Annette M. May

Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations

The protein cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of immune responses, and its loss causes fatal autoimmunity in mice. We studied a large family in which five individuals presented with a complex, autosomal dominant immune dysregulation syndrome characterized by hypogammaglobulinemia, recurrent infections and multiple autoimmune clinical features. We identified a heterozygous nonsense mutation in exon 1 of CTLA4. Screening of 71 unrelated patients with comparable clinical phenotypes identified five additional families (nine individuals) with previously undescribed splice site and missense mutations in CTLA4. Clinical penetrance was incomplete (eight adults of a total of 19 genetically proven CTLA4 mutation carriers were considered unaffected). However, CTLA-4 protein expression was decreased in regulatory T cells (Treg cells) in both patients and carriers with CTLA4 mutations. Whereas Treg cells were generally present at elevated numbers in these individuals, their suppressive function, CTLA-4 ligand binding and transendocytosis of CD80 were impaired. Mutations in CTLA4 were also associated with decreased circulating B cell numbers. Taken together, mutations in CTLA4 resulting in CTLA-4 haploinsufficiency or impaired ligand binding result in disrupted T and B cell homeostasis and a complex immune dysregulation syndrome.

Cytogenetics of extramedullary manifestations in multiple myeloma

Extramedullary disease in patients with multiple myeloma is a rare event, occurring mostly in advanced disease or relapse. Outcome is poor and prognostic factors predicting the development of extramedullary disease have not been defined. We investigated cytogenetic alterations of myeloma cells in different extramedullary manifestations by adapting the fluorescence in situ hybridization (FISH) technique in combination with cytoplasmic immunoglobulin staining to study the cytogenetics of plasma cell tumours on paraffin embedded material. Thirty six patients were investigated: 19 with extramedullary disease, 11 with skeletal extramedullary disease and six with solitary extramedullary plasmacytoma. The first two groups showed the following results: del(17p13) 32% vs. 27%, del(13q14) 35% vs. 27%, MYC‐overrepresentation 28% vs. 18% and t(4;14) 37% vs. 18%. We detected an overall higher incidence of del(17p13) in both groups compared to data from bone marrow samples of multiple myeloma reported to date (range 7–16%). The solitary extramedullary plasmacytomas presented overall less cytogenetic aberrations than the other groups. Most important, three patients with extramedullary disease and one with skeletal extramedullary disease presented different FISH findings in the extramedullary tumour compared to their bone marrow plasma cells. del(17p13), occurring additional in three of four cases, seems a strong marker for extramedullary progression of myeloma.

Large registry analysis to accurately define second malignancy rates and risks in a well-characterized cohort of 744 consecutive multiple myeloma patients followed-up for 25 years

Additional malignancies in multiple myeloma patients after first-line and maintenance treatment have been observed, questioning whether specific risks exist. Second primary malignancies have also gained attention since randomized data showed associations to newer drugs. We have conducted this large registry analysis in 744 consecutive patients and analyzed: 1) frequency and onset of additional malignancies; and 2) second primary malignancy- and myeloma-specific risks. We assessed the frequency of additional malignancies in terms of host-, myeloma- and treatment-specific characteristics. To compare these risks, we estimated cumulative incidence rates for second malignancies and myeloma with Fine and Gray regression models taking into account competing risks. Additional malignancies were found in 118 patients: prior or synchronous malignancies in 63% and subsequent in 37%. Cumulative incidence rates for second malignancies were increased in IgG-myeloma and decreased in bortezomib-treated patients (P<0.05). Cumulative incidence rates for myeloma death were increased with higher stage and age, but decreased in IgG-subtypes and due to anti-myeloma treatment (P<0.05). Cytogenetics in patients acquiring second primary malignancies were predominantly favorable, suggesting that indolent myeloma and long disease latency may allow the manifestation of additional malignancies. An assessment of the Surveillance, Epidemiology, and End Result Program of the National Cancer Institute and our data with long-term follow up of 25 years confirmed a prevalence of second malignancy of 10% at 25 years, whereas death from myeloma decreased from 90% to 83%, respectively. Our important findings widen our knowledge of second malignancies and show that they are of increasing relevance as the prognosis in myeloma improves and mortality rates decrease.

Immune response as a possible mechanism of long-lasting disease control in spontaneous remission of MLL/AF9-positive acute myeloid leukemia

Spontaneous complete remission (CR) is a rare, poorly understood phenomenon in acute myeloid leukemia (AML). We describe the 10-year follow-up of a patient with MLL-AF9-positive AML (Müller et al. Eur J Haematol 73:62–66, 2004), including ex vivo antileukemic immune responses which may contribute to the long-lasting spontaneous CR (tantamount to cure). We could demonstrate strong in vitro cytotoxic activity mediated by the patient’s serum (cryopreserved at diagnosis 2001) against myeloid cell lines. We also addressed cellular cytotoxic activity against myeloid leukemia cells. When the patient’s natural killer (NK) cells (obtained in 2007) were tested against the K562 cell line, upregulation of CD107 occurred, implying that long-term CR in this patient could be due to NK cell-mediated disease control.

Intratibial Injection of Human Multiple Myeloma Cells in NOD/SCID IL-2Rγ(Null) Mice Mimics Human Myeloma and Serves as a Valuable Tool for the Development of Anticancer Strategies

Background We systematically analyzed multiple myeloma (MM) cell lines and patient bone marrow cells for their engraftment capacity in immunodeficient mice and validated the response of the resulting xenografts to antimyeloma agents. Design and Methods Using flow cytometry and near infrared fluorescence in-vivo-imaging, growth kinetics of MM cell lines L363 and RPMI8226 and patient bone marrow cells were investigated with use of a murine subcutaneous bone implant, intratibial and intravenous approach in NOD/SCID, NOD/SCID treated with CD122 antibody and NOD/SCID IL-2Rγ(null) mice (NSG). Results Myeloma growth was significantly increased in the absence of natural killer cell activity (NSG or αCD122-treated NOD/SCID). Comparison of NSG and αCD122-treated NOD/SCID revealed enhanced growth kinetics in the former, especially with respect to metastatic tumor sites which were exclusively observed therein. In NSG, MM cells were more tumorigenic when injected intratibially than intravenously. In NOD/SCID in contrast, the use of juvenile long bone implants was superior to intratibial or intravenous cancer cell injection. Using the intratibial NSG model, mice developed typical disease symptoms exclusively when implanted with human MM cell lines or patient-derived bone marrow cells, but not with healthy bone marrow cells nor in mock-injected animals. Bortezomib and dexamethasone delayed myeloma progression in L363- as well as patient-derived MM cell bearing NSG. Antitumor activity could be quantified via flow cytometry and in vivo imaging analyses. Conclusions Our results suggest that the intratibial NSG MM model mimics the clinical situation of the disseminated disease and serves as a valuable tool in the development of novel anticancer strategies.

Detection of MYD88 L265P mutations in formalin-fixed and decalcified BM biopsies from patients with lymphoplasmacytic lymphoma

The diagnosis of bone marrow (BM) infiltration by Waldenström macroglobulinemia (WM)/lymphoplasmacytic lymphoma (LPL) poses a diagnostic challenge in hematopathology. No definitive morphology or immunophenotype is able to distinguish between infiltration of paraffin-embedded BM sections by WM/LPL and other indolent lymphomas, in particular those of the splenic marginal zone (SMZL) which may also show plasmacytic maturation. An oncogenic gain-of-function mutation (L265P) in the human MYD88 gene has been found to be present in most cases of WM/LPL, yet is absent in most other cases of B-cell chronic lymphoproliferative disorders (LPD), including SMZL. Here, we compare two newly developed diagnostic protocols for detection of this mutation in paraffin-embedded archival tissues which are particularly applicable to decalcified BM biopsies. Sanger sequencing can easily detect levels of BM infiltration above 15% by WM lymphoplasmacytic cells, while the allele-specific PCR can detect the L265P mutation in BM infiltrations below 1% of lymphoma cells. We show that these methods are easily applicable to archival BM specimens and markedly improve diagnostic accuracy of BM infiltrations by indolent B-cell lymphomas.

From CLL to Multiple Myeloma - Spleen Tyrosine Kinase (SYK) influences multiple myeloma cell survival and migration

Arnaud Petit Andr e Baruchel Jacqueline Clavel Benôıt Brethon Yves Bertrand Patrick Lutz Fabienne Nacka St ephane Ducassou Unit of Paediatric Haematology and Oncology, Bordeaux University Hospital, Bordeaux-Cedex, Unit of Paediatric Haematology and Oncology, Armand Trousseau Hospital, Unit of Paediatric Haematology and Immunology, Robert Debr e Hospital, Paris, Institut National de la Sant e et de la recherche M edicale (INSERM), Unit754, Registry of Malignant Hemopathies in childhood, Villejuif, Institute of Paediatric Haematology and Oncology, 1, place du Professeur Joseph Renault, Lyon, Unit of Paediatric Haematology and Oncology, Hautepierre Hospital, Strasbourg, and Clinical Investigation Centre in Paediatrics CIC 1401, Bordeaux University Hospital, Bordeaux-Cedex, France E-mail: stephane.ducassou@chu-bordeaux.fr

Decitabine in combination with donor lymphocyte infusions can induce remissions in relapsed myeloid malignancies with higher leukemic burden after allogeneic hematopoietic cell transplantation

The combination of 5-azacytidine (AZA) with donor lymphocyte infusions (DLIs) can induce remissions in patients with relapsed myeloid malignancies after allo-HCT. As decitabine (DAC) is known to be effective also in AML/MDS with leukocytosis, we investigated the combination of DAC with DLIs for relapse after allo-HCT. Between 2006 and 2016, 26 patients (median age 59 years) with AML (n = 18), MDS (n = 6), or MPN (n = 2) and overt hematological relapse after allo-HCT were treated. Median duration from allo-HCT to relapse was 306 days (range, 76-4943). Eighteen patients received DAC + DLIs, 8 DAC-only (median number cycles of DAC: 2, range 1-13, median number of DLIs: 2, range 1-10). The incidence of acute and chronic GvHD in patients receiving DLI was 17% (3/18) and 6% (1/18), respectively. CR/CRi was achieved in 15% (4/26), PR in 4% (1/26), and stable disease in 58% (15/26) of patients. Eight patients received a second allo-HCT. Median overall survival was 4.7 months. Elevated PD-L1 protein expression in bone marrow cells was detected in 4/8 patients with >20% blast infiltration prior to DAC, without a clear association with response. In conclusion, the DAC + DLI regimen proved feasible and effective in relapsed myeloid malignancies after allo-HCT, with efficacy not restricted to patients with low leukemic burden.

Biallelic Expression of Mucin-1 in Autosomal Dominant Tubulointerstitial Kidney Disease: Implications for Nongenetic Disease Recognition

BACKGROUND Providing the correct diagnosis for patients with tubulointerstitial kidney disease and secondary degenerative disorders, such as hypertension, remains a challenge. The autosomal dominant tubulointerstitial kidney disease (ADTKD) subtype caused by MUC1 mutations (ADTKD-MUC1) is particularly difficult to diagnose, because the mutational hotspot is a complex repeat domain, inaccessible with routine sequencing techniques. Here, we further evaluated SNaPshot minisequencing as a technique for diagnosing ADTKD-MUC1 and assessed immunodetection of the disease-associated mucin 1 frameshift protein (MUC1-fs) as a nongenetic technique. METHODS We re-evaluated detection of MUC1 mutations by targeted repeat enrichment and SNaPshot minisequencing by haplotype reconstruction via microsatellite analysis in three independent ADTKD-MUC1 families. Additionally, we generated rabbit polyclonal antibodies against MUC1-fs and evaluated immunodetection of wild-type and mutated allele products in human kidney biopsy specimens. RESULTS The detection of MUC1 mutations by SNaPshot minisequencing was robust. Immunostaining with our MUC1-fs antibodies and an MUC1 antibody showed that both proteins are readily detectable in human ADTKD-MUC1 kidneys, with mucin 1 localized to the apical membrane and MUC1-fs abundantly distributed throughout the cytoplasm. Notably, immunohistochemical analysis of MUC1-fs expression in clinical kidney samples facilitated reliable prediction of the disease status of individual patients. CONCLUSIONS Diagnosing ADTKD-MUC1 by molecular genetics is possible, but it is technically demanding and labor intensive. However, immunohistochemistry on kidney biopsy specimens is feasible for nongenetic diagnosis of ADTKD-MUC1 and therefore, a valid method to select families for further diagnostics. Our data are compatible with the hypothesis that specific molecular effects of MUC1-fs underlie the pathogenesis of this disease.

Acute subepicardial infarction associated with severe septic shock--insight in myocardial perfusion.

Isolated infarctions of the subepicardial myocardium without changes in subendocardium are extremely rare. We present an autoptic case with an acute subepicardial infarction of the right- and left-ventricular myocardium. A 53-year-old male was admitted to hospital with acute upper abdominal pain. Clinical examination revealed an acute infero-lateral myocardial infarction. The patient succumbed to acute heart failure a few hours later. Autopsy revealed numerous pulmonary abscesses due to suppurative lobular pneumonia with consecutive pericardial effusion. Furthermore, we diagnosed an acute myocardial infarct encompassing the entire right and left ventricles but limited to the subepicardial myocardium only. Microscopically, we observed fibrin microemboli in the subepicardial microvessels. The existence of an isolated subepicardial myocardial infarct challenges our understanding of myocardial perfusion.