bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence of Light-Chain Monoclonal Gammopathy of Undetermined Significance (LC-MGUS) among Olmsted County, Minnesota Residents Aged 50 Years or Greater.
Authors: Rajkumar S. Vincent , Kyle Robert , Plevak Matthew , Clark Raynell , Larson Dirk , Therneau Terry , Bradwell Arthur , Dispenzieri Angela , Melton L. Joseph , Katzmann Jerry
Published: April 2020

Background: Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant plasma cell disorder that carries a 1% per year risk of progression to multiple myeloma (MM) or related malignancy. The prevalence and natural history of MGUS, in which by definition intact immunoglobulin heavy chain (IgH) is expressed, has been well described. However, up to 20% of myeloma (MM) is characterized by complete lack of IgH expression (Light-chain MM); the prevalence of a corresponding precursor entity, light chain MGUS (LC-MGUS) has not been determined. We report the first prevalence estimates of LC-MGUS in the general population from a large, well-defined geographic population using modern laboratory techniques.

Methods: The cohort for this study was derived from one previously assembled by us to estimate the prevalence of MGUS (N Engl J Med2006;354:1362-9). The original cohort used to estimate the prevalence of MGUS consisted of 21,463 of the 28,038 enumerated residents aged 50 or over of Olmsted County Minnesota as of January 1, 1995. The sensitive serum free light chain (FLC) assay (The Binding Site Limited, Birmingham, U.K.) was performed on stored serum samples from these 21,463 persons. IgH expression was determined by immunofixation on all FLC results that had an abnormal kappa/lambda ratio (<0.26 or >1.65). LC-MGUS was defined as the presence of an abnormal FLC ratio and a negative immunofixation for IgH expression.

Results: Adequate stored serum samples were available in 20,733 (97%) of the 21,463 persons. To date, the FLC assay has been performed and results were available for analysis on samples from 16,637 persons. An abnormal FLC ratio was observed in 572 persons. IgH expression was detected in 255 of these cases on immunofixation; these persons are considered as having MGUS, and were excluded from the estimation of LC-MGUS prevalence. This resulted in 317 persons out of 16,637 who had an abnormal FLC ratio without evidence of IgH expression, resulting in an estimated prevalence of LC- MGUS of 2%. Of the 317 cases of LC-MGUS identified in this study, 217 were kappa and 100 were lambda; in 35 cases the presence of the corresponding monoclonal light chain was apparent on immunofixation. The median age of the cohort of LC-MGUS was 62 years; males=151, females =166. The involved FLC level ranged from 0.118–270.0 mg/dL. The FLC ratio ranged from 0.014–0.253 (lambda) and 1.67–511.01 (kappa). So far, progression to multiple myeloma has occurred in 4 patients, a rate much higher than what is expected based on the prevalence of myeloma in the general population. Two additional patients have developed CLL.

Conclusions: LC-MGUS is prevalent in 2% of the general population aged 50 years of age or older. The natural history of this disorder needs to be determined.
Impact of Whole-Body Magnetic Resonance Imaging on Staging in Patients with Newly Diagnosed Plasma Cell Disease.
Authors: Zoz Martin , Baldauf Andreas , Schipp Anne , Hillengass Jens , Ho Anthony D. , Goldschmidt Harmut , Hansmann Jochen , Moehler Thomas
Published: April 2020

INTRODUCTION: MRI has a high sensitivity in determining changes in bone marrow induced by metastatic disease or primary neoplasms of the bone marrow. Whole-Body Magnetic Resonance Imaging (WB-MRI) is a novel imaging technique that displays nearly the complete skeletal system in one exam. We investigated the differences between conventional skeletal survey and WB-MRI and their impact on staging of patients with newly diagnosed plasma cell disease.

METHOD AND MATERIALS: In 41 consecutive patients with newly diagnosed MGUS (n=5), Multiple Myeloma (n=34) or AL-Amyloidosis (n=2) conventional radiographs and WB-MRI (coronar T1 tse and T2 tirm sequences and sagittal T2 star sequences, 1,5 T MRI with parallel imaging, Siemens Avanto®) were performed. Radiographs and scans were evaluated for diffuse and focal bone marrow involvement in consent by two experienced radiologists blinded for patient name and study time. Staging was performed including clinical data according to the Salmon/Durie classification system and the Durie/Salmon PLUS classification system with inclusion of WB-MRI.

RESULTS: In 24 (59%) patients there were no lesions in conventional radiography or MRI. In 17 (41 %) patients results of MRI and conventional radiography were discrepant. 4 (10%) patients had lesions only in MRI, 3 (7%) only in conventional radiography and 10 (24%) in both techniques but in different localization. In 16 (38%) patients with radiological signs of osteopenia there was no diffuse infiltration in MRI or vice versa. We systematically analysed the consequences of WB-MRI on staging of patients based on the newly proposed staging system Durie/Salmon PLUS. Replacing conventional radiographs by WB-MRI resulted in reclassification of 12 patients. 3 patients were reclassified from MGUS or stage I into stage II or III. 9 patients were downstaged from stage III or II into stage I or MGUS.

CONCLUSION: WB-MRI is a valuable technique for the initial work-up of patients with Multiple Myeloma. In comparison with conventional skeletal survey there are often differing results with impact on clinical staging and influence on therapy decision. WB-MRI can give additional information in patients with unclear staging situation before onset of therapy. Further evaluation of WB-MRI within prospective studies is warranted in particular with respect to prognostic impact regarding overall prognosis as well as regarding local complications. Until then WB-MRI should be used complementary to conventional radiography or CT-techniques that provide accurate imaging of the bone.
Differential Effects of Hsp70 Inhibition within the Tumor and Microenvironment in Multiple Myeloma.
Authors: Braunstein Marc J. , Liu Na , Klueppelberg Uwe , Scott Craig , Behrman Shannon , Tamucin Can , Walter Peter , Wipf Peter , Brodsky Jeffrey , Batuman Olcay
Published: April 2020

Multiple myeloma (MM) remains fatal despite prolonged survival by recent advances in treatment, including the use of proteasome inhibitors. It has been proposed that proteasome inhibitors target MM by modulating the NF-κB pathway. Alternatively, proteasome inhibitors may target MM cells because the proteasome helps alleviate the unfolded protein response (UPR) that results from the accumulation of aberrant proteins in the endoplasmic reticulum (ER), which in turn may trigger apoptosis. Indeed, the UPR is induced in neoplastic plasma cells, possibly because of increased immunoglobulin (IG) synthesis that exceeds the protein folding capacity of the ER. Consequently, the degree to which inhibition of the proteasome induces apoptosis may be related to the concentration of unfolded light chains within the ER in MM. We therefore examined whether inhibition of a protein-folding mediator, the Hsp70 molecular chaperone, results in synergistic induction of MM cell apoptosis when combined with a proteasome inhibitor. To this end, the effects of MAL3-101, a novel inhibitor of Hsp70 function, both alone and in combination with a proteasome inhibitor (MG132) on three MM cell lines as well as primary patient MM cells were examined. Dose-response and time course studies in MM cell lines U266, RPMI-8226, and NCI-H929 showed increasing apoptosis and inhibition of proliferation after 16 hours of exposure to MAL3-101 (IC50: 0.9 μM) or to MG132 (IC50: 7 μM). Strikingly, when sub-effective concentrations of MG132 and MAL3-101 were combined, a strong, synergistic apoptotic response was observed in the NCI cell line after 16 hours, and synergistic effects were observed in all cell lines after 36 hours of exposure to the two drugs. Next, we studied MM cells and endothelial progenitor cells (EPCs) derived from the bone marrow of five untreated patients. These two cell populations, which were shown to bear clonotypic similarities, also showed sensitivity to dual targeting. However, these effects occurred at drug concentrations different than those found to be most potent in the cell lines. Moreover, semi-quantitative RT-PCR studies indicated that RPMI cells but not U266 cells exhibited a strong UPR induction after 16 hours of exposure to 7 μM MG132. These data correlated with the greater degree of IG secretion observed in RPMI cells compared to U266 cells as assessed by pulse-chase analysis. Taken together, these results suggest that the apoptotic response of MM cells via targeting the UPR and ER stress pathways may be dependent on basal protein production, including IG synthesis. Studies relating the secretion “index” to UPR induction and sensitivity to Hsp70 and proteasome inhibition in primary tumor cells as well as in microvascular cells from MM patients are ongoing.
Single Institution Study of Abnormalities Involving Chromosomes 13 and 17 Detected by Classical Cytogenetics and Fluorescent In Situ Hybridization in Myeloma and Their Relationship to Survival in Patients Undergoing Autologous Stem Cell Transplant.
Authors: Rajan Arun , Stein Constance , Gentile Teresa , Shah Chirag
Published: April 2020

Multiple myeloma is associated with a variety of chromosomal anomalies each of which may affect survival to varying degrees. Recent data indicate that patients with chromosome 13 and 17 anomalies have a poor prognosis. In the current single institution study we analyzed the presence of anomalies involving chromosomes 13 and 17 detected by classical cytogenetics (karyotype analysis) (CC) and interphase fluorescent in situ hybridization (FISH) in patients who underwent autologous stem cell transplantation for multiple myeloma and correlated this with mean survival. Our patients had undergone autologous stem cell transplantation between April 1998 and March 2006. Of the 79 patients studied, data on CC and FISH was available on 43 patients and 29 patients respectively. 34 of 43 patients had normal CC and 11 of 29 patients had normal FISH. The mean survival of patients with normal cytogenetics was 45.36 months and for those with a normal FISH result it was 50 months. 9 of 43 patients had an abnormal CC result and 18 of 29 patients had an abnormal FISH result. Mean survival with an abnormal CC result was 30.40 months compared to 40.83 months with an abnormal FISH result. Anomalies of chromosomes 13 and/or 17 were seen in 6 of 9 patients by CC and 15 of 18 patients by FISH. Mean survival in these two groups was 30.25 months and 42.80 months respectively. These results indicate that normal cytogenetics and fluorescent in situ hybridization results at the time of diagnosis are associated with favorable outcome. The presence of abnormalities detected by conventional cytogenetics is associated with poorer outcome, particularly for deletion 13q and deletion 17p. Patients with interphase FISH abnormalities for chromosomes 13 and 17 have better survival than those with abnormalities for these chromosomes detected by conventional cytogenetics. Abnormalities in conventional cytogenetics may suggest actively proliferating disease. Thus interphase FISH should not substitute for conventional cytogenetic analysis in the diagnosis of myeloma.
Bortezomib Therapy Response Is Independent of Cytogenetic Abnormalities in Relapsed/Refractory Multiple Myeloma.
Authors: Chang Hong , Trieu Young , Qi Xiaoying , Xu Wei , Stewart Keith , Reece Donna
Published: April 2020

Myeloma patients with unfavorable molecular cytogenetics have a poor prognosis irrespective of treatment with conventional chemotherapy or autologous stem cell transplant. To investigate whether bortezomib, a new proteasome inhibitor, is active in relapsed/refractory myeloma patients with genetic risk factors, we evaluated the outcome of 65 patients and correlated the clinical response with 13q deletion, translocations t(11;14) and t(4;14) and CKS1B amplification as detected by interphase cytoplasmic fluorescence in situ hybridization (cIg-FISH). Thirty-seven of 61 (61%) evaluable patients had an objective response to bortezomib with median progression free (PFS) and overall survivals (OS) of 9.5 and 15.1 months, respectively. Of 43 cases with evaluable bone marrows, cIg-FISH determination of del(13q), t(4;14), t(11;14) and CKS1B amplification was done on 40, 41, 41 and 37 cases and the frequency of their detection was 35%, 15%, 15%, and 32% respectively. Two different abnormalities coexisted in 12 patients: 13q deletion with CKS1B amplification in 7, t(11;14) with CKS1B amplification in 2, t(4;14) with CKS1B amplification in 1, 13q deletion with t(11;14) in 1 and 13q deletion with t(4;14) in 1 patient. There was no statistically significant difference in response to bortezomib for patients with or without 13q deletion (77% vs. 50%), t(4;14) (67% vs. 56%), t(11;4) (33% vs. 62%), or CKS1B amplification (67% vs. 57%). Furthermore, there was no statistically significant difference in PFS or OS following bortezomib therapy between patients with or without these molecular cytogenetic abnormalities. Our data suggest that, in this pilot study, bortezomib is an effective salvage therapy for refractory/relapsed myeloma, irrespective of genetic risk factors.
Radiation Recall Dermatitis after Bortezomib Therapy.
Authors: Amin Alpesh J. , Maroules Michael
Published: April 2020

Introduction: Radiation recall dermatitis (RRD) is an inflammatory skin reaction at a site of previous radiation therapy after the administration of a medication, usually a chemotherapeutic agent. A radiation recall reaction has not been previously reported with bortezomib.

Case Report: A seventy-year-old man with stage III IgD multiple myeloma was treated with bortezomib after previous treatments with pulse decadron and cyclophosphamide. The patient was treated ten years earlier for a chest wall sarcoma with a local resection and radiation therapy to the tumor bed. After receiving bortezomib therapy for multiple myeloma, the patient developed erythema, desquamation, and ulceration only at the site of radiation therapy ten years prior. The patient received a dose reduced second infusion, causing a similar reaction.

Discussion: RRD is an inflammatory skin reaction that develops in a previously irradiated area after administration of certain promoting agents, most often with chemotherapy. Unlike radiation enhancement, there may be a long interval between the eruption and the administration of the causative agent. A number of drugs have been associated with this phenomenon, particularly the anthracyclines. Bortezomib has not been previously described as a causative agent for RRD. Erythema is the most common sign, which may be painful. Vesiculation, desquamation, and ulceration have also been reported. Histologically, the involved areas reveal epidermal dysplasia, necrotic keratinocytes, mixed inflammatory reaction, and sometimes psoriasiform dermatitis. Dose reduction, and the use of corticosteroids have been used to prevent recurrence of RRD, although rechallenge with the same agent may not lead to RRD again. The pathogenesis of this local phenomenon is not well understood, although an idiosyncratic drug hypersensitivity reaction has been proposed.
Uptake of New Therapeutics in the Treatment of Multiple Myeloma (MM) in Germany (GER) - Results from a Representative Multicentre Treatment Survey 2006.
Authors: Angermund Ralf , Knauf W. , Freund M. , Nowrousian M. , Einsele H. , Goldschmidt H. , Berdel W. , Poenisch W. , Straka C. , Kellermann L. , Pliskat H.
Published: April 2020

Background: Over the past decade new treatment options and drugs significantly altered the treatment paradigm and treatment guidelines for patients with MM. We reported in 2005 initial results from a treatment survey in GER (Freund ASH2005# 5158; Angermund, ASH2005, #5156). Bortezomib (Velcade®, Vel) at that time (6 months (m) after approval) was the 2nd most frequently (freq) used drug after dexamethasone (dex) in the approved indication (3rd line) and played a minor role in 2nd line therapy. The survey performed in 1st quarter 2006 (1.5 years (y) after the initial survey), used identical methods and questionnaires. These results allow for monitoring of changes in treatment patterns.

Methods: The method of this representative analysis was provided elsewhere (Freund ASH2005# 5158). The data presented here are a subset analysis based on 66 sites and 428 patients.

Results: 230 male and 195 female patients, 43% at the time of analysis for decision on primary therapy, 27% for secondary treatment and 30% for further treatment were included in this analysis. The centers participating were 11% university hospitals (UH), 38% non-university hospitals with specialized (SH) and 20% without specialized (NSH) haematology department, and 32% office-based haematologists (OBH). The main treatment in primary therapy did not change (table) whereas Vel gets more commonly used. The increase in Vel in all treatment lines is mainly due to SH (64 (2006) vs. 52 % (2004); OBH: 32 (2006) vs. 32% (2004); NSH: 0 (2006) vs. 2 % (2004); UH 4 (2006) vs 14% (2004)). In 2nd line Vel was the 2nd most prescribed drug (31%) (1st dex 41%; 2004: Vel 8% ranked 10th) mainly in UH/SH in patients below 60 y and after primary high dose chemotherapy (HDCT) (35% Vel vs. 28 % all) and with longer remission period (median 9 m Vel, median 7 m all). Within treatment ≥ 3rd line, Vel was the most freq used drug followed by dex (42% and 38% respectively). For patients currently in 1st remission Vel was most freq planned as next therapy (45%) followed by lenalidomide (len) (19%) and dex (12%). Vel was mostly used by SH following melphalan or VAD/VID treatment (multivariate analysis). Len and thalidomide (thal) (both drugs not approved in GER at that time) are rarely used in MM therapy (0% len vs. 10% thal in ≥ 2nd line).

Conclusion: New approved treatment options like Vel are quickly integrated into treatment behaviour in GER, mainly used in SH within approved indication, whereas not approved drugs like len or thal play only a minor role. 9 m after approval for 2nd line therapy, Vel increased uptake within ≥ 2nd line indicating this drug’s possibility to become a future treatment standard in pretreated patients. In order to detect the dynamic of change of treatment of MM, further follow up is planned.
Renal Recovery Following Light Chain Removal by Extended Haemodialysis in a Patient with Cast Nephropathy.
Authors: Hutchison Colin A. , Cook Mark , Bradwell Arthur R. , Cockwell Paul
Published: April 2020

Cast nephropathy is the main cause of renal impairment in patients with multiple myeloma (MM). Serum free light chain (sFLC) removal by extended hemodialysis on a protein leaking membrane may aid renal recovery. A patient presenting with MM, high serum FLC concentrations and dialysis dependent acute renal failure was studied. A renal biopsy showed monoclonal kappa FLC cast nephropathy. He was dialysed for 2–8h on daily / alternate days using the Gambro HCO 1100 dialyser. sFLC were measured at frequent intervals in the serum and dialysate fluids. Albumin and urea concentrations were measure pre- and post-dialysis. Over 22 days the patient underwent 14 dialysis sessions with an average sFLC reduction of 38% (15.2–61.8%). A total of 16.5g of kappa was removed in the dialysate fluid with an average concentration of 18.3mg/L (3.3–27.3). Figure 1 demonstrates serum reductions in kappa concentrations pre- and post-dialysis and timing of chemotherapy. The average serum reductions were: albumin 2.1g/L, urea 56% and creatinine 44%. By day 22 the patient was independent of dialysis. Four months later renal function is stable with an eGFR of 35ml/min (Cockcroft-Gault). Further studies are needed to determine whether this method would benefit many patients with acute renal failure and light chain cast nephropathy.

Figure Figure
Identification and Characterization of Cancer Stem Cells in Multiple Myeloma.
Authors: Sze Daniel , Loh Yen S. , Mo Suilin , Kaplan Warren , Brown Ross D. , Ho Phoebe Joy , Gibson John , Joshua Douglas E.
Published: April 2020

There is increasing evidence that the Cancer Stem Cell (CSC), a small percentage of cancer cells that have the characteristic to self-renew, proliferate, differentiate and replenish the bulk of the tumor, may be the leading cause of cancer relapse after chemotherapy. A distinct subpopulation within cancer cells known as the side population (SP), distinguished via Hoechst 33342 staining, has been repeatedly reported to be enriched with cells possessing CSC characteristics. In this study, we first examine if SP can be identified in four established myeloma cell lines. Hoechst-33342 stained cells were analyzed through FACS LSRII using a blue and red dual-wavelength analysis after UV excitation to detect the important red-blue differential dye emission feature of the SP cells. An average of 26.5% of SP cells was found in RPMI8226, 21.4% in KMS-11, 5.4% in U266 and 10.1% in OPM-2 within the viable cells compartment in four separate experiments. In addition, 4.9% of SP cells were also identified in the plasma cell leukemia cell line ARH77.

SP and non-SP cells of RPMI8226 and KMS-11 were then sorted and respective RNAs were extracted, amplified and labeled for subsequent Affymetrix microarray scanning. The gene-expression profiles of SP and non-SP cells from RPMI8226 and KMS-11 were compared before selecting statistically significant genes of interest. Two genes were found to be highly upregulated in SP cells. The first one was the Receptor for Hyaluronan-Mediated Motility (RHAMM) gene of 29.0 and 3.9 fold increase in RPMI8226 and KMS-11 respectively. Increased expression of RHAMM has been previously reported to correlate with human B cell malignancies. The second upregulated gene was the human inhibitor of apoptosis protein-family gene, Apollon, of 4.4 and 3.2 fold increase in RPMI8226 and KMS-11 respectively. Another gene - interferon gamma receptor 1 (IFNGR1) gene was found to be down-regulated in SP cells, of 4.3 and 1.2 fold decrease in RPMI8226 and KMS-11 that may provide an explanation of their escape from immune response via T cells.

We then examine if SP cells that represent putative CSC exist in primary myeloma cells. Seven myeloma patients’ bone marrow samples were investigated and 0.1 to 5.2 %, of an average of 1.4% of SP cells were identified in the CD38++ plasma cells compartments in 6 out of 7 bone marrow samples. This study reports the existence of SP in both myeloma cell lines and primary myeloma cases. Further studies will be required to confirm the protein expression of selected genes selected from the gene-expression profiles of SP versus non-SP from myeloma cells for the insights into the characteristic and tumorigenicity of CSC in myeloma.
A Phase II Study of Alemtuzumab in the Treatment of Plasma Cell Disorders.
Authors: Simpson Lijo , Kumar Shaji , Allred Jacob , Lacy Martha , Dispenzieri Angela , Hayman Suzanne , Zeldenrust Steven , Rajkumar S. Vincent , Lust John A. , Greipp Philip R. , Gertz Morie Abraham , Witzig Thomas E.
Published: April 2020

Background: While the therapeutic armamentarium for plasma cell disorders have expanded recently with introduction of thalidomide, lenalidomide and bortezomib, these diseases are still incurable and more agents are needed. We have previously demonstrated expression of CD 52 on plasma cells in MM, MGUS and AL amyloidosis. Based on this data we conducted a Phase II Study of Campath (Alemtuzumab; Monoclonal Anti-CD52 Antibody) in Primary Systemic Amyloidosis and Relapsed or Refractory Multiple Myeloma.

Methods: The study was an IRB approved, Phase II protocol, with a two stage design and interim analysis at 14 patients, to assess the efficacy and toxicity of Alemtuzumab. The regimen involved a dose escalation during week one (Day I 3 mg SQ; Day II 10 mg SQ; Day III 30 mg SQ; then 30 mg SQ three times a week for a planned total of 17 additional weeks). All patients received concurrent Co-Trimoxazole, Fluconazole and Valacyclovir prophylaxis. The planned accrual was 32 patients.

Results: A total of 14 patients were accrued, 8 patients with MM and 6 patients with AL. There were 8 females and 6 males in the study. The median age was 62 years (range: 50–78) years. The patients were heavily pretreated with the median number of treatments being 4. There were 5 grade 3–4 toxicities (CMV viremia (2 pts), skin rash (2 pts), and febrile neutropenia (1 pt)). Seven patients had evidence of disease progression and were removed from study. One patient completed the treatments with a mixed response. One patient had a sudden death while on study. The median duration of therapy was 6 weeks (range 3 weeks to 17 weeks). The accrual was stopped after 14 patients when the interim analysis showed a lack of anti-tumor efficacy and significant toxicity.

Conclusion: This phase II study in patients with relapsed / refractory myeloma or amyloidosis demonstrated no evidence of clinical activity with use of Campath. In addition it was also associated with significant toxicity in this population. We do not recommend use of campath as a primary treatment modality in these patients. The lack of efficacy despite demonstrated expression of CD52 on clonal plasma cells once again highlights the need for combining treatments with multiple mechanisms in order to improve the response and outcome in these patients. Whether it can synergize with other therapies at lower doses without significant toxicity remains unknown.
The Feature of Distribution and Clonality of TCR Vα and Vβ Repertoire in Cord Blood.
Authors: Chen Shaohua , Li Yangqiu , Yang Lijian , Cen Dongzhi , Chen Si , Li Chuang
Published: April 2020

Analysis of T cell receptor (TCR) Vα and Vβ repertoire is one of the sensitive methods to understand the distribution and clonality of T cells from different samples, which is used to identify clonal expansion T cells that response to tumor associated antigens. Cord blood has been used successfully as a source of hematopoietic stem cells for transplantation. Recently, stem cells transplantation was demonstrated more important as immunotherapy against malignance cells. And cord blood T cells are resourceful for production the specific CTL to use in leukemia immunotherapy. But the feature of distribution and clonality of TCR Vα and Vβ subfamily T cells in cord blood is not yet clearly defined. In the present study, the CDR3 of 29 TCR Vα and 24 Vβ subfamily genes were analyzed in T cells from 10 cases of cord blood, which obtained at delivery from full-term healthy pregnancies, using RT-PCR and genescan technique. Peripheral blood of nine cases healthy adult volunteers served as controls. The results showed that 15–19 of 29 Vα and 9–15 of Vβ subfamily T cells could be identified in the most cases, whereas only 2–4 of Vα subfamilies expressed was found in 3 cases. The most frequently expressed Vα subfamilies were Vα 3 and Vα 10 (100%), Vα 4, 5, 6, 8, 12, 15, 17 and Vα 21 (70%), Vα1, 13, 25, and Vα26 (60%), with a lower expression rate found in Vα16, 24 and Vα 28 (10%). Vα 9 and Vα 29 were not detected in both CB and healthy adults. In analysis of TCR Vβ repertoire, Vβ3, 5, 8 and Vβ9 genes could be identified in all samples, Vβ7 and Vβ13 could be found in 90% of samples, whereas Vβ4, 6, 11, 12, 18, 23 and Vβ24 were absent in all samples, which could be detected in normal peripheral blood samples. Genescan analysis showed that all PCR products of TCR Vα and Vβ subfamilies from cord blood displayed a Gaussian distribution of CDR3 lengths (multi-peaks), which are corresponding to polyclonal rearrangement pattern, except for tow cases, which displayed oligoclonal peak in Vα 24 or Vα28 respectively. In contrast, some Vα subfamily products from 8 of 9 cases of healthy adult volunteers contained at least an oligoclonal peak in different Vα subfamilies respectively, however, multi-peaks were found in all PCR products of TCR Vβ from normal peripheral blood. In conclusion, more than 20% of TCR Vα or Vβ subfamily T cells were absent in cord blood. The majority of TCR Vα and Vβ subfamily T cells in cord blood displayed polyclonality. Occasional oligoclonal peaks are identified in some cases.
Study of Mechanisms Underlying Fetal Transplantation Tolerance: Relative Organ Distribution of B Cells and CD45 Isoform Expression Patterns.
Authors: Skopal-Chase Jessica L. , Pixley John S. , Bhat Anupama S. , Torabi Alireza , Aiazzi Nicole M. , Thain David S. , Zanjani Esmail D.
Published: April 2020

Transplantation of hematopoietic stem cells into fetal sheep early in gestation (day (d) 55) (term gestation = 145d) results in long-term multi-lineage hemopoietic chimerism. We have proposed that the fetus is immunologically receptive at this time. However, our understanding of the mechanisms underlying this establishment of fetal tolerance remains poorly defined. We hypothesized that examination of developmental events in fetal immune ontogeny might provide insights into mechanisms underlying fetal tolerance formation. To this end we have previously reported the early appearance of cells phenotypically consistent with T regulatory cells (CD4+/CD25+) and multilineage expression of the CD45 isoform CD45R early in gestation (d40–65). Here, we extend our observations on B lineage cell distribution and relative CD45 isoform expression during gestation. The two commercially-available ovine CD45 antibodies are CD45 (immunoprecipitates proteins of MW 225, 210, and 190 kD, (Immunology.1985; 55:347–353)) and CD45R (immunoprecipitates a single protein of MW 220 kD (Cell. Immunol.1987;110:46–55)). Using dual-color flow cytometry, cells from fetal thymus, intestine, lung, spleen, bone/bone marrow, and peripheral blood (PB) were analyzed for expression of surface IgM, CD45 and CD45R from d39 to two days after birth. Surface IgM+ cells in the thymus were observed to be as high as 28% at d39 of gestation (uniformly CD45R+/CD45−). This rapidly dissipated to less than 1% after d65 of gestation, although IgM+ cells were observed as late as two days after birth. All IgM+ cells expressed CD45R in all organs tested but relative CD45 expression varied by gestational age and organ. Subsequent sampling noted rapid onset of CD45 coexpression on >70% of detectable thymic surface IgM+ cells throughout gestation. Similarly, splenic IgM+ cells co-expressed high levels of CD45 while PB IgM+ cells had delayed CD45 expression (beyond d65). Bone/bone marrow showed variable coexpression of CD45 throughout gestation. By contrast, surface IgM+ cells in lung and intestine maintained CD45R expression without coexpression of CD45 prior to birth. We have previously reported humoral tolerance to retroviral vector transgene products transplanted at d55–60 (Blood.2001; 97:3417–3423). Further understanding of CD45 isoform expression patterns and relative organ distribution of surface IgM+ cell populations during the time of immune receptivity in the sheep may provide insight into fetal transplantation tolerance.
Unique MicroRNA Profiles in Childhood Acute Lymphoblastic Leukemia.
Authors: Schotte Diana , Chau Jacqueline C.K. , Sylvester Giselle , Liu Gwen , Peters Susan T.C.J.M. , Pieters Rob , Den Boer Monique L. , Chen Chang-Zheng
Published: April 2020

MicroRNAs (miRNAs), an abundant class of ~22-nt endogenous regulatory RNAs that control gene expression at the posttranscriptional levels, have been implicated to play roles in the normal hematopoiesis and pathogenesis of leukemias. To identify “leukemic miRNAs”--miRNAs that may be oncogenes or tumor suppressors in human leukemias, we systematically cloned miRNAs from the blast cells of childhood acute lymphoblastic leukemia (ALL) patients with either a poor prognostic mixed-lineage leukemia rearrangement phenotype (MLL) or a prognostically more favorable precursor B-cell ALL phenotype (B-ALL). We have identified 87 known and 43 new human mature miRNAs, which potentially encoded by 101 known and 94 new human miRNA genes, respectively. Many newly identified miRNAs are not conserved in mouse, suggesting that systematic miRNA cloning analysis can facilitate the discovery of many novel human leukemia specific miRNAs. Interestingly, quantification of miRNA expression by real-time PCR analyses revealed that miRNAs are generally expressed at higher levels in MLL and B-ALL leukemia cells when compared to that in normal CD34+ bone marrow cells. We selected 21 highly differentially expression miRNA candidates and determined their expression in a larger group of 14 MLL patients and 16 B-ALL patients. We found that 9 miRNAs, including 4 newly identified miRNAs, were significantly differentially expressed (p < 0.05) between MLL and B-ALL subtypes. These findings demonstrate that the expression of both known and newly identified miRNA genes differs in genetically and prognostically different subgroups of ALL and normal CD34+ progenitor cells, suggesting that systematic miRNA cloning analyses and subsequent expression profiling analysis may facilitate the identification of specific signatures for leukemia classification and tumor suppressors or oncogenes that contribute to the pathogenesis of MLL and B-ALL.
Safety and Efficacy of Anti-Fibrinolytics in Orthopedic Surgery: A Meta-Analysis of Randomized Trials.
Authors: Kagoma Yoan K. , Nematullah Adeela , Lim Wendy P. , Crowther Mark A.
Published: April 2020

Background: Minimizing blood transfusions following elective orthopedic procedures is desirable given the risks associated with transfusion, including cost, infection, and allergic reactions. Anti-fibrinolytic agents may reduce perioperative blood loss but are used infrequently possibly due to a lack of data supporting their efficacy and concerns about increased risks for venous thromboembolism (VTE). We performed a meta-analysis to determine whether anti-fibrinolytic agents reduce perioperative blood loss in patients undergoing total hip replacement (THR) or total knee arthroplasty (TKA).

Data sources: MEDLINE (1966 to May Week 3 2006), EMBASE (1980 to 2006 Week 21), Cochrane Database of Systematic Reviews, ACP Journal Club, Database of Abstracts of Reviews of Effects, and Cochrane Central Register of Controlled Trials (the latter four databases to May 29, 2006) were searched. Additional trials were identified by manual searches. Authors and/or pharmaceutical companies were contacted if required. Study selection was based on the following criteria: prospective randomized clinical trials in which tranexamic acid, epsilon-aminocaproic acid, or aprotinin were administered. Two independent reviewers abstracted transfusion requirements, total blood loss, and venous thromboembolism. Study quality was assessed using the Jadad Quality Assessment scale. Data was pooled using the Mantel-Haenszel method and a random effects model.

Results: In 25 studies involving 1332 patients, the weighted mean difference (WMD) in total blood loss in patients treated with anti-fibrinolytic agents compared to patients receiving placebo was −409.50 mL (95%CI, −548.17 to −270.82; P< 0.00001, I2 = 89.6%). In 1557 patients, the relative risk (RR) for transfusion was 0.52 (95%CI, 0.42 to 0.64; P<0.00001, I2 = 58.0%). VTE did not appear to be increased in patients who received anti-fibrinolytic agents (RR 0.972 (95%CI, 0.885 to 1.06).

Conclusions: Patients undergoing TKR or THA who receive anti-fibrinolytic agents appear to have reductions in blood loss and need for transfusion. Whether these medications increase VTE risk cannot be determined due to limited data. Study characteristics were heterogeneous and the optimal timing, dose, and type of anti-fibrinolytic therapy have yet to be determined. Further studies in this area are warranted.
Protease Signaling in Severe Systemic Inflammation.
Authors: Niessen Frank , Ahamed Jasimuddin , Andrade-Gordon Patricia , Ruf Wolfram
Published: April 2020

The tissue factor (TF) pathway drives disseminated intravascular coagulation in sepsis. Although extensive in vitro data demonstrate proinflammatory effects of protease activated receptor (PAR) signaling, it remains unclear whether PAR signaling plays a significant role in severe systemic inflammation. In a murine model of severe, 75–90% lethal endotoxemia, we addressed the interplay between intravascular coagulation activation (based on thrombin-antithrombin complexes) and inflammatory exacerbation (based on inflammatory cytokines IL6, IL1b and MCP1) downstream of PAR signaling. TF cytoplasmic domain deleted mice displayed increased TF expression in macrophages and enhanced systemic coagulation activation. However, these mice resolved inflammation earlier than wild-type controls. Thus, intravascular coagulation activation per se does not predict survival. Rather, genetic deletion of PAR2 demonstrated that PAR2 signaling increased survival in TF cytoplasmic domain-deleted mice. PAR2 deficiency alone initially attenuated inflammatory cytokine production, but did not prevent progressive exacerbation of coagulation and inflammation, further evidence for protective PAR2 signaling in advanced, severe endotoxemia. In contrast, PAR1-deficiency had no effect on initial inflammation and coagulation, but at late stages attenuated inflammation and prolonged survival. In addition, PAR1-deficiency produced a synergistic survival benefit with TF cytoplasmic domain deletion. These experiments show that coagulation protease signaling both regulates and amplifies severe systemic inflammation. Strategies to target TF cytoplasmic domain signaling may therefore be beneficial to reduce sepsis lethality in combination with therapeutic blockade of proinflammatory PAR1 signaling.
Activation-Induced Cytidine Deaminase Acts as a Mutator in BCR-ABL1-Transformed Acute Lymphoblastic Leukemia Cells.
Authors: Feldhahn Niklas , Klein Florian , Hofmann Wolf-Karsten , Rowley Janet D. , Jumaa Hassan , Müschen Markus
Published: April 2020

Acute lymphoblastic leukemia (ALL) cells are derived from B and T cell precursors and typically carry rearranged immunglobulin (Ig) or T cell receptor (TCR) variable (V) region genes devoid of somatic mutations. The Philadelphia chromsome (Ph) encoding the oncogenic BCR-ABL1 kinase defines a subset of ALL with a particularly unfavorable prognosis. Here we show that oncogenic BCR-ABL1 kinase activity induces aberrant somatic hypermutation in Ph-positive ALL cells. Under physiological conditions, somatic hypermutation is restricted to mature germinal center B cells and depends on expression of the DNA-deaminating enzyme AID. Comparing Ph-positive and Ph-negative ALL cells, AID expression was found in 24 of 28 Ph-positive but only 3 of 80 Ph-negative ALLs. As shown by RT-PCR and Western blot, expression of AID in Ph-positive ALL cells reached similar levels as in germinal center B cells.

Forced expression of BCR-ABL1 in Ph-negative ALL cells and usage of the BCR-ABL1-kinase inhibitor STI571 revealed that BCR-ABL1 kinase activity is required and sufficient to induce aberrant expression of AID in Ph-positive ALL. Consistent with aberrant AID expression in Ph-positive ALL, Ig VH region genes were mutated in most Ph-positive but unmutated in Ph-negative cases. Of note, also non-Ig genes including BCL6 and MYC harbored somatic mutations in Ph-positive but not Ph-negative ALL cells. Likewise, Ph-positive T cell lineage ALL cells express AID and carry somatically mutated TCRβ V region genes. As demonstrated by ligation-mediated PCR, AID introduced DNA-single-strand breaks also within the tumor suppressor gene CDKN2B in Ph-positive ALL cells, which was sensitive to both inhibition of BCR-ABL1 kinase activity and silencing of AID expression by RNA interference. These findings identify AID as a BCR-ABL1-induced mutator in Ph-positive ALL cells, which may be relevant with respect to the particularly unfavorable prognosis of this leukemia subset.
Escape from Suppression: Tumor-Specific Effector Cells Out-Compete Regulatory T Cells Following Stem Cell Transplantation.
Authors: Mirmonsef Paria , Tan Gladys , Zhou Gang , Pyhel Tricia , Borrello Ivan M. , Levitsky Hyam
Published: April 2020

Autologous hematopoietic stem cell (HSC) transplantation is an accepted therapy for many hematological malignancies. High dose chemo-radiation reduces tumor burden but also ablates lymphohematopoiesis. Subsequent infusion of cellular grafts containing HSC and mature lymphocytes “rescues” the host from this otherwise lethal ablation, and initiates immune reconstitution. In many systems, tumor-specific T cells are functionally tolerant in the presence of established tumor. Paradoxically, however, the infusion of these lymphocytes into irradiated tumor-bearing syngeneic recipients unmasks effector function manifested as prolonged progression-free survival when compared to recipients treated with lymphocytes from non-tumor bearing donors. We have recently demonstrated that this tolerant tumor-specific T cell population from mice with established tumor is in fact a heterogeneous mixture of naive, effector, and regulatory T cells (Tregs), which as a whole are rendered functionally unresponsive through dominant suppression. The apparent reversal of tolerance in the post-transplant setting prompted a more detailed examination of the fate of these individual components during immune reconstitution. Here, we show that CD4+ T cells specific for a model tumor antigen are hyporesponsive to antigen when isolated from mice harboring an established systemic B cell lymphoma. Upon transfer into irradiated lymphoma-bearing mice, however, these cells undergo robust antigen-driven clonal expansion, and their ability to produce interferon gamma (IFNγ) is restored. Notably, in spite of the presence of tumor in the transplant recipients, tolerance to tumor antigen was not established in the early post-transplant period, even for mice receiving naive T cells in the graft. Tumor-specific CD4+CD25+Foxp3+ Tregs isolated from the donors were found to undergo a modest tumor-antigen-driven expansion in transplant recipients. When isolated from recipients, such cells maintained expression of Foxp3 and their capacity to suppress naive T cells when cultured in vitro. However, the presence of tumor-specific Tregs failed to significantly inhibit the expansion of naive or effector T cells specific for tumor in vivo, when examined 2 weeks post BMT. Indeed, the expansion of tumor-specific effector T cells significantly exceeded the expansion of Tregs, resulting in a nearly five-fold increase in the effector:Treg ratio. At the ratios present during this phase of immune reconstitution, the frequency of Tregs was insufficient to suppress effector cell function (proliferation and IFNγ production) when studied in vitro. This accounts for the reversal of tolerance identified in the population as a whole and its capacity to mediate tumor rejection.
A Signal Hierarchy Model of Alloreactive T Cell Trafficking for the Organ Manifestation in Acute Graft-Versus-Host Disease.
Authors: Beilhack Andreas , Schulz Stephan , Baker Jeanette , Beilhack Georg F. , Nishimura Ryosei , Landan Gilad , Baker Enosh M. , Herman Edward I. , Contag Christopher H. , Negrin Robert S.
Published: April 2020

Acute graft-versus-host disease (aGVHD) is caused by alloreactive effector T cells attacking the gastrointestinal tract, liver and skin after allogeneic hematopoietic cell transplantation (aHCT). The mechanism by which alloreactive T cells target these organs and not others remains elusive. Recently, we reported that different secondary lymphoid organs (SLOs), as alloreactive priming sites, can imprint distinct homing phenotypes on evolving alloreactive effector cells in vivo. However, preventing access to selected lymphoid organs (via the use of blocking antibodies or recipient mice lacking Peyer’s patches (PP), PP and lymph nodes (LN) or spleens) did not alter the aGVHD organ manifestation. These findings not only suggested a high redundancy of SLOs as induction sites of aGVHD, but also questioned whether homing instruction of alloreactive T cells by these sites can explain the mechanism of aGVHD target organ manifestation.

To test the homing instruction model we transplanted transgenic luciferase+ (luc+) FVB/N (H-2q, Thy1.1+) splenocytes into conditioned (2×400rad) Balb/c recipients (H-2d, Thy1.2+). On day+3 we isolated luc+ donor lymphocytes from peripheral LN, mesenteric LN, or spleens and transferred them into conditioned secondary allogeneic recipients. 16 hours later, bioluminescence imaging revealed that allogeneic luc+ T cells irrespective of their original priming site targeted the intestinal tract and liver. Subsequently, we compared aHCT of conditioned with non-conditioned secondary Balb/cRag−/− cγ-Chain−/− recipients.

Surprisingly, we found allogeneic luc+ T cells accumulating in SLOs in non-conditioned recipients in contrast to intestinal and hepatic tissues in conditioned recipients.

These in vivo findings establish that alloreactive effector cells migrate to aGVHD target tissues because of attraction to these sites rather than specific instruction by SLOs. Therefore, we propose a signal hierarchy model of alloreactive cell trafficking whereby inflammatory signal/ligand interactions dominate over organ-specific homing receptor/ligand interactions.
Regulatory CD4+CD25+ T Cells Modulated the Immune Response of a Mild Hemophilia A Subject to an Immunodominant FVIII Epitope within a Distinct Time Window.
Authors: James Eddie A. , Kwok William W. , Thompson Arthur R. , Pratt Kathleen P.
Published: April 2020

A mild hemophilia A subject with a missense mutation, A2201P, developed high titer neutralizing antibodies against infused factor VIII (FVIII) following post−operative FVIII administration. This missense mutation is in the C2 domain of FVIII. His baseline clotting activity fell from 10% to 3% of normal; FVIII infusions were discontinued, and his factor VIII clotting activity improved to its baseline within 2 mo. His inhibitor titer to FVIII declined over the ensuing year during which 12 samples were studied for T−cell epitopes, antibody titer, or both. His HLA haplotype was DR0101/DR1503, allowing use of fluorescent−labeled MHC Class II tetramers specific for DR0101. These were loaded with synthetic 20−mer peptides spanning the FVIII C2 domain to identify specific T cells responding to epitopes on FVIII by FACS. Proliferation assays were also carried out stimulating T cells with FVIII, the recombinant wild−type FVIII C2 domain, the C2 domain with mutation A2201P, and peptides. Tetramer staining and proliferation assays of total CD4+ and CD4+CD25− fractions indicated that CD4+CD25+ regulatory T cells modulated his immune response to FVIII between 10 and 19 weeks following the initial bleed. Peptides containing the wild type A2201, but not P2201, elicited strong T−cell responses. Markedly decreased staining of the total CD4 fractions compared to CD4+CD25− fractions in response to a peptide containing A2201 was observed between weeks 10 and 19 (see Figure showing staining at week 10). T−cell proliferation assays were carried out for cloned, expanded T cells that were selected using the tetramers. These T cell clones proliferated in response to FVIII, wild−type C2 protein, and peptides with the native A2201 sequence, whereas a peptide containing the mutation A2201P abrogated tetramer binding and proliferation. The T−cell epitope corresponded to a predicted MHC Class II binding motif for DR0101, underscoring the importance of studying T−cell responses within the context of each individual’s HLA haplotype. The epitope also overlapped a B−cell epitope identified by peptide mapping of the subject’s anti−FVIII C2 domain IgG fraction. Tetramer−guided T−cell epitope mapping provides greater sensitivity and specificity and markedly less ambiguous information than traditional mapping methods based on proliferation in response to peptides. FACS sorting of stained T cells allowed cloning of single T cells that retained the original specificity for the factor VIII peptides comprising this immunodominant T−cell epitope.

Response to peptide 2194-2213 10 weeks after inhibitor first appeared Response to peptide 2194-2213 10 weeks after inhibitor first appeared