Multiple Sclerosis and MEN2 Neoplasia in a Female Patient: A Unique Co-Existence with Expanded Immunological Interest and Therapeutical Challenges, before and after Patient's COVID-19 Infection.

Multiple sclerosis (MS) and its various comorbidities that may be observed are of great interest due to the complexity of MS pathophysiology and all of the immunological changes that follow. The incidence of cancer in MS has been investigated for several years, as not only does it affect ongoing therapeutical decisions, but also, certain disease-modifying treatments (DMTs) may increase the risk of tumorigenesis. For the first time, we present a case of a female patient with pediatric-onset MS (POMS) and multiple endocrine neoplasia 2B (MEN2B) and analyze the immunological impact of these diseases on the therapeutical choice, under the umbrella of her COVID-19 infection and the SARS-CoV-2 pandemic as a whole. We also review the existing literature regarding the immunogenetic and immunological correlations between these two extremely rare diseases and discuss the most suitable treatment for our case, which seems to be an anti-CD20 agent due to a better outcome in putative MS worsening and tumor progression, when killer immunoglobulin-like receptors' (KIR) expression is reduced in natural killer (NK) cells. We also broaden our concerns on this comorbidity issue, at the same time focusing on the future research needed in this unexplored field of the comorbidity of MS and cancers.

HLA-genotyping by next-generation-sequencing reveals shared and unique HLA alleles in two patients with coexisting neuromyelitis optica spectrum disorder and thymectomized myasthenia gravis: Immunological implications for mutual aetiopathogenesis?

The exact immunopathogenesis, genetic mechanisms and triggering factors underlying myasthenia gravis (MG) and neuromyelitis optica (NMO) remain unknown and the coexistence may underline an aetiopathogenetic link be- tween these two diseases. We report the cases of two thymectomized patients with acetylcholine receptor (AChR) antibody (Ab)-positive MG who eventually developed AQP4-NMO. Next-Generation Sequencing (NGS) analysis showed that patient-1 had two HLA alleles previously associated with MG, mainly HLA-A*01:01:01 and HLA-DRB1*03:01, present in a haplotype in Caucasian MG patients (HLA-A1-B8-DR3-DQ2). Patient-2, expressed HLA-C*07:01:01, a well characterized MG risk factor and HLA-DQB1*05:02:01, previously described both in MG and NMO patients. Finally, we observed two common alleles in patient 1 and 2, HLA-DQA1*05:01:01 and HLA-DPB1*04:02:01. We believe that this study provides clinical evidence of the role of specific HLA alleles in rare forms of combined human peripheral and CNS autoimmunity, a fact that enhances the aim towards tailor-made therapeutic decision making.

Evaluation of HLA-G Expression in Multipotent Mesenchymal Stromal Cells Derived from Vitrified Wharton's Jelly Tissue.

BACKGROUND: Mesenchymal Stromal Cells (MSCs) from Wharton's Jelly (WJ) tissue express HLA-G, a molecule which exerts several immunological properties. This study aimed at the evaluation of HLA-G expression in MSCs derived from vitrified WJ tissue. METHODS: WJ tissue samples were isolated from human umbilical cords, vitrified with the use of VS55 solution and stored for 1 year at -196 °C. After 1 year of storage, the WJ tissue was thawed and MSCs were isolated. Then, MSCs were expanded until reaching passage 8, followed by estimation of cell number, cell doubling time (CDT), population doubling (PD) and cell viability. In addition, multilineage differentiation, Colony-Forming Units (CFUs) assay and immunophenotypic analyses were performed. HLA-G expression in MSCs derived from vitrified samples was evaluated by immunohistochemistry, RT-PCR/PCR, mixed lymphocyte reaction (MLR) and immunofluorescence. MSCs derived from non-vitrified WJ tissue were used in order to validate the results obtained from the above methods. RESULTS: MSCs were successfully obtained from vitrified WJ tissues retaining their morphological and multilineage differentiation properties. Furthermore, MSCs from vitrified WJ tissues successfully expressed HLA-G. CONCLUSION: The above results indicated the successful expression of HLA-G by MSCs from vitrified WJ tissues, thus making them ideal candidates for immunomodulation.

Non-Inherited Maternal Antigens Identify Acceptable HLA Mismatches: A New Policy for the Hellenic Cord Blood Bank.

BACKGROUND: During pregnancy, the maternal-fetal contact may lead to the development of tolerance against the maternal human leukocyte antigen (HLA) that is not inherited by the fetus. These non-inherited maternal antigens (NIMAs) define acceptable HLA mismatches; therefore, the number of HLA phenotypes that are suitable matches for patients who need a hematopoietic stem cell transplant could be increased. Cord blood unit (CBU) transplantations to patients mismatched for a HLA loci, but similar to the ΝΙΜAs of the CBU, have a prognosis similar to 6/6-matched ones. METHODS: The Hellenic Cord Blood Bank (HCBB) identified the maternal HLA of 380 cord blood donors, specifying the NIMA haplotypes of the related cryostored CBUs. RESULTS: The HCBB extended the pool of HLA phenotypes through the generation of unique virtual phenotypes (VPs). A "VP database" was set up, using Microsoft Office-Access™, in order to provide NIMA-matched CBUs for potential recipients. The effectiveness of VPs' matching was tested in 80 Greek patients. CONCLUSION: This methodology may contribute to the increase of the number of available CBUs for patients, in the case where there is no available CBU, or in case an additional one is needed. Through this method, the CBUs could be used faster and more effectively, rather than being cryostored for long periods of time.

A new mathematical model for the interpretation of translational research evaluating six CTLA-4 polymorphisms in high-risk melanoma patients receiving adjuvant interferon.

Adjuvant therapy of stage IIB/III melanoma with interferon reduces relapse and mortality by up to 33% but is accompanied by toxicity-related complications. Polymorphisms of the CTLA-4 gene associated with autoimmune diseases could help in identifying interferon treatment benefits. We previously genotyped 286 melanoma patients and 288 healthy (unrelated) individuals for six CTLA-4 polymorphisms (SNP). Previous analyses found no significant differences between the distributions of CTLA-4 polymorphisms in the melanoma population vs. controls, no significant difference in relapse free and overall survivals among patients and no correlation between autoimmunity and specific alleles. We report new analysis of these CTLA-4 genetic profiles, using Network Phenotyping Strategy (NPS). It is graph-theory based method, analyzing the SNP patterns. Application of NPS on CTLA-4 polymorphism captures allele relationship pattern for every patient into 6-partite mathematical graph P. Graphs P are combined into weighted 6-partite graph S, which subsequently decomposed into reference relationship profiles (RRP). Finally, every individual CTLA-4 genotype pattern is characterized by the graph distances of P from eight identified RRP's. RRP's are subgraphs of S, collecting equally frequent binary allele co-occurrences in all studied loci. If S topology represents the genetic "dominant model", the RRP's and their characteristic frequencies are identical to expectation-maximization derived haplotypes and maximal likelihood estimates of their frequencies. The graph-representation allows showing that patient CTLA-4 haplotypes are uniquely different from the controls by absence of specific SNP combinations. New function-related insight is derived when the 6-partite graph reflects allelic state of CTLA-4. We found that we can use differences between individual P and specific RRPs to identify patient subpopulations with clearly different polymorphic patterns relatively to controls as well as to identify patients with significantly different survival.

A multi-factorial genetic model for prognostic assessment of high risk melanoma patients receiving adjuvant interferon.

PURPOSE: IFNa was the first cytokine to demonstrate anti-tumor activity in advanced melanoma. Despite the ability of high-dose IFNa reducing relapse and mortality by up to 33%, large majority of patients experience side effects and toxicity which outweigh the benefits. The current study attempts to identify genetic markers likely to be associated with benefit from IFN-a2b treatment and predictive for survival. EXPERIMENTAL DESIGN: We tested the association of variants in FOXP3 microsatellites, CTLA4 SNPs and HLA genotype in 284 melanoma patients and their association with prognosis and survival of melanoma patients who received IFNa adjuvant therapy. RESULTS: Univariate survival analysis suggested that patients bearing either the DRB1*15 or HLA-Cw7 allele suffered worse OS while patients bearing either HLA-Cw6 or HLA-B44 enjoyed better OS. DRB1*15 positive patients suffered also worse RFS and conversely HLA-Cw6 positive patients had better RFS. Multivariate analysis revealed that a five-marker genotyping signature was prognostic of OS independent of disease stage. In the multivariate Cox regression model, HLA-B38 (p = 0.021), HLA-C15 (p = 0.025), HLA-C3 (p = 0.014), DRB1*15 (p = 0.005) and CT60*G/G (0.081) were significantly associated with OS with risk ratio of 0.097 (95% CI, 0.013-0.709), 0.387 (95% CI, 0.169-0.889), 0.449 (95% CI, 0.237-0.851), 1.948 (95% CI, 1.221-3.109) and 1.484 (95% IC, 0.953-2.312) respectively. CONCLUSION: These results suggest that gene polymorphisms relevant to a biological occurrence are more likely to be informative when studied in concert to address potential redundant or conflicting functions that may limit each gene individual contribution. The five markers identified here exemplify this concept though prospective validation in independent cohorts is needed.

High frequency of human leukocyte antigen class II DRB1*1602 haplotype in Greek patients with myelodysplastic syndrome and of DRB1*1501 in the low-risk subgroup.

Myelodysplastic syndromes (MDS) comprise a heterogenous group of clonal hematopoietic disorders in which the immune-mediated pathogenetic mechanisms are under investigation. Overrepresentation of human leukocyte antigen (HLA)-DR2 and its serologic split HLA-DR15 has been associated with low-risk MDS in certain ethnic groups and has been proposed as a predictive factor for a favorable response to immunomodulatory treatment. Because the HLA-DRB1*15 haplotype does not predominate in the Greek population, we investigated the frequency of HLA-DRB1 alleles among 114 patients of Greek origin suffering from various types of MDS: 36 refractory anemia (RA), 24 refractory anemia with ringed sideroblasts (RARS), 19 refractory anemia with excess of blasts (RAEB), 14 refractory anemia with excess of blasts in transformation (RAEB-t), 14 chronic myelomonocytic leukemia, and 7 hypoplastic MDS patients. HLA-DRB1 molecular typing was performed with polymerase chain reaction-sequence specific oligonucleotides and results were compared with that from a previously reported control Greek population. HLA-DRB1*1602 was the only allele that was significantly overrepresented in Greek MDS patients as a whole, whereas HLA-DRB1*1501 allele frequency was significantly higher in Greek patients with low-risk myelodysplasia. Our results suggest the possible value of HLA-DR15 and HLA-DR16 as determinants for immunomodulatory interventions, at least for Greek patients with low-risk MDS.

Evaluation of six CTLA-4 polymorphisms in high-risk melanoma patients receiving adjuvant interferon therapy in the He13A/98 multicenter trial.

PURPOSE: Interferon is approved for adjuvant treatment of patients with stage IIb/III melanoma. The toxicity and uncertainty regarding survival benefits of interferon have qualified its acceptance, despite significant durable relapse prevention in a fraction of patients. Predictive biomarkers that would enable selection of patients for therapy would have a large impact upon clinical practice. Specific CTLA-4 polymorphisms have previously shown an association with response to CTLA-4 blockade in patients with metastatic melanoma and the development of autoimmunity. EXPERIMENTAL DESIGN: 286 melanoma patients and 288 healthy controls were genotyped for six CTLA-4 polymorphisms previously suggested to be important (AG 49, CT 318, CT 60, JO 27, JO30 and JO 31). Specific allele frequencies were compared between the healthy and patient populations, as well as presence or absence of these in relation to recurrence. Alleles related to autoimmune disease were also investigated. RESULTS: No significant differences were found between the distributions of CTLA-4 polymorphisms in the melanoma population compared with healthy controls. Relapse free survival (RFS) and overall survival (OS) did not differ significantly between patients with the alleles represented by these polymorphisms. No correlation between autoimmunity and specific alleles was shown. The six polymorphisms evaluated where strongly associated (Fisher's exact p-values < 0.001 for all associations) and significant linkage disequilibrium among these was indicated. CONCLUSION: No polymorphisms of CTLA-4 defined by the SNPs studied were correlated with improved RFS, OS, or autoimmunity in this high-risk group of melanoma patients.

Correlation of molecular human leukocyte antigen typing and outcome in high-risk melanoma patients receiving adjuvant interferon.

BACKGROUND: Interferon is approved for adjuvant treatment of patients with stage IIB/III melanoma. The identification of predictive markers that would permit selection of patients would be beneficial. Specific human leukocyte antigen (HLA) class I and II antigens have previously shown an association with response to therapy or overall survival of patients with metastatic melanoma. METHODS: A total of 284 high-risk melanoma patients participating in a randomized trial and 246 healthy controls were molecularly typed for HLA class I and II. Specific allele frequencies were compared between the healthy and patient populations, as well as presence or absence of these in relation to recurrence. Alleles related to autoimmune disease were also investigated. RESULTS: No significant differences were found between the distribution of HLA genotype in the melanoma population compared with healthy controls. Correlations between nonrecurrence and the presence of HLA-Cw 06 allele were noted present in 19.3% of melanoma patients. The median relapse-free survival of the Cw 06-positive cohort was 100.2 months versus 37.3 months in the Cw 06-negative cohort (P = .013). The median overall survival for the Cw 06-positive cohort has not yet been reached, versus 78.9 months in the Cw 06-negative cohort (P = .025). HLA-Cw 06 was present in 29.79% of patients in the autoimmunity group and 15.38% of patients in the nonautoimmunity group (P = .049). CONCLUSIONS: : No allele was associated with absence of recurrence in patients receiving adjuvant interferon with the exception of HLA-Cw 06, an allele correlated with psoriasis. HLA-Cw 06-positive patients have better relapse-free and overall survival.

Distribution of killer cell immunoglobulin-like receptors in the Greek population.

The interaction between killer cell immunoglobulin-like receptors (KIR) expressed on natural killer (NK) cells, and human leukocyt antigen (HLA) molecules expressed on target cells is known to regulate the cytolytic activity. A wide range of KIR genotypes is observed in the population, as the number of KIR loci can vary. In the present study we have determined the frequencies and combinations of 13 KIR genes and two CD94:NKG2 receptor genes and their distribution in the two haplotype groups in a panel of 233 unrelated healthy Greek Caucasians. We have typed genomic DNA for the presence of the putative KIR loci KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, and KIR3DS1 using modified polymerase chain reaction sequence-specific primers. The frequency of KIR loci combined with the linkage disequilibrium values suggest that the Greek population shares several general features with other Caucasoid populations studied before, but still distinguishes itself by the increased or decreased frequency of several alleles. The majority of the 45 different KIR genotypes seen in Greeks have been observed in Caucasoid populations studied before. Nevertheless, two of these genotypes, although met in other populations, have not been found in Caucasians before. One individual possesses a novel profile with no KIR inhibitory gene. The A haplotypes remain the most prevalent, with AA1 being the most common genotype, and the number of inhibitory KIRs being more variable than the number of noninhibitory KIRs in most haplotypes.

Natural killer (NK) cell receptors' repertoire in couples with recurrent spontaneous abortions.

PROBLEM: Natural killer (NK) cell receptors (NKRs) have been suggested to protect trophoblast, but their function at the fetomaternal interface remains unknown. To investigate if the outcome of pregnancy depends on women's NKRs, we studied the NKR repertoire in couples with recurrent spontaneous abortions (RSA). METHODS: Twenty-six childless couples with > or = 2 abortions, characterized by alloimmune abnormalities, and 26 control couples were genotyped for five killer immunoglobulin-like receptors (KIR) and two CD94/NKG receptors, known to have as ligands human leukocyte antigen (HLA) class I molecules with trophoblastic expression: inhibitory 2DL1,2,3 and activating 2DS1,4 KIRs, inhibitory NKG2A and activating NKG2C. Detected repertoires of women and partners were compared between the two groups. RESULTS: Less aborters than controls were found to have all three inhibitory KIRs (30.77% versus 69.23%, P = 0.01), some of them had only one inhibitory KIR (19.23% versus 3.85%, P = 0.08) and most of them were lacking inhibitory KIRs possessed by their husbands (57.69% versus 15.38%, P = 0.001). CONCLUSIONS: Women with alloimmune abortions have a limited inhibiting KIR repertoire and such miscarriages may occur because trophoblastic HLA class I molecules are recognized by decidual NK cells lacking the appropriate inhibitory KIRs.