Somatic mutations in the HLA genes of patients with hematological malignancy.

Somatic mutations and genomic alterations are frequent events in the clonal evolution of hematologic malignancies. Recent studies have reported copy neutral loss of heterozygosity (LOH) for the mismatched human leukocyte antigen (HLA) haplotype in patients relapsed after haploidentical hematopoietic cell transplantation (HCT) for a hematologic malignancy. Herein, we report 15 cases of somatic mutations in the HLA genes of patients with a variety of hematologic diseases, including acute myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, myelodysplastic syndrome, and non-Hodgkin's lymphoma, encountered at our institute over the past decade. While two of the cases were identified in patient relapse specimens collected post-HCT, 13 cases were found in peripheral blood specimens submitted for HLA typing prior to transplantation. Ten patients exhibited acquired LOH for all or part of one HLA haplotype. Five other cases involved somatic mutations in the nucleotide sequences of common HLA-A or HLA-B alleles. Since they are not systematically evaluated prior to HCT, acquired mutations in HLA genes are likely under reported. Beyond the implications for accurate HLA typing and donor selection, alternations that result in the loss of HLA expression may allow escape from immune surveillance and adversely impact transplant outcome.

Associations between human leukocyte antigen type and nasopharyngeal carcinoma in Caucasians in the United States.

A genetic component to nasopharyngeal carcinoma (NPC) has been suggested by associations of the malignancy with human leukocyte antigens (HLAs) in Southern Chinese populations, among which NPC is a major cancer. Data from other races are inconclusive. We have investigated associations between NPC and HLA antigens at the HLA-A, B, C, and DQ loci and alleles at the DRB1 locus in a population-based, multicenter investigation in the United States. Data from 82 cases and 140 controls are presented, making this the largest study population analyzing data from Caucasians to date. HLA frequencies from study cases were also compared with external control groups from the 11th International Histocompatibility Workshop and the National Marrow Donor Program. Logistic regression methods were used to investigate the effects of the joint occurrence of multiple HLA types and to assay for differences in HLA-associated risk in different age groups. A meta-analysis was undertaken to compare and summarize our results with previously published findings. The meta-analysis found a protective association with A2 antigen in non-Chinese [odds ratio (OR), 0.63; P < 0.001], a protective association with A11 across all races (OR, 0.54; P < 0.001), and an increased risk associated with B5 in Caucasians (OR, 2.81; P < 0.001). The present study also found independent associations, in a logistic regression model, between NPC and DRB1*1501 (OR, 0.33), DRB1*0405 (OR, 7.57), and Cw3 (OR, 0.42), although these data must be interpreted cautiously due to multiple-testing considerations. Associations were found to be more pronounced in younger patients for A2, A11, A28, B8, and B51.

Probability of finding HLA-matched unrelated marrow donors.

Bone marrow transplantation has become the treatment of choice for certain hematologic diseases. However, only 30-40% of patients who might benefit from this procedure have a suitable family donor. Consequently, many centers have begun to explore the use of unrelated volunteer donors. Initial results have demonstrated the feasibility of this approach. As a result, a national effort has begun to recruit HLA-typed volunteers in order to establish a registry of individuals who would be willing to serve as bone marrow donors. This manuscript explores the potential impact of establishing such a registry. We find that a registry of attainable size could more than double the number of marrow transplants now being performed. However, even with a registry of enormous size, it will still not be possible to identify an HLA-matched donor for some patients.

Two homozygous typing cells defining a subgroup of HLA-Dw6.

Homozygous typing cells (HTC) and responder cells from a panel of randomly selected unrelated caucasians were used in a study of the HLA-Dw6 antigen complex. Using HTC characterized by the Eighth International Histocompatibility Workshop, the Dw6 specificity was shown to be a broadly defined, heterogeneous cluster that could be subdivided. An HTC from our laboratory, 8W146, and a second locally identified cell, EMJ, were used to define one clearly distinguishable subcluster of Dw6, provisionally termed "6.1." HTC 8W146 and EMJ were mutually nonreactive in mixed leukocyte culture and showed strong association (r = 0.85) when used as stimulators against the caucasian cell panel. The calculated gene frequency of the 8W146/EMJ determinant in this population was 0.024. In an informative family, the 6.1 specificity could be shown to segregate independently of other Dw6 subgroups. DR typing of 8W146 and EMJ showed both to be positive for MT-1 and MT-2 but negative for DRw "3 + 6."

DQw3 variants defined by cloned alloreactive T cells.

The polymorphism of HLA class II molecules expressing the serologically defined alloantigen DQw3 was studied using cloned proliferative T lymphocytes. Two clones, IG9 and IC3, were selectively primed against DQw3-associated determinants and tested against a panel of 92 HLA-D homozygous cells. Both clones were specific for DQw3, but each showed a distinct response pattern. Clone IG9 recognized a DQw3-associated determinant expressed on a subset of DR4 and DR5 haplotypes and on all DRw6, 7, w8, and w9 haplotypes tested. In contrast, clone IC3 recognized a distinct DQw3-associated determinant expressed only on a subset of DR4 haplotypes. In monoclonal antibody inhibition experiments, anti-DQ, but not anti-DR or anti-DP antibodies, blocked reactivity of both clones IG9 and IC3, further demonstrating that the determinants defined by these clones are associated with DQ molecules. In DNA hybridization studies using a DQ beta probe, a correlation was observed between restriction site polymorphisms in the DQ beta gene, designated DQw"3.1" and "3.2," and the expression of the T-cell-defined IG9 and IC3 determinants. It is, thus, possible to demonstrate by cloned T-cell reactivity functionally relevant recognition sites on DQw3+ molecules that are associated with structural polymorphisms defined by molecular and genomic analysis.

Recognition by a murine monoclonal antibody of a unique epitope specific for the human alloantigen HLA-B8.

A cytotoxic murine monoclonal antibody recognizing a specific HLA alloantigen was produced from the spleen cells of a BALB/c immunized with partially purified class I glycoproteins from an HLA-A1,B8 homozygous B-lymphoblastoid cell line. The antibody, designated P8.1, was tested against cells from 521 unrelated donors. It reacted with each of the 83 donors known to be HLA-B8 positive and with no HLA-B8 negative donors (sensitivity, 100%; specificity, 100%). Immunoprecipitation with antibody P8.1 and polyacrylamide gel electrophoresis confirmed that the antigen recognized was a class I structure. Although most murine monoclonal anti-HLA antibodies previously described have recognized "public" or supertypic specificities, the identification of a monoclonal antibody specific for a "private" HLA alloantigen indicates first that the BALB/c mouse has the appropriate immune response repertoire for recognizing certain HLA allospecificities and second that HLA-B8 can be defined by a single unique epitope.

Definition of LD "4 X 7": a unique HLA-D specificity defined by two homozygous typing cells.

Two homozygous typing cells (HTC) from the Eighth International Histocompatibility Workshop were used to define a unique HLA-D specificity, designated LD "4 X 7". Typing cell 8W316 (Seattle) was obtained from a donor of Japanese descent; cell 8W324 (Leiden) was obtained from a European Caucasian donor. The HLA-D specificities defined by these two HTC showed a significant correlation (R = 0.65) in unselected panel studies and clear segregation as a single determinant within families. LD "4 X 7" was found in low frequency in Caucasians (1.0%) but with detected with a frequency of 24% in Japanese. The cells of the donors of HTC 8W316 and 8W324 express DRw9, and the LD "4 X 7" antigen fills an HLA-D locus "blank" on DRw9-positive haplotypes. The frequency of LD "4 X 7" noted in this study corresponds to the frequency of DRw9 reported for Japanese and Caucasian ethnic groups, a further indication that LD "4 X 7" and DRw9 are associated.

The HLA system in Inupiat and Central Yupik Alaskan Eskimos.

We have studied HLA antigen profiles of the Inupiaq and Yupik-speaking peoples, two of the four Eskimo linguistic groups residing in Alaska. A relatively restricted polymorphism of HLA-A and -B locus antigens was noted. Only 35% of A locus specificities and 37% of B locus specificities tested for were detected in each population. The most common A locus alleles were A2, A24, and A28; the most common B locus alleles were B51(5), B27, B35, Bw60(40), Bw61(40), and Bw62(15). The antigens Cw3 (75 and 69%) and DR4 (81 and 67%) were found in high frequency in both groups. HLA-DR1, DR2, and DR7 were detected infrequently, while DR3 was not detected at all. DR4 was frequently associated in both Inupiats and Yupiks with Dw4, a specificity that was thought to occur only in Caucasian populations. A statistically significant difference between Inupiats and Yupiks was found for polymorphism at the A locus, but no significant differences were found for polymorphisms at the B, C, D, or DR loci. Analysis of HLA linkage disequilibrium revealed the presence of several novel haplotypes not previously described in other populations, suggesting that the selective factors responsible for positive associations observed in these Native Alaskans were probably distinct.

Marrow transplantation from donors other than HLA identical siblings.

As of 31 December 1979, 39 patients in Seattle have received marrow grafts from donors other than HLA genotypically identical siblings. Sixteen transplants were between siblings, 21 from a parent to a child, one from a paternal uncle, and one from an unrelated donor. Ten patients had aplastic anemia and 29 had a hematological malignancy. As of 1 February 1980, only one of the ten patients transplanted for aplastic anemia is currently alive (greater than 1048 days) with a normal marrow and without graft-versus-host disease. This surviving patient was untransfused and received marrow from an HLA phenotypically identical mother. There were five episodes of graft rejection among the ten aplastic patients. Among the 29 patients transplanted for hematological malignancy, 12 (42%) are surviving from greater than 64 to greater than 995 days. Twelve of 29 patients were transplanted while in remission and eight (75%) are alive from greater than 148 to greater than 790 days. The two most frequent causes of death were relapse of leukemia and interstitial pneumonia. Only two patients died from complications clearly related to graft-versus-host disease. Five of the surviving patients were phenotypically identical with their donor for HLA, while seven were incompatible for some HLA determinants. One patient--donor pair was incompatible for HLA-D and DR as a result of HLA-B/D recombination, and six pairs were incompatible for HLA-A and/or B.

A supertypic HLA-DR specificity (DR4+5) defined by murine monoclonal antibody.

The hybridoma technique was used to produce a monoclonal antibody specific for a polymorphic determinant on human la antigens. BALB/c mice were immunized with a B-lymphoblastoid cell line derived from an HLA-DR3/DR4 positive donor. Hybridoma culture supernates were screened in two stages by means of the microcytotoxicity assay. Supernates were first tested against the la-positive immunizing cell line and against la-negative cells. Cultures identified as producing antibody cytoxic for the immunizing cell line but not for la-negative cells were subcultured and the supernates then tested against a selected panel of HLA-D homozygous cell lines (HCL). In this manner, it was possible to identify a microwell of hybrid cells producing an antibody that reacted with a polymorphic HLA-DR determinant. Cells from this culture, designated 17.15, were cloned twice by limiting dilution. Specificity was evaluated with a panel of 39 HCL and B cells from 70 normal donors. Antibody 17.15 recognized a supertypic DR "4 + 5" specificity, which was also present on DRw9 and some DR7 positive cells. These findings suggest that the cross-reactivity groups DR "4 + 5" (MB-3) and DR "4 + 7" (MT-3) defined by alloantisera may share a common supertypic specificity that is recognized by monoclonal antibody 17.15.

The HLA system in the Korean population.

The frequencies of HLA-A, B, C, DR, and DQ antigens, HLA-D (HTC-defined) haplotypes, and the HLA-linked genetic markers glyoxalase I (GLO), factor B (Bf), C2 and C4 were studied in 162 healthy unrelated Koreans. Antigens A2, A24, A26, B44, B51, Bw62, B35, Cw1, Cw3, DR2, DR4, DRw6, DR7, and DRw8 were observed at frequencies of 15% or greater, and GLO-2, BfS, C4A*3, C2C, C4A*4, C4B*1, and C4B*2 were also frequently observed. The antigens A23, A25, B18, Bw42, Bw47, and B21 were not observed at all. HLA-DR4 was the most common class II antigen and was associated with a series of HLA-D-defined haplotypes including Dw4, Dw10, Dw13, and Dw15. The HLA-DRw6, DR2,Dw8, and DRw8 haplotypes were also found frequently. DR2 haplotypes were either Dw2 or Dw12, while all DRw8 haplotypes tested corresponded to the DB7 or Dw "8.3" specificity that has been described in other Oriental populations. Significant linkage disequilibrium was found between the alleles A2,Cw1; A30,B13; A30,Cw6; A30,DR7; Cw1,Bw22; Cw5,B12; Cw6,B13; Cw6,DR7; B7,DR1; B12,Dw6; B12,DR7; B12,Dw7; B13,DR7, B17,DR3; Bw22,C4B*6; DRw6,BfF; and C4A*4,C4B*2. A comparison of gene frequencies and commonly observed haplotypes between Koreans, Chinese, Japanese, and Caucasians showed that while Koreans share several characteristics in common with other Oriental populations, there are allelic frequencies and haplotypes in Koreans that are distinct.

HLA-Cw*0409N is associated with HLA-A*2301 and HLA-B*4403-carrying haplotypes.

The associations of HLA-B*4402 and HLA-B*4403 with alleles of HLA-A and HLA-Cw were investigated in panels of HLA-B*4403 and HLA-B*4402 homozygous individuals and in selected individuals carrying HLA-Cw*04 and HLA-B*4403. Some of these individuals were genotyped and also carried (HLA-DRB1*0701, DQB1*02). Among the latter, we studied individuals carrying the conserved extended haplotype (CEH) [HLA-Cw*04, B*4403, FC31, DRB1*0701, DQB1*02]. Four different common (HLA-Cw*, B*44) haplotypes were identified that extended to the HLA-A locus: HLA-A*0201, Cw*0501, B*4402; HLA-A*2902, Cw*1601, B*4403; HLA-A*2301, Cw*0401, B*4403; and HLA-A*2301, Cw*0409N, B*4403. We identified eight unrelated examples of the allele HLA-Cw*0409N. HLA-A*2301 was associated with both HLA-Cw*0401 and HLA-Cw*0409N, suggesting that HLA-Cw*0409N may have arisen from a mutation in a CEH. We estimate that approximately 2 to 5 in 1000 Caucasian individuals carry the allele HLA-Cw*0409N, making it one of the most frequent null HLA alleles known to date. Our findings demonstrate the first example of three different HLA-Cw-determined subtypes of a common or CEH carrying a shared HLA-B allele, in this case HLA-B*4403.

A protective association between the HLA-A2 antigen and nasopharyngeal carcinoma in US Caucasians.

Analyzing data from Caucasians participating in a multicentered population-based case-control study of nasopharyngeal carcinoma and HLA type in the US, we found persons with the A2 antigen to have a significantly lower risk than those with other antigens at the A locus [odds ratio (OR), 0.46; 95% confidence interval (CI), 0.21-0.96]. The protective association was stronger among presumptive homozygotes for A2. Similar results were obtained when cases were compared with US Caucasians typed either as part of the Collaborative Transplant Study, or by the 9th International Histocompatibility Workshop. These results are supported by a statistical summary of odds ratios for A2 from a number of previous studies in non-Chinese (summary OR, 0.69; 95% CI, 0.54-0.88). The odds ratio for patients with squamous-cell carcinomas was 0.56; among 7 patients with undifferentiated tumors the OR was 0.14. Results from in vitro studies of immune response to Epstein-Barr virus have found that the HLA-A2 antigen efficiently presents the EBV gene product LMP-2, which has been detected in NPC tumor cells. This offers a rationale for the observed protective association between the HLA-A2 antigen and nasopharyngeal carcinoma.

Six variants of HLA-B27 identified by isoelectric focusing.

Six variant forms of HLA-B27 were identified among 68 unrelated B27-positive donors by isoelectric focusing (IEF) gel analysis. Each of the six IEF variants was distinguished by charge heterogeneity of desialated B27 heavy chains immunoprecipitated with specific monoclonal antibody (MAb). Charge differences varied from single to several charge units, indicating that these variants may have substantially different amino acid compositions. Informative family study showed that three B27 variant molecules were genetically determined. The uniqueness of these variant molecules was also demonstrable using a panel of alloantisera and MAbs recognizing B27-associated epitopes. Six distinct serological reactivity patterns were observed. Five of these serological patterns correlated with four of the IEF-defined variants, two of these patterns being associated with one IEF variant form. The sixth serological pattern was shared by the remaining two IEF variants. Combining the results of the electrophoretic and serological analyses, it is apparent that there are more than six structural variants within the B27 alloantigen family. Some B27 variant forms were found only in individuals of particular racial origin, indicating that unique genetic variations might occur in different racial groups. In a preliminary analysis of patients with ankylosing spondylitis, no apparent correlation was observed between any specific B27 variants and disease susceptibility.

A monoclonal antibody recognizing a determinant shared by HLA-A2 and HLA-Aw69 (A28* variant).

A cytotoxic murine monoclonal antibody, designated P5.1, was tested against 613 unrelated donors and found to react with 401 who were positive for HLA-A2 (sensitivity = 100%) and with 8 of 82 positive for HLA-A28. The latter split of A28 corresponds to the "A28* variant" that in the Ninth International Histocompatibility Workshop (9WS) was designated Aw69(28*). The epitope recognized by antibody P5.1 is distinct from the alloantisera-defined determinants that characterize HLA-A2 and A28. Immunoprecipitation of specific antigens with selected monoclonal antibodies and isoelectric focusing gel electrophoresis demonstrated that A2, Aw68(28) and Aw69(28*) are distinct polypeptides. Thus, the A2-A28 antigen family consists of at least three different alleles definable using alloantiserums specific for A2 and A28, and monoclonal antibodies such as P5.1 recognizing the A2,Aw-69(28*)-epitope.

Association of aspirin-sensitive asthma with HLA-DQw2.

Patients with ASA-sensitive asthma form a clinically homogeneous subgroup of asthmatics characterized by nonatopic eosinophilia, sinusitis, nasal polyps, and frequent steroid dependency. Twenty-six Caucasian patients with ASA-sensitive asthma and 22 Caucasian patients with uncomplicated asthma were typed for HLA Class I and II antigens. A significant increase in HLA-DQw2 (relative risk, 4.06) was found in ASA-sensitive asthmatics. Asthmatic patients who were not ASA-sensitive had HLA frequencies that did not differ significantly from healthy Caucasian control subjects. These findings suggest that ASA-sensitive asthma represents a disease entity unique from other forms of asthma. Presumably, DQw2 or an associated genetic factor is involved in the pathogenesis of ASA-sensitive asthma.

HLA and leprosy in Koreans.

HLA antigens in 157 unrelated Koreans with leprosy have been identified and compared with 162 healthy Korean controls. The patient group consisted of 124 with lepromatous leprosy and 33 with tuberculoid leprosy. Although no significant differences were detected between the two patient groups, several antigens were found to be increased in the combined patient group compared to healthy controls. Two Class I antigens were increased: HLA-A11 (22% vs 12%) and Aw33 (27% vs 14%). Four Class II antigens were increased: HLA-DR1 (16% vs 7%), DR2 (39% vs 21%), DRw9 (14% vs 6%) and DQw1 (74% vs 55%). HLA-DR4 (28% vs 48%), DRw53 (46% vs 69%) and DQw3 (50% vs 75%) in contrast were significantly decreased in patients. Interaction of DR1, DR2, DRw9 and DQw1 as risk factors was analyzed. HLA-DR2 appeared to be the strongest risk factor. No evidence for synergy between DR1, DR2 and DRw9 was detected. DQw1 was not significantly increased in patients in the absence of DR1 and DR2, and thus it was not apparent in this study that DQw1 was an independent risk factor.

Maternal-fetal disparity in HLA class II alloantigens and the pregnancy-induced amelioration of rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis frequently remits during pregnancy, for unknown reasons. Since an immune response to paternally inherited fetal HLA can occur during normal pregnancy and since rheumatoid arthritis is an autoimmune disorder with a known HLA class II antigen association, we tested the hypothesis that maternal-fetal disparity in HLA alloantigens might be associated with the pregnancy-induced remission of rheumatoid arthritis. METHODS: We studied 57 pregnancies of 41 women with rheumatoid arthritis, 18 prospectively and 39 retrospectively. Serologic and DNA techniques were used to study HLA class I and II antigens. For newborns, typing was performed from cord-blood samples obtained at delivery. For four young children, typing was performed from DNA extracted from hair samples. RESULTS: We found significantly more maternal-fetal disparity in HLA-DR and DQ antigens in pregnancies characterized by the remission or improvement of rheumatoid arthritis than in pregnancies characterized by active disease. Further studies using DNA-typing techniques to define allelic variants of HLA-DR and DQ antigens confirmed this observation. Maternal-fetal disparity in alleles of HLA- DRB1, DQA, and DQB occurred in 26 of 34 pregnancies characterized by remission or improvement (76 percent), as compared with 3 of 12 pregnancies characterized by active arthritis (25 percent) (odds ratio, 9.7; P = 0.003). The difference between the two groups was most marked for alleles of HLA-DQA. CONCLUSIONS: Amelioration of rheumatoid arthritis during pregnancy is associated with a disparity in HLA class II antigens between mother and fetus. These findings suggest that the maternal immune response to paternal HLA antigens may have a role in the pregnancy-induced remission of rheumatoid arthritis.

Remission of rheumatoid arthritis during pregnancy and maternal-fetal class II alloantigen disparity.

Rheumatoid arthritis (RA) is an autoimmune disorder known to be associated with specific class II genes. Although it has been known since 1938 that the majority of women with RA experience disease improvement or remission during pregnancy, the reasons remain unknown. Pregnancy represents an immunologic challenge and maternal immune recognition of the semi-allogeneic fetus occurs as part of normal pregnancy. We hypothesized that maternal immune response to fetal HLA antigens might be associated with the effect of pregnancy on arthritis activity. To test this hypothesis, we studied HLA antigens in mother-child pairs comparing maternal-fetal HLA antigen sharing for pregnancies where arthritis improved with those where disease was active. No significant difference was observed in the two groups for class I HLA antigens. Fetal-maternal disparity for HLA-DR and HLA-DQ antigens was observed significantly more frequently in pregnancies with remission or improvement compared with those in which disease was active. These observations suggest that maternal immune response to fetal paternally-inherited class II HLA antigens may be important in RA remission observed during pregnancy.