. During genomic PCR, in this 4-primer system, the outer set of primers containing the MID and the 454 adaptor sequences are incorporated into an amplicon generated by the inner HLA target-specific primers each containing a common sequence tag at the 5' end of the forward and reverse primers. Pools of the resulting amplicons are used for emulsion PCR and clonal sequencing on the 454 Life Sciences GS FLX System, followed by genotyping with Conexio software. We have genotyped 192 samples with 100% concordance to known genotypes using 8 primer pairs (covering exons 2 and 3 of HLA-A, B and C, and exon 2 of DRB1, 3/4/5 and DQB1) and 96 MIDs in a single GS FLX run. An average of 166 reads per amplicon was obtained. We have also genotyped 96 samples at high resolution (14 primer pairs covering exons 2, 3, and 4 of the class I loci and exons 2 of DRB1, 3/4/5, DQA1, DQB1, DPB1, and exon 3 of DQB1), recovering an average of 173 sequence reads per amplicon.

Homozygosity for killer immunoglobin-like receptor haplotype A predicts complete molecular response to treatment with tyrosine kinase inhibitors in chronic myeloid leukemia patients

Several recent reports suggest a possible role for killer immunoglobulin-like receptors (KIR) in the onset of chronic myeloid leukemia (CML) and response to therapy with tyrosine kinase inhibitors (TKIs). To explore this hypothesis, we studied KIRs and their human leukocyte antigen class I ligands in 59 consecutive patients with chronic-phase CML (mean age, 53 years; range, 23-81 years) and a group of 121 healthy control participants belonging to the same ethnic group as the patients. The 2-year cumulative incidence of complete molecular response, obtained after a median of 27 months (range, 4-52 months), was 51.2%. An increased frequency of the activating receptor KIR2DS1 (pm = 0.05) and a reduced frequency of the KIR-ligand combination KIR2DS2/2DL2 absent/C1 present (pm = 0.001) were significantly associated with CML. Moreover, KIR repertoires in patients appeared to influence response to TKI therapy. Homozygosity for KIR haplotype A (pm = 0.01), a decreased frequency of the inhibitory KIR gene KIR2DL2 (pm = 0.02), and low numbers of inhibitory KIR genes (pm = 0.05) were all significantly associated with achievement of complete molecular remission. These data suggest that a decrease in properly stimulated and activated NK cells might contribute to the occurrence of CML and indicate homozygosity for KIR haplotype A as a promising immunogenetic marker of complete molecular response that could help clinicians decide whether to withdraw treatment in patients with CML.

Treatment of Relapse After Allogeneic Hematopoietic Stem-Cell Transplantation for Myelodysplastic Syndrome: A Large-Scale Study On Behalf of the Société Française De Greffe De Moelle Et De Thérapie Cellulaire (SFGM-TC)

AbstractAbstract ▪593▪This icon denotes a clinically relevant abstract Background:Treatment of patients with myelodysplastic syndrome (MDS) relapsing after allo-SCT remains disappointing and prognostic factors for outcome are still unclear. Several therapeutic approaches are offered to those patients including: palliative and supportive care, intensive chemotherapy (ICT), demethylating agents (DMA) and immunotherapy with either donor lymphocyte infusion (DLI) or second allo-SCT. The aim of this study was to identify predictive factors for outcome and to investigate the impact of different treatment groups on survival. Methods:We report a retrospective study on 137 consecutive MDS patients who relapsed after allo-SCT between Jul 1999 and March 2011 in 19 French and Belgian centers. Data quality was ensured using computerized discrepancy errors and vigorous on-site data verification of every single file. HLA matching was double-checked by the French Bone Marrow Donor Registry. Results:At diagnosis, 61 patients presented RA/RARS/RCMD, 27 RAEB1 and 49 RAEB2 according the WHO classification. Cytogenetic was unfavorable in 47 patients (35%) while 76 patients (57%) had IPSS Int-2 or High and 52 (39%) had progressed to a more advanced disease before allo-SCT. At transplant, 62 patients (47%) were considered responders (in CR or PR), while 70 were transplanted with progressive disease (untreated, stable without hematological improvement, relapsed or refractory disease). Patients received either myeloablative (MAC) (n=38) or nonmyeloablative (RIC) (n=99) conditioning. Source of stem cells was BM (n=40) or PBSC (n=97) from sibling (n=89) or allele well-matched unrelated (10/10) (n=48) donors. Median age at relapse was 57 years (range, 19–70). Patients aged <18 years and those who received graft from either cord blood or mismatched donor were excluded.Post-transplant relapse occurred after a median time of 6 months (range, 0.8–102). Of the 109 evaluable patients 62 (57%) had >= 10% of marrow blasts, of whom 38 had >= 20%. At the time of relapse, 61 patients were still under immunosuppressive treatment which was quickly stopped in all of them. For the analysis, patients were divided into three groups according to ultimate salvage received treatment as follows: palliative and supportive care (PSC-group) (n=42, 31%), cytoreductive treatment alone (CRT-group) [DMA (n=18, 13%) and ICT (n= 11, 8%)] and immunotherapy preceded or not by a CRT (IT-group) [DLI (n=48, 35%) and second allo-SCT (n=18, 13%)]. With a median follow-up of 33 months from the relapse, estimated 2-year overall survival was 2.4%, 6.7% and 30.3% for the PSC-group, CRT-group and IT-group, respectively (p<.0001). In multivariate analysis, 3 independent factors have been identified: the absence of immunotherapy HR= 1.45 [95% IC= 1.25–1.69, p<.0001], early relapse within 6 months after transplant, HR=2.71 [95% IC= 1.66–4.45, p<.0001] and marrow blasts at relapse >= 10%, HR=2.56 [95% IC= 1.55–4.22, p<.0001]. Conclusion:This study shows that salvage immunotherapy (DLI or second allo-SCT) provides the best results and should, whenever possible, be offered to patients with MDS who relapse after allo-SCT especially those with low tumor burden.Patients who received cytoreductive treatment alone (be it chemotherapy or DMA) had less satisfactory outcome. Our results emphasize the need to perform prospective protocols combining cytoreductive treatments and immunotherapy. Disclosures:No relevant conflicts of interest to declare.

Adult ALL Patients Treated with Pediatric Inspired Trials Have Inacceptable Early Death but Better Relapse Free Survival Compared to Adult Trials: A Tunisian Single Center Experience

Abstract 4297 Background:In Tunisia children with acute lymphoblastic leukemia (ALL) are treated according to the EORTC 58951 protocol with complete remission rate (CR) of 84% and long-term survivals reaching 80% at 5 years. Survivals of adult ALL remain disappointing mainly because of the high rate of early deaths, the limited number of allografted patients performed only for those aged < 40y, and lack of access to the international bone marrow donors registry. Are the improvements made in the last decades in developed countries in the treatment of adult ALL transposable for patients diagnosed in developing countries? The aim of the present study is to report the experience of a Tunisian hematology center in the management of adult ALL. Patients and methods:Fifty one adult ALL patients were treated with three consecutive trials in the hematology department of Aziza Othmana university hospital of Tunis between January 2005 and July 2011.18 patients were treated with Tunisian LALA03 protocol (pediatric –inspired induction) between 2005 and 2007: BFM-like HR induction (PND 60mg/m2x22d, VCRx4, DNR 30mg/m2x4, ASPx8). HR patients receive 6 consolidations blocs (GRAALL03 blocs) + CNS RT+ maintenance therapy. SR patients receive EORTC AR2 consolidations (IB, interval therapy, IIAIIB) +CNS RT +maintenance therapy. This protocol led to an inacceptable rate of induction death of 38.8%. 24 and 9 patients were respectively treated with Hyper-CVAD regimen from 2008 to 2010 and GRAALL05 since 2010. Results:Male/female ratio was is 2.6. Median age was 37.5 years (21–59). Immunophenotyping revealed B-cell precursor and T-lineage ALL in respectively 60% and 35% of patients. 1 patient had biphenotypic leukemia and the immunophenotype was not precised in one case. Median baseline WBC count was 18.2 × 109/L (0.3–265 ×109/L). Three patients presented with central nervous system (CNS) disease at diagnosis. 21.5% of patients have Philadelphia positive ALL. Only 14 patients had SCT from a matched sibling donor. Complete remission rate after induction was 74.5%. Eight patients (15.6%) had early death during induction from septic shock and pneumonia in 5 and 3 cases respectively. Median overall survival (OS) was 18 months. 3 years OS was 30%. By univariate analysis, age less than 32 years (p=0.01) and bone marrow transplantation (p=0.04) had positive impact on OS. For the whole study population 3 year relapse free survival (RFS) was 20%. Despite a lower RFS with Hyper CVAD there was no significant difference between the three trials but a trend to better outcome with pediatric inspired trials i.e. LALA03 and GRAALL05. Conclusion:Pediatric-inspired protocols are associated with increased early death but prolonged remission duration compared to Hyper-CVAD. Hyper-CVAD is well tolerated but is associated to a high rate of relapse in our patients mainly because of the small number of SCT. The outcome of adult ALL in Tunisia is still unsatisfactory and can be improved with pediatric-like regimens. Effective therapy for adult ALL is feasible in our country. Controlling death induction by supportive measures and better management of myelosuppression and offering a stem cell transplant to a greater number of patients could be an option to improve on the RFS and OS. Disclosures:No relevant conflicts of interest to declare.

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Aim When doing search for matched unrelated donor (MUD) in the international bone marrow donor registries, it is recommended to start the search with HLA class I and II high resolution typing of the patient. However, performing high resolution typing specially for HLA class I; is not available in many laboratories in contrast to HLA class II high resolution, due to cost of equipment, reagent and technical expertise and TAT. The Aim of this study was to determine the correlation between the results of HLA class I typing obtained by One Lambda rSSO Lab Type method (suggested allele level typing) and high resolution HLA sequence based typing (SBT). Methods A total of 50 samples previously SBT typed specimen were also typed by rSSO Lab Type (One Lambda, Inc.). The correlation was made of the ability of the rSSO Lab Type low resolution typing suggested first or second allele choice in each locus to predict the correct HLA allele. Results See [ Table 1 ]. Conclusions When we obtain the potential matched donors list for the search that was performed with low resolution HLA class I and high resolution HLA class II typing of the patient, in our hand we found the Lab Type rSSO method to be fast and reliable for screening (inclusion/exclusion) of possible match donor. Once a potential matched donor is identified based on the alleles identified by the Lab Type rSSO. An actual high resolution SBT typing would have been ready for confirmation of the matching level.

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Marrow donor registries continue to evolve their processes and technology with the goal of reducing time required to find a match for a patient in need. Critical factors in achieving this goal include facilitating donor recruitment, collecting samples that meet requirements for the HLA typing technologies, and integrating cost efficiently into laboratory procedures. Non-invasive saliva sample collection with the Oragene family of products delivers a high quality and reliable solution for collection, stabilization and transportation of DNA. Saliva samples are currently being used as a reliable source of DNA for technologies such as SSO, SSP, SBT. Here we demonstrate in a comparison with blood that saliva collected using the Oragene family of products is a reliable sample for next generation sequencing. Saliva was collected using the Oragene collection device and then stored at room temperature until processing. Blood was collected into an EDTA tube and processed immediately by purifying on a silica column. Saliva samples were purified using PrepIT ® L2P from DNA Genotek. In this study we used the Raindance HLASeq™ research panel to tile amplicons across the entire HLA locus, which includes all Class I and Class II genes. The generated amplicons were then sequenced on the Ion Torrent™ Personal Genome Machine™ using the Ion 318™ chip. DNA extracted from saliva collected using Oragene was successfully amplified using the Raindance HLASeq panel and sequenced using the Ion Torrent PGM. The generated sequences were then compared between the paired samples and against results for the same donors assessed using traditional technologies. These results demonstrate a high concordance between paired blood and saliva samples. The results confirm that saliva collected using the Oragene family of products is an excellent source of DNA for the Raindance HLASeq panel and the high quality DNA is suitable for HLA typing by next generation sequencing. Iwasiow: DNA Genotek Inc.: Employee. Tayeb: DNA Genotek: Employee.