Expression of LRP and MDR1 in locally advanced breast cancer predicts axillary node invasion at the time of rescue mastectomy after induction chemotherapy.

BACKGROUND: Axillary node status after induction chemotherapy for locally advanced breast cancer has been shown on multivariate analysis to be an independent predictor of relapse. However, it has been postulated that responders to induction chemotherapy with a clinically negative axilla could be spared the burden of lymphadenectomy, because most of them will not show histological nodal invasion. P-glycoprotein expression in the rescue mastectomy specimen has finally been identified as a significant predictor of patient survival. MATERIALS AND METHODS: We studied the expression of the genes encoding multidrug resistance associated protein (MDR1) and lung cancer associated resistance protein (LRP) in formalin-fixed, paraffin-embedded tumor samples from 52 patients treated for locally advanced breast cancer by means of induction chemotherapy followed by rescue mastectomy. P-glycoprotein expression was assessed by means of immunohistochemistry before treatment in 23 cases, and by means of reverse-transcriptase-mediated polymerase chain reaction (RT-PCR) after treatment in 46 (6 failed). LRP expression was detected by means of immunohistochemistry, with the LRP-56 monoclonal antibody, in 31 cases before treatment. Immunohistochemistry for detecting the expression of c-erb-B2, p53, Ki67, estrogen receptor and progesterone receptor are routinely performed in our laboratory in every case, and the results obtained were included in the study. All patients had received between two and six cycles of standard 5-fluorouracil, doxorubicin and cyclophosphamide (FAC) chemotherapy, with two exceptions [one patient received four cycles of a docetaxel-adriamycin combination, and the other four cycles of standard cyclophosphamide-methotrexate-5-fluorouracil (CMF) polychemotherapy]. Response was assessed in accordance with the Response Evaluation Criteria In Solid Tumors (RECIST). By these, 2 patients achieved a complete clinical response, 37 a partial response, and the remaining 13 showed stable disease. This makes a total clinical response rate of 75.0%. None achieved a complete pathological response. RESULTS: MDR1 mRNA expression detected by RT-PCR was associated with the presence of invaded axillary nodes at surgery in 18/22 cases (81.8%), compared with 13/24 (54.2%) in the group with undetectable MDR1 expression. This difference was statistically significant (P < 0.05). LRP expression in more than 20% of tumor cells before any treatment was associated with axillary nodal metastasis after chemotherapy and rescue mastectomy in 17/23 cases, compared with 3/8 in nonexpressors. Again, this difference was highly significant (P < 0.01). LRP expression before treatment and MDR1 mRNA expression after treatment were significantly interrelated (P < 0.001), which might reflect the presence of chemoresistant clones liable to metastasize to the regional nodes. Persistence of previously detected MDR1-positivity after treatment (7/9 compared with 0/2 cases) was significantly associated with axillary node metastasis (P < 0.05). Finally, in a logistic regression multivariate model, histology other than ductal, a Ki67 labeling index of at least 20% and the combination of LRP and MDR1 positivity emerged as independent predictors of axillary node invasion at the time of rescue mastectomy. CONCLUSION: The expression of different genes involved in resistance to chemotherapy, both before and after treatment with neoadjuvant, is associated with the presence of axillary node invasion at rescue surgery in locally advanced breast cancer. This might reflect the presence of intrinsically resistant clones before any form of therapy, which persist after it, and could be helpful both for prognosis and for the choice of individual treatment.

[Study of the most often encountered allele of the HLA-B27 gene in Tuvinian and Russian inhabitants of Western Siberia]. / Izuchenie naibolee chasto vstrechaiushchikhsia allelei gena HLA-B27 u tubintsev i russkikh zhitelei zapadnoi Sibiri.

HLA-B27 gene frequencies and allelic polymorphism were studied in two Siberian ethnic groups: Russians from Novosibirsk (western Siberia) and Tuvinians from Kyzyl (southern Siberia). The HLA-B27 frequencies were determined by means of serologic typing of HLA antigens in 198 Tuvinians and 288 Russians. Molecular typing was performed via hybridization of oligonucleotide probes with amplified DNAs obtained from 30 HLA-B27-positive Russians and 11 HLA-B27-positive Tuvinians. The HLA-B27 gene frequencies in Tuvinians and Russians were 5.5 and 10.4%, respectively. Molecular variants of the HLA-B27 gene were studied in Tuvinians for the first time. The proportions of the HLA-B2705 and HLA-B2704 alleles were found to be 64 and 36%, respectively, in the population studied. The presence of the HLA-B2704 allele indicates a Mongoloid origin of Tuvinians. In the Russian population of Novosibirsk, the HLA-B2704 allele was not found, whereas the proportions of the HLA-B2705 and HLA-B2702 alleles were 76.2 and 23.8%, respectively, which is characteristic of Caucasoid populations.

The MICA-A9 triplet repeat polymorphism in the transmembrane region confers additional susceptibility to the development of psoriatic arthritis and is independent of the association of Cw*0602 in psoriasis.

OBJECTIVE: To investigate the relative contribution of HLA antigens in the susceptibility to psoriasis and to localize additional genetic factors involved in psoriatic arthritis (PsA). METHODS: DNA from 45 patients with psoriasis, 65 with PsA, and 177 healthy control subjects was examined by polymerase chain reaction (PCR) using sequence-specific oligonucleotide probes to determine HLA-C. To examine whether MICA (class I major histocompatibility complex chain-related gene A) confers additional susceptibility, trinucleotide repeat polymorphism in the transmembrane region of the MICA gene was investigated by radioactive PCR. Further analysis of MICA was made by PCR-single-strand conformational polymorphism to determine the allelic variant corresponding to MICA transmembrane polymorphism. RESULTS: Our results reveal new findings: 1) the frequency of the Cw*0602 allele was significantly increased in both patient groups: psoriasis (corrected P [Pcorr] < 10(-5), relative risk [RR] 6.2), PsA (Pcorr < 10(-6), RR 6.3), 2) the trinucleotide repeat polymorphism MICA-A9 was present at a significantly higher frequency in PsA patients (Pcorr < 0.00035, RR 3.2), whereas a similar distribution was found in both the control and psoriasis population, 3) this polymorphism corresponds to the MICA-002 allele and was found to be overrepresented in patients with the polyarticular form (Pcorr < 0.0008, RR 9.35), 4) the increase in MICA-A9 in PsA patients is independent of linkage disequilibrium with Cw*0602, 5) this allele confers additional relative risk (RR 3.27, etiologic fraction 0.44; etiologic fraction is the proportion of disease cases among the total population that are attributable to 1 allele when the relative risk is > 1) in PsA patients who carry Cw*0602. CONCLUSION: The data obtained in this study are consistent with the polygenic inheritance of psoriasis. Cw*0602 appears to be the stronger genetic susceptibility factor for psoriasis. Independent of the HLA-C association, MICA-A9 polymorphism corresponding to the MICA-002 allele is a possible candidate gene for the development of PsA.

HLA-B27 polymorphism and worldwide susceptibility to ankylosing spondylitis.

HLA-B27 is strongly associated to ankylosing spondylitis (AS) and represents a family of eleven B27 alleles (B*2701-11). Our aim was to analyze the distribution of B27 subtypes by PCR/SSOP and genomic sequencing in a large group of populations (n = 17). 711 B27-positive samples from Caucasoid, Asian, African, Amerindian and Polynesian populations were selected to ascertain transracial gene mapping of the B27 subtypes. 476 of these were AS patients, chosen to investigate the contribution of B27 alleles to AS susceptibility. Some significant new findings have arisen from this study: 1) B*2705 was the predominant subtype in circumpolar and subarctic areas. B*2702 was found to be practically restricted to Caucasian populations, showing a higher frequency in Middle-East (Jews) and North Africa (Arabs/Berbers) groups. 2) B*2703 appears associated with AS in Western Africans. This is of remarkable interest since it was suggested that B*2703 would be negatively disease-associated. 3) Although B*2706 appears negatively associated with AS in Thais, we identified two patients from northern China carrying it. This may be a reflection of a disease heterogeneity and could indicate that more than one pathogenic agent can be involved in AS. B*2709 has been recently described as negatively associated with AS in Sardinians. The molecular changes His114Asp (B*2706) and Asp116His (B*2709) could modify the genetic susceptibility to AS.

HLA-B27 structure, function, and disease association.

The polymorphism of HLA-B27 alleles is located in the peptide-anchoring motif. In recent years, fundamental insights have been made into the molecular aspects of HLA-B27-restricted presentation. Subtle differences in peptide binding fine specificity are especially interesting for closely related HLA-B27 alleles that have differential association with ankylosing spondylitis. Bacterial infection has been suggested to play a role in the pathogenesis of HLA-B27-associated disease. Remarkable progress has been made in identifying peptides derived from bacteria that can be presented by HLA-B27. Despite the mechanisms proposed to explain B27-associated diseases, there are no clear correlations between peptide sequence, differential binding to B27 subtypes, and recognition by peptide-specific T cell receptors. Furthermore, new transgenic models have now been developed that we hope will allow a clearer view of the function of B27 and the mechanisms involved in the pathogenesis of spondyloarthropathies.

Molecular evolution of the N-formyl peptide and C5a receptors in non-human primates.

N-formyl peptides (FMLP) and complement fragment C5a are neutrophil chemoattractants. In humans, a single-copy gene was identified for the C5a receptor, and the receptor for FMLP (FPR1) is encoded by a single gene that shows 53% amino acid similarity to the C5aR. Two other human FPR1 homologues, FPR-like 1 (FPR2/FPRL1) and FPR-like 2 (FPRL2) have been cloned. The human C5aR, FPR1, FPRL1, and FPRL2 are physically linked. By direct sequencing or by sequencing plasmid clones we studied the C5aR and FPR genes from four non-human primates (chimpanzee, gorilla, orangutan, and macaque). The sequences showed 95% - 99% similarity to the human homologues, with the major divergences observed in macaque. In these genes, the transmembrane and the cytoplasmic domains are highly conserved, while the highest divergence corresponded to the extracellular loops involved in ligand binding. Additionally, we constructed a physical map of these genes in non-human primates. In all species the four genes were physically linked and we defined the relative orientation of the four genes in primates: C5aR>FPR1>FPR2 (FPRL1)>FPRL2.

Characterization of interleukin-8 receptors in non-human primates.

Interleukin-8 is a chemokine with a potent neutrophil chemoattractant activity. In humans, two different cDNAs encoding human IL8 receptors designated IL8RA and IL8RB have been cloned. IL8RA binds IL8, while IL8RB binds IL8 as well as other alpha-chemokines. Both human IL8Rs are encoded by two genes physically linked on chromosome 2. The IL8RA and IL8RB genes have open reading frames (ORF) lacking introns. By direct sequencing of the polymerase chain reaction products, we sequenced the IL8R genes of cell lines from four non-human primates: chimpanzee, gorilla, orangutan, and macaca. The IL8RB encodes an ORF in the four non-human primates, showing 95% - 99% similarity to the human IL8RB sequence. The IL8RA homologue in gorilla and chimpanzee consisted of two ORF 98% - 99% identical to the human sequence. The macaca and orangutan IL8RA homologues are pseudogenes: a 2 base pair insertion generated a sequence with several stop codons. In addition, we describe the physical linkage of these genes in the four non-human primates and discuss the evolutionary implications of these findings.

Characterization of B27 haplotypes by oligotyping and genomic sequencing in the Mexican Mestizo population with ankylosing spondylitis: juvenile and adult onset.

The aim of this study was to investigate the contribution of the different B27 subtypes in the Mexican Mestizo population with juvenile and adult AS. No differences in the distribution of B27 subtypes were found between both populations, B*2705 being the predominant subtype followed by B*2702. Transracial gene mapping was performed in order to find out the origin of the B27 alleles of the Mexican Mestizos. A PCR with SSOPs was used to analyze the polymorphism in exons 2 and 3 of HLA-B27 and HLA-C related alleles. This population shares with the Spanish Caucasians B*2705 and B*2702, which are absent in Central and South American Indians. AS and healthy Mexican mestizo donors were analyzed to ascertain B27/Cw haplotypes. The B27/Cw linkage arrangements seen in mestizos are similar to those reported for Caucasian Spaniards with three different haplotypes positively associated with AS in both populations, B*2705/Cw*0102, B*2705/Cw*02022, and B*2702/Cw*02022, suggesting that B27 in Mexicans may be due to a recent Caucasoid admixture with the Spanish genes. Finally, a strategy for sequence analysis of exons 2 and 3 from genomic DNA of HLA-B27 alleles was developed. A novel HLA-B27-like allele typed serologically as B27 was identified and sequenced by this method in a healthy Mexican mestizo, corresponding to the B*7301 variant found with low frequency in different populations. Analysis of the association of B*7301 to AS would require an extensive study in different populations and could provide insights into the molecular structure of the alleles involved in the disease.

Molecular analysis of HLA-B27 haplotypes in Caucasoids. Frequencies of B27-Cw in Jewish and Spanish populations.

PCR in combination with SSO probes was used to analyze the polymorphism in exons 2 and 3 of HLA-B27 subtypes and HLA-C-related alleles in two genetically distant Caucasian groups: Spanish and Jewish populations. AS patients and healthy B27 donors from both populations were analyzed in order to ascertain B27-Cw haplotypes. Three different ancestral haplotypes were found to be represented in both populations: B*2705/Cw*0102, B*2705/Cw*02022, and B*2702/Cw*02022. The B*2705 (92.5%) was the most frequent allele found in the Spanish population, carried by B*2705/Cw*0102 (60.9%) and B2705/Cw*02022 (30.4%) haplotypes. In contrast, B*2702 (59.4%) was the most prevalent allele found in the Jewish population and was carried by the B*2702/Cw*02022 (63.3%) haplotype. No different allelic and haplotypic distributions were among healthy and AS patients in either Spanish or Jewish populations. The differences found in the distribution of B27 haplotypes among Spanish and Jewish Caucasian populations are consistent with the genetic distance of these ethnic groups. When the Jewish population was subdivided into Ashkenazi (A) and non-Ashkenazi (NA) groups, no significant differences were observed in the distribution of B*2702/Cw*02022 haplotype. Minor differences were observed in the underrepresented B*2705 haplotypes. The present results reflect the ancestral affinities of A and NA Jewish populations. A possible HLA-B27 evolutive pathway in Caucasians is proposed according to the data available for the B27/Cw ancestral haplotypes in Spanish and Jewish groups.