Mutational landscape in Waldenström macroglobulinemia evaluated using a next-generation sequencing lymphoma panel in routine clinical practice.

Next-generation sequencing (NGS) affords comprehensive insights into the genomic landscape of lymphomas. We examined the mutational pattern in patients with Waldenström macroglobulinemia (WM) or lymphoplasmacytic lymphoma (LPL) as well as the diagnostic and clinical utility of a tailored NGS lymphoma panel. A consecutive series of 45 patients was reviewed and NGS analysis was performed as part of a routine diagnostic setup. The custom designed NGS panel assayed all coding sequences of 59 genes of known clinical significance in lymphoid neoplasms. The most frequently mutated genes were MYD88, CXCR4, BIRC3, CD79B, and ARID1A. Additional somatic mutations were detected in 17 genes with four mutations categorized as pathogenic or likely pathogenic. BIRC3 and TP53 mutations were associated with adverse clinical phenotypes. NGS performance for the MYD88L265P variant was 96% when compared to qPCR. In conclusion, targeted NGS provided important diagnostic and prognostic information in a routine clinical setting.

Next generation sequencing in routine diagnostics of mature non-Hodgkin's B-cell lymphomas.

INTRODUCTION: Integration of molecular characterization of lymphomas in clinical diagnostics may improve subclassification and risk-stratification, and we implemented a next generation sequencing (NGS) analysis as part of routine diagnostic work-up of all mature B-cell non-Hodgkin's lymphoma (B-NHL). Here, we present data of mutational profiles with potential complementary diagnostic, prognostic, and predictive value detected in our consecutive non-selected cohort of B-NHL patients. METHODS: NGS results from 298 patients with both newly diagnosed and relapsed/refractory disease were included as a single center study. NGS was performed as routine analysis together with standard diagnostic work-up using a custom-made amplicon PCR-based multiplex NGS panel covering all coding exons and consensus splice sites in 59 genes. RESULTS: Mutations were detected in 94% of the 298 samples. Most lymphomas could be classified definitively, but 24 cases were classified as small B-cell lymphomas without defining characteristics. Of these, 50% (12/24 cases) could retrospectively be assigned a likely diagnostic subtype according to mutational findings. CONCLUSION: Implementation of a 59 gene exome sequencing panel added diagnostic value to 50% of unclassified cases and provided in 94% of the cases possible biomarkers for disease monitoring as well as potential diagnostic, prognostic, and predictive markers for future studies.

Multi-site pre-therapeutic biopsies demonstrate genetic heterogeneity in patients with newly diagnosed diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, both regarding clinical presentation, response to treatment and outcome. Recently, subclassification of DLBCL based on mutational profile has been suggested, and next generation sequencing (NGS) analysis may be relevant as part of the diagnostic workflow. This will, however, often be based on analysis of one tumor biopsy. Here, we present a prospective study where multi-site sampling was performed prior to treatment in patients with newly diagnosed DLBCL. Two spatially different biopsies from 16 patients were analyzed using NGS with an in-house 59-gene lymphoma panel. In 8/16 (50%) patients, mutational differences were found between the two biopsy sites, including differences in TP53 mutational status. Our data indicate that a biopsy from the extra-nodal site may represent the most advanced clone, and an extra-nodal biopsy should be preferred for analysis, if safely accessible. This will help ensure a standardized stratification and treatment decision.

Mismatch repair deficiency testing in Lynch syndrome-associated urothelial tumors.

Introduction: Lynch syndrome-associated cancer develops due to germline pathogenic variants in one of the mismatch repair (MMR) genes, MLH1, MSH2, MSH6 or PMS2. Somatic second hits in tumors cause MMR deficiency, testing for which is used to screen for Lynch syndrome in colorectal cancer and to guide selection for immunotherapy. Both MMR protein immunohistochemistry and microsatellite instability (MSI) analysis can be used. However, concordance between methods can vary for different tumor types. Therefore, we aimed to compare methods of MMR deficiency testing in Lynch syndrome-associated urothelial cancers. Methods: Ninety-seven urothelial (61 upper tract and 28 bladder) tumors diagnosed from 1980 to 2017 in carriers of Lynch syndrome-associated pathogenic MMR variants and their first-degree relatives (FDR) were analyzed by MMR protein immunohistochemistry, the MSI Analysis System v1.2 (Promega), and an amplicon sequencing-based MSI assay. Two sets of MSI markers were used in sequencing-based MSI analysis: a panel of 24 and 54 markers developed for colorectal cancer and blood MSI analysis, respectively. Results: Among the 97 urothelial tumors, 86 (88.7%) showed immunohistochemical MMR loss and 68 were successfully analyzed by the Promega MSI assay, of which 48 (70.6%) were MSI-high and 20 (29.4%) were MSI-low/microsatellite stable. Seventy-two samples had sufficient DNA for the sequencing-based MSI assay, of which 55 (76.4%) and 61 (84.7%) scored as MSI-high using the 24-marker and 54-marker panels, respectively. The concordance between the MSI assays and immunohistochemistry was 70.6% (p = 0.003), 87.5% (p = 0.039), and 90.3% (p = 1.00) for the Promega assay, the 24-marker assay, and the 54-marker assay, respectively. Of the 11 tumors with retained MMR protein expression, four were MSI-low/MSI-high or MSI-high by the Promega assay or one of the sequencing-based assays. Conclusion: Our results show that Lynch syndrome-associated urothelial cancers frequently had loss of MMR protein expression. The Promega MSI assay was significantly less sensitive, but the 54-marker sequencing-based MSI analysis showed no significant difference compared to immunohistochemistry. Data from this study alongside previous studies, suggest that universal MMR deficiency testing of newly diagnosed urothelial cancers, using immunohistochemistry and/or sequencing-based MSI analysis of sensitive markers, offer a potentially useful approach to identification of Lynch syndrome cases.

Test of the FlashFREEZE unit in tissue samples freezing for biobanking purposes.

Availability of molecularly intact biospecimens is essential in genetic diagnostics to obtain credible results. Integrity of nucleic acids (particularly RNA) may be compromised at various steps of tissue handling, and affected by factors such as time to freeze, freezing technique and storing temperature. At the same time, freezing and storing of the biological material should be feasible and safe for the operator. Here, we compared quality of DNA and RNA from biospecimens derived from different organs (breast, colon, adrenal glands, testes, rectum and uterus) frozen either using dry ice-cooled isopentane or with FlashFREEZE unit, in order to verify if the latter is suitable for routine use in biobanking. Implementing FlashFREEZE device would enable us to limit the use of isopentane, which is potentially toxic and environmentally harmful, whilst facilitate standardization of sample freezing time. We considered factors such RNA and DNA yield and purity. Furthermore, RNA integrity and RNA/DNA performance in routine analyses, such as qPCR, next generation sequencing or microarray, were also assessed. Our results indicate that freezing of tissue samples either with FlashFREEZE unit or isopentane ensures biological material with comparable expression profiles and DNA mutation status, indicating that RNA and DNA of similar quality can be extracted from both. Therefore, our findings support the use of the FlashFREEZE device in routine use for biobanking purposes.

Inactivating BTK mutations in large B-cell lymphoma in a real-world cohort: Strong correlation with BCL2 translocation.

Inactivating mutations in Bruton's tyrosine kinase (BTK) in patients with follicular lymphoma (FL) have recently been reported. These mutations were found in BTK inhibitor-treatment naïve patients. Here, we report the BTK mutation status in a real-world cohort of patients with non-Hodgkin lymphoma. We found primary BTK mutations in 7.7% of patients with large B-cell lymphoma (LBCL) and in 14.1% of patients with FL. All patients with BTK-mutated LBCL were BCL2 translocation positive, and the correlation between BCL2 translocation and BTK mutation persisted even when patients with known transformation from FL were excluded.

The value of next-generation sequencing in routine diagnostics and management of patients with cytopenia.

INTRODUCTION: We performed a single-center study of real-world health data to investigate the direct clinical consequence of targeted next-generation sequencing (NGS) results integrated in the clinicopathological evaluation of patients with cytopenia suspected of myelodysplastic syndrome (MDS). METHODS: The study included 87 newly referred patients, who had a bone marrow examination, which included targeted NGS analysis. NGS was requested at the discretion of either examining pathologist or hematologist. Data were collected retrospectively from patient files including pathology reports with integrated NGS results. RESULTS: The NGS results had a diagnostic impact in 67 cases (77%) when combining both histopathological and final clinical evaluation and provided prognostic value in 19 cases (22%). NGS supported a confident or tentative histopathological diagnosis in 52 cases (60%). Twenty cases (23%) had a final diagnosis of either Clonal Cytopenia of Undetermined Significance (CCUS) or Idiopathic Cytopenia of Undetermined Significance (ICUS). In 4 cases, NGS results affected the choice of principal treatment strategy, including considerations of allotransplantation. Twenty-one patients (24%) could be discharged to primary care physician. CONCLUSION: In a multidisciplinary clinicopathological real-world setting, NGS analysis of bone marrow samples from selected patients contributed substantially to the diagnostic evaluation and management of patients with cytopenia suspected of MDS. Consequently, we have now included NGS analysis in most routine bone marrow examinations from patients with MDS or unexplained cytopenia.

Epigenetic therapy in combination with a multi-epitope cancer vaccine targeting shared tumor antigens for high-risk myelodysplastic syndrome - a phase I clinical trial.

BACKGROUND: Standard care for patients with high-risk myelodysplastic syndrome (MDS) is hypomethylating agents such as azacitidine (AZA), which can induce expression of methylated tumor-associated antigens and therefore potentiate immunotherapeutic targeting. METHOD: In this phase 1 trial, we combined AZA with a therapeutic peptide vaccine targeting antigens encoded from NY-ESO-1, MAGE-A3, PRAME, and WT-1, which have previously been demonstrated to be upregulated by AZA treatment. RESULT: Five patients who had responded to AZA monotherapy were included in the study and treated with the vaccine. The combination therapy showed only few adverse events during the study period, whereof none classified as serious. However, no specific immune responses could be detected using intracellular cytokine staining or ELISpot assays. Minor changes in the phenotypic composition of immune cells and their expression of stimulatory and inhibitory markers were detected. All patients progressed to AML with a mean time to progression from inclusion (TTP) of 5.2 months (range 2.8 to 7.6). Mean survival was 18.1 months (range 10.9 to 30.6) from MDS diagnosis and 11.3 months (range 4.3 to 22.2) from inclusion. Sequencing of bone marrow showed clonal expansion of malignant cells, as well as appearance of novel mutations. CONCLUSION: The patients progressed to AML with an average time of only five months after initiating the combination therapy. This may be unrelated to the experimental treatment, but the trial was terminated early as there was no sign of clinical benefit or immunological response. Why the manuscript is especially interesting This study is the first to exploit the potential synergistic effects of combining a multi-peptide cancer vaccine with epigenetic therapy in MDS. Although our results are negative, they emphasize challenges to induce immune reactivity in patients with high-risk MDS.

Bone Involvement as a Primary Rare Manifestation of Waldenstrom Macroglobulinemia: A Case Report and Prevalence in a Nationwide Population-Based Cohort Study.

Bone involvement is a rare extranodal manifestation in patients with malignant lymphoproliferative diseases and has also been noted as a rare event in patients with Waldenstrom macroglobulinemia (WM). However, the actual prevalence has not been previously reported. We describe an unusual case of a patient with WM who presented with lower back pain and focal bone lesions at initial diagnosis. Magnetic resonance imaging (MRI) revealed multiple vertebral fractures. Positron emission tomography (PET) detected only nodal changes without pathological skeletal-related metabolic activity. Lymph node and bone marrow biopsies combined with an immunoglobulin M (IgM) M component revealed the diagnosis of WM. A next-generation sequencing (NGS) analysis using a targeted lymphoma panel of 59 recurrently mutated genes in lymphoid neoplasms showed mutations in the MYD88 and CD79B genes. After treatment with rituximab and bendamustine, the patient achieved a partial remission and pain relief. After 3 years of stable disease, a spontaneous subcapital fracture at the base of the femoral neck and new vertebral compression fractures occurred. Whole-body low-dose computed tomography (WB-LDCT) and bone density (dual energy X-ray absorptiometry (DEXA)) scan revealed marked osteopenia. After insertion of a hip prosthesis, examination of the removed hip showed infiltration of clonal lymphoplasmacytic cells. Our case confirms that one must be aware that bone involvement in patients with WM can occur as a rare manifestation. Interestingly, the MYD88/CD79B-mutated (MCD) genotype in diffuse large B-cell lymphoma is characterized by extranodal involvement and may also be involved in the pathogenesis of skeletal-related disease in the present case. As a follow-up to this unusual case, we have carried out an analysis based on the Danish Lymphoma Registry (LYFO) covering the entire national population in the period 2000 - 2020. The registry study included a cohort of 2,459 patients with WM and lymphoplasmacytic lymphoma. Our data revealed that primary bone involvement at diagnosis occurs in 1.75% of adults with WM. To the best of our knowledge, this is the first report of the prevalence of skeletal-related disease in a large nationwide cohort and defines bone involvement as an exceedingly rare event in WM.

Improved Variant Detection in Clinical Myeloid NGS Testing by Supplementing a Commercial Myeloid NGS Assay with Custom or Extended Data Filtering and Accessory Fragment Analysis.

BACKGROUND: Commercial myeloid next-generation sequencing (NGS) panels may facilitate uniform generation of raw data between laboratories. However, different strategies for data filtering and variant annotation may contribute to differences in variant detection and reporting. Here, we present how custom data filtering or the use of Oncomine extended data filtering improve detection of clinically relevant mutations with the Oncomine Myeloid Research Assay. METHODS: The study included all patient samples (n = 264) analyzed during the first-year, single-site, clinical use of the Ion Torrent Oncomine Myeloid Research Assay. In data analysis, the default analysis filter was supplemented with our own data filtering algorithm in order to detect additional clinically relevant mutations. In addition, we developed a sensitive supplementary test for the ASXL1 c.1934dupG p.Gly646fs mutation by fragment analysis. RESULTS: Using our custom filter chain, we found 96 different reportable variants that were not detected by the default filter chain. Twenty-six of these were classified as variants of strong or potential clinical significance (tier I/tier II variants), and the custom filtering discovered otherwise undetected tier I/tier II variants in 25 of 132 patients with clinically relevant mutations (19%). The remaining 70 variants not detected by the default filter chain were classified as variants of unknown significance. Among these were several unique variants with possible pathogenic potential judged by bioinformatic predictions. The recently launched Oncomine 5.14 extended filter algorithm detects most but not all of the tier I/tier II variants that were not detected by the default filter. The supplementary fragment analysis for the ASXL1 c.1934dupG p.Gly646fs confidently detected a variant allele frequency of down to 4.8% (SD 0.83%). The assay also detected the ASXL1 c.1900_1922del23 mutation. CONCLUSION: Detection of clinically relevant variants with the Oncomine Myeloid Research NGS assay can be significantly improved by supplementing the default filter chain with custom data filtering or the recently launched Oncomine 5.14 extended filter algorithm. Our accessory fragment analysis facilitates easy testing for frequent ASXL1 mutations that are poorly or not covered by the NGS assay.

High-grade B-cell lymphomas with MYC and BCL2 translocations lack tumor-associated macrophages and PD-L1 expression: A possible noninflamed subgroup.

We investigated the intratumoral source of PD-L1 expression and the infiltration of tumor-associated macrophages (TAMs) in large B-cell lymphomas (LBCLs) with or without MYC-translocation, as well as possible correlations to BCL2-and BCL6-translocations and cell of origin (COO). One-hundred and twenty-six patient samples were studied in a cohort enriched for MYC-translocated tumors with 34 samples carrying this translocation. Demonstration of intratumoral distribution and cellular source of PD-L1 was enabled by immunohistochemical (IHC) dual staining specifically highlighting PD-L1 expression in lymphoma B-cells with antibodies against PD-L1 and PAX5. Additional IHC with antibodies against CD68 and CD163 identified TAMs. We found that CD68-positive TAMs were the main source of PD-L1 protein expression in contrast to lymphoma B cells which rarely expressed PD-L1. Semiquantitative IHC demonstrated a significant correlation between CD68 and PD-L1 protein expression. Unsupervised hierarchical analysis of PD-L1, CD68, and CD163 IHC data subsequently demonstrated three potential clusters defined by expression of the three biomarkers. Cluster A consisted of patient samples with significantly lower expression of PD-L1, CD68, and CD163, but also significantly higher prevalence of BCL2-translocation and MYC-BCL2-double-hit (DH) compared to the other two clusters. In cluster C we found a significant accumulation of BCL6 translocated tumors. This cluster in contrast had the highest protein expression of PD-L1, CD68, and CD163. Cluster B tumors had an intermediate expression of the three biomarkers, but no accumulation of the specific genetic translocations. Our data, which were based on morphological analysis, immunophenotyping and genotyping by fluorescence in situ hybridization were in line with new concepts of LBCL taxonomy integrating genetic, phenotypical, and immunological characteristics with identification of new subgroups where MYC translocation and MYC-BCL2 DH may identify a noninflamed subtype. These findings may furthermore hold significant predictive value especially regarding immune checkpoint blockade therapy, but further molecular characterization should be done to substantiate this hypothesis.

Marked variability in clinical presentation and outcome of patients with C1q immunodeficiency.

OBJECTIVE: Globally approximately 60 cases of C1q deficiency have been described with a high prevalence of Systemic Lupus Erythematosus (SLE). So far treatment has been guided by the clinical presentation rather than the underlying C1q deficiency. Recently, it was shown that C1q production can be restored by allogeneic hematopoietic stem cell transplantation. Current literature lacks information on disease progression and quality of life of C1q deficient persons which is of major importance to guide clinicians taking care of patients with this rare disease. METHODS: We performed an international survey, of clinicians treating C1q deficient patients. A high response rate of >70% of the contacted clinicians yielded information on 45 patients with C1q deficiency of which 25 are published. RESULTS: Follow-up data of 45 patients from 31 families was obtained for a median of 11 years after diagnosis. Of these patients 36 (80%) suffer from SLE, of which 16 suffer from SLE and infections, 5 (11%) suffer from infections only and 4 (9%) have no symptoms. In total 9 (20%) of the C1q deficient individuals had died. All except for one died before the age of 20 years. Estimated survival times suggest 20% case-fatality before the age of 20, and at least 50% of patients are expected to reach their middle ages. CONCLUSION: Here we report the largest phenotypic data set on C1q deficiency to date, revealing high variance; with high mortality but also a subset of patients with an excellent prognosis. Management of C1q deficiency requires a personalized approach.

Novel CFI mutation in a patient with leukocytoclastic vasculitis may redefine the clinical spectrum of Complement Factor I deficiency.

Factor I is an important regulator of the complement system. Lack of Factor I causes uncontrolled activation of the complement system leading to consumption of C3. Complete deficiency of Factor I is a rare condition and only around 40 cases has been reported in the literature. The clinical presentation of Factor I deficiency varies and includes severe recurrent bacterial infections, glomerulonephritis and autoimmune diseases. The patient, a 28-years old woman with consanguineous parents, presented with recurrent leukocytoclastic vasculitis in the lower extremities with no associated systemic involvement, and without increased infection tendency. Initial testing showed low C3 concentration and a detailed complement evaluation absence of complement Factor I. Sequencing revealed a homozygous missense mutation in exon 2 of the CFI gene (SCV000221312). Even though the clinical symptoms of CFI mutations vary among patients sole association with leukocytoclastic vasculitis redefines the clinical spectrum of complete Factor I deficiency.

Recurrent severe invasive pneumococcal disease in an adult with previously unknown hyposplenia.

BACKGROUND: The risk of life-threatening and invasive infections with encapsulated bacteria is increased in patients with hyposplenia or asplenia. We report a case of recurrent invasive pneumococcal meningitis in a woman with previous unknown hyposplenia. She was vaccinated after the first episode of meningitis and developed sufficient levels of pneumococcal antibodies. The pneumococcal strains isolated were serotype 7 F and 17 F. To our knowledge, there has been no previously reported case of recurrent invasive pneumococcal disease in a pneumococcal vaccinated adult with hyposplenia and apparently sufficient antibody response. CASE PRESENTATION: We report the course of a 38-year-old Caucasian woman presenting with recurrent episodes of invasive pneumococcal disease (IPD) and previously unknown hyposplenia. Hyposplenia was discovered during the second episode of IPD and no underlying medical condition was found. Despite immunization against S. pneumoniae and measurement of what was interpreted as protective levels of serotype-specific IgG antibodies after vaccination, the patient suffered from a third episode of IPD. CONCLUSIONS: Individuals with predisposing medical conditions or a history of severe infections with encapsulated bacteria should be screened for spleen dysfunction. If splenic function is impaired, prevention against severe invasive infection with encapsulated bacteria are a major priority.

Immunodeficiency among children with recurrent invasive pneumococcal disease.

BACKGROUND: Recurrent invasive pneumococcal disease (rIPD) occurs mostly in children with an underlying disease, but some cases remain unexplained. Immunodeficiency has been described in children with rIPD, but the prevalence is unknown. We used a nationwide registry of all laboratory-confirmed cases of rIPD to identify cases of unexplained rIPD and examine them for immunodeficiency. METHODS: Cases of rIPD in children 0-15 years of age from 1980 to 2008 were identified. Children without an obvious underlying disease were screened for complement function, T-cell, B-cell, natural killer--cell counts and concentration of immunoglobulins. B-cell function was evaluated by measuring antibody response to polysaccharide-based pneumococcal vaccination and the extent of fraction of somatic hypermutation. Toll-Like receptor (TLR) signaling function and mutations in key TLR-signaling molecules were examined. RESULTS: In total, rIPD were observed in 54 children (68 cases of rIPD of 2192 IPD cases). Children with classical risk factors for IPD were excluded, and among the remaining 22 children, 15 were eligible for analysis. Of these 6 (40%) were complement C2-deficient. Impaired vaccination response was found in 6 children of whom 3 were C2 deficient. One patient had a severe TLR signaling dysfunction. No mutations in IRAK4, IKBKG or MYD88 were found. CONCLUSION: Of an unselected cohort of children with rIPD at least 11% were C2 deficient. Data suggest that screening for complement deficiencies and deficient antibody response to pneumococcal vaccines in patients with more than 1 episode of IPD is warranted.

Genotype/phenotype correlations in complement factor H deficiency arising from uniparental isodisomy.

We report a male infant who presented at 8 months of age with atypical hemolytic uremic syndrome (aHUS) responsive to plasma therapy. Investigation showed him to have complement factor H (CFH) deficiency associated with a homozygous CFH mutation (c.2880delT [p.Phe960fs]). Mutation screening of the child's parents revealed that the father was heterozygous for this change but that it was not present in his mother. Chromosome 1 uniparental isodisomy of paternal origin was confirmed by genotyping chromosome 1 SNPs. CD46 SNP genotyping was undertaken in this individual and another patient with CFH deficiency associated with chromosome 1 uniparental isodisomy. This showed a homozygous aHUS risk haplotype (CD46GGAAC) in the patient with aHUS and a homozygous glomerulonephritis risk haplotype (CD46AAGGT) in the patient with endocapillary glomerulonephritis. We also showed that FHL-1 (factor H-like protein 1) was present in the patient with aHUS and absent in the patient with glomerulonephritis. This study emphasizes that modifiers such as CD46 and FHL-1 may determine the kidney phenotype of patients who present with homozygous CFH deficiency.

Primary complement C5 deficiencies - molecular characterization and clinical review of two families.

Inherited deficiency states of the terminal complement component C5 are rare and often associated with increased risk of recurrent Neisseria infections. More than 50 cases with primary C5 deficiency have been reported. In spite of this, the molecular basis has only been documented in a few cases. In the present study we investigated two unrelated Caucasian probands with C5 deficiency originating from Norway and Denmark, respectively, and found three previously undescribed mutations. With these data, thirteen mutations associated with C5 deficiency have been described. By genetic screening of the family of the Norwegian patient, previously diagnosed as homozygous C5 deficient and suffering four Neisseria infections, an additional case of C5 deficiency was discovered, who had experienced one episode of Neisseria infections. Detailed review of the clinical history of the patients and their healthy relatives did not reveal any differences between C5 deficient and sufficient individuals with regard to clinical presentation, apart from the susceptibility to Neisseria infections. Of note, one of the patients described here, and several C5 deficient patients from the literature had Neisseria meningitidis serotype B infections, which is not covered by the current vaccines. These data support the clinical guidelines for patients treated with C5 inhibitors, who are functional C5 deficient by the treatment.

Low C4 gene copy numbers are associated with superior graft survival in patients transplanted with a deceased donor kidney.

Complement C4 is a central component of the classical and the lectin pathways of the complement system. The C4 protein exists as two isotypes C4A and C4B encoded by the C4A and C4B genes, both of which are found with varying copy numbers. Deposition of C4 has been implicated in kidney graft rejection, but a relationship between graft survival and serum C4 concentration as well as C4 genetic variation has not been established. We evaluated this using a prospective study design of 676 kidney transplant patients and 211 healthy individuals as controls. Increasing C4 gene copy numbers significantly correlated with the C4 serum concentration in both patients and controls. Patients with less than four total copies of C4 genes transplanted with a deceased donor kidney experienced a superior 5-year graft survival (hazard ratio 0.46, 95% confidence interval: 0.25-0.84). No significant association was observed in patients transplanted with a living donor. Thus, low C4 copy numbers are associated with increased kidney graft survival in patients receiving a kidney from a deceased donor. Hence, the degree of ischemia may influence the clinical impact of complement.

Anti-glomerular basement membrane glomerulonephritis and thrombotic microangiopathy in first degree relatives: a case report.

BACKGROUND: Anti-glomerular basement membrane glomerulonephritis and thrombotic microangiopathy are rare diseases with no known coherence. CASE PRESENTATION: A daughter and her biological mother were diagnosed with pregnancy-induced thrombotic microangiopathy and anti-glomerular basement membrane glomerulonephritis, respectively. Both developed end-stage renal disease. Exploration of a common aetiology included analyses of HLA genotypes, functional and genetic aspects of the complement system, ADAMTS13 activity and screening for autoantibodies.The daughter was heterozygous carrier of the complement factor I G261D mutation, previously described in patients with membranoproliferative glomerulonephritis and atypical haemolytic uremic syndrome. The mother was non-carrier of this mutation. They shared the disease associated complement factor H silent polymorphism Q672Q (79602A>G). CONCLUSION: An unequivocal functional or molecular association between these two family cases was not found suggesting that the patients probably share another, so far undiagnosed and unknown, predisposing factor. It seems highly unlikely that two infrequent immunologic diseases would occur by unrelated pathophysiological mechanisms within first degree relatives.

Complement plays a central role in Candida albicans-induced cytokine production by human PBMCs.

In experimental studies, the role of complement in antifungal host defense has been attributed to its opsonizing capability. In this study, we report that in humans an activated complement system mainly augments Candida albicans-induced host proinflammatory cytokine production via C5a-C5aR signaling, while phagocytosis and intracellular killing of Candida are not influenced. By blocking the C5a-C5aR signaling pathway, either with anti-C5a antagonist antibodies or with the C5aR antagonist W-54001, C. albicans-induced IL-6 and IL-1ß levels were significantly reduced. Recombinant C5a augmented cytokine production. In addition, using serum from patients with various complement deficiencies, we demonstrated a crucial role of C5, but not C6 or the membrane attack complex, in C. albicans-induced IL-6 and IL-1ß production in monocytes. These findings reveal a central role of anaphylatoxin C5a in augmenting host proinflammatory cytokine production upon contact with C. albicans, and define the role of the complement system in anti-Candida host defense in humans.