The role of cryopreservation techniques in manufacturing, transport, and storage of Car-T therapy products.

Several clinical trials have proved the efficacy and safety of T-cells chimeric antigen receptor (CAR-T cells) in treatment of malignant lymphoma and the first products were registered in the European Union in 2018. The shelf-life of CAR-T cell products in the liquid state is short, so cryopreservation offers a significant benefit for logistics in manufacturing and patient management. Direct shipment of the cryopreserved CAR-T cell therapy products to the clinical department is feasible, nevertheless, intermediate storage in the hospital cryostorage facility gives significant advantage in planning of their administration to patients. Moreover, some manufacturers prefer transport of the starting material cryopreserved at the collection site. The cryopreservation protocol used for starting material by the authors is based on combining dimethyl sulphoxide (DMSO) with hydroxyethyl starch (HES) and slow controlled cooling in cryobags housed in metal cassettes. This achieves the mononuclear cell post-thaw viability of 98.8 ± 0.5 % and recovery of 72.8, ± 10.2 %. Transport of the starting material to the manufactures and return transport of the CAR-T therapy product is performed by authorized courier companies. Intermediate cryostorage of the final CAR-T cell therapy product is performed in a separate dry-storage liquid nitrogen container. On the day of infusion, the cryopreserved products are transported to the clinical department in a dry shipper. On the wards the product is removed from the cassette, inserted into a sterile plastic bag, thawed in a 37 degree C water bath followed by immediate intravenous administration. The authors discuss the adherence of the used technology to good manufacturing practice (GMP) principles and genetic safety assurance rules. Doi: 10.54680/fr23310110112.

Prognostic factors affecting the outcome after allogeneic haematopoietic stem cell transplantation for myelodysplastic syndrome.

In the present study, we retrospectively analysed the results of HSCT in 47 consecutive patients with MDS diagnosed at our department between 2002 and 2019, with a focus on possible predictive factors influencing overall survival (OS), the development of relapse, infections, and the occurrence of graft versus host disease (GvHD). In a univariate analysis, the pre-transplantation value of blasts in the marrow < 5% (p = 0.006), the revised International Prognostic Scoring System (IPSS-R) (p = 0.041), and karyotype (p = 0.009) were predictive of OS. Neither the elevation of serum ferritin (> 1000 ug/ml) nor increased C-reactive protein (CRP) (> 5 mg/l) was associated with shorter OS. In contrast, elevated serum lactate dehydrogenase (LDH) (> 213 U/l) was associated with shorter OS (p = 0.04).

Lenalidomide and dexamethasone in treatment of patients with relapsed and refractory multiple myeloma - analysis of data from the Czech Myeloma Group Registry of Monoclonal Gammopathies.

Lenalidomide (LEN) is an immunomodulator with clinical activity against myeloma cells. Based on the pivotal phase 3 trials MM-009 and MM010, the combination of lenalidomide and dexamethasone(DEX) was approved for patients with multiple myeloma who received at least one prior therapy. Here, we evaluated LEN/DEX therapy in unselected population and subsequently in selected sub-groups of patients with relapsed/refractory multiple myeloma followed in the Registry of Monoclonal Gammopathies of the Czech Myeloma Group. Altogether 858 patients were treated with LEN/DEX in the Czech Republic and Slovakia until end of 2017. The analyzed sub-groups were defined as patients with high risk cytogenetic aberrations and patients with relapsed and refractory MM. The overall response rate (ORR; partial remission or better response, PR) in the whole group of patients was 46.3% for all lines of therapy, 26.4% for high-risk group and 32.1% for relapsed and refractory group. Medians of overall survival (OS) in the same cohorts were as follows: 25.6, 15.7 and 18.5 months, progression free survival (PFS) was: 11.2, 6.4 and 9.0 months respectively. The most common adverse events were hematologic and infectious. In conclusion we found that our results correlated with those found in other studies in terms of response rates, survival measures, and also of treatment toxicity.

Simplified novel prognostic score for real-life older adults with multiple myeloma-registry-based analysis.

The main goal was to find a simple prognostic to evaluate overall survival of patients older than 65 years of age with myeloma. Retrospective registry-based analysis from the Registry of Monoclonal Gammopathies was conducted. Patients over 65 years with symptomatic myeloma were included. The four major parameters with impact on survival were identified: male gender, age > 75, creatinine > 152 µmol/L, and ECOG performance status 2-4. The patients were scored as good (0 points), intermediate good (1 point), intermediate poor (2 points), poor (3-4 points). Patients (1410 MM) were included. Median OS (months) was 65.7 (95% CI 49.8-81.7) for good, 51.0 (44.1-57.8) for intermediate good, 32.2 (26.2-38.2) for intermediate poor, and 18.9 (15.1-22.7) for poor. The differences in OS were statistically significant (p < 0.0001). Good score was used as reference for hazard ratios, which for each other score were 1.43 (1.09-1.84) for intermediate good, 2.58 (2.00-3.33) for intermediate poor, and 3.88 (2.94-5.10) for poor. Time to progression showed medians (months) 20.5 (17.4-62.4) for good, 19.3 (17.0-21.7) for intermediate good, 19.6 (16.2-23.0) for intermediate poor, and 13.0 (10.8-15.2) for poor. The suggested scoring system provides readily available information about the prognosis of MM patients above 65 years.

Viridans group streptococci bloodstream infections in neutropenic adult patients with hematologic malignancy: Single center experience.

Viridans group streptococci bloodstream infections (VGS BSI) remain a significant cause of mortality and morbidity in patients with severe neutropenia. The goal of our study was to evaluate clinical course and microbiological susceptibility of VGS BSI at our center. Retrospective analysis of all microbiologically documented bloodstream infections caused by VGS during the 9-year time period (from January 2006 until December 2014) was carried out. Only patients with severe neutropenia (< 500/µL) were included in the study. Clinical outcome and microbiological susceptibility pattern of isolates were recorded. Fifty-one individual patients with episode of VGS BSI were identified. The most frequent agent was Streptococcus mitis (23/51 cases, 45.1%). 88.2% (45/51) of patients were on recommended ciprofloxacin prophylaxis. 20/51 (39.2%) of patients suffered from mucositis at the time of diagnosis (10 patients had oral mucositis, 2 patients had bowel mucositis, and 8 patients both). Twenty-six patients (51.0%) had clinically relevant lung damage caused by VGS BSI (i.e., acute lung injury or acute respiratory distress syndrome). Twenty-four (47.0%) patients presented with bilateral lung infiltrated upon chest imaging, and two (4.0%) patients had unilateral lung infiltrates. Three patients (5.9%) died due to VGS BSI until day 28 of observation. No difference in signs of shock syndrome was observed in the patients during transplantation procedures compared to patients without transplantation as well as in a group received previous high-dose chemotherapy with cytosinarabinoside or in patients with mucositis. Only 3/51 of isolates (5.9%) were resistant to penicillin. All isolates were susceptible to empirical treatment. While the penicillin resistance of VGS remains low in middle Europe, initial antibiotic therapy of febrile neutropenia are still effective in most cases. The mortality and complication rates of VGS BSI were comparable to other studies, and no specific risk factor of shock presence could be identified.

[Ganciclovir treatment failure in adult allogeneic hematopoietic stem cell transplant recipients with cytomegalovirus infection--a single centre experience]. / Klinická rezistence lidského cytomegaloviru pri lécbe gancyklovirem u pacientu po alogenní transplantaci hematopoetických bunek--zkusenosti jednoho centra.

OBJECTIVE: To determine the incidence of infection with ganciclovir-resistant cytomegalovirus (CMV) in adult allogeneic hematopoietic stem cell transplant (HSCT) recipients. Clinical resistance or treatment failure was defined as persistent DNAemia or increasing viral load in peripheral blood after 2 weeks of virostatic treatment. The association between the treatment failure and viral resistance was analysed. The presence of ganciclovir-resistant CMV strains was confirmed by genotypic testing able to detect mutations conferring resistance. METHODS: In 2012 and 2014, 40 patients who underwent allogeneic HSCT for hematologic malignancies and were treated for human CMV reactivation/disease were followed up prospectively. In patients with treatment failure, CMV DNA was isolated and analysed by nucleotide sequence analysis of the UL 97 and UL 54 genes conferring resistance to the virostatic agent. RESULTS: The treatment failure occurred in seven patients, but ganciclovir resistance conferring mutations were only detected in two of them (mutations L595F and M460I in the UL 97 gene). Another mutation in the UL 97 gene (N510S) was found in a patient with recurrent CMV replication who needed to be retreated but did not meet the criteria for treatment failure. CONCLUSION: The low incidence of genetically confirmed ganciclovir-resistant CMV isolates in HSCT recipients with relatively common clinical treatment failure suggests that the mechanism underlying slower viral clearance is often other than mutations conferring ganciclovir resistance to the virus.

Monoclonal gammopathy of undeterminated significance: introduction and current clinical issues.

Monoclonal gammopathy of undetermined significance (MGUS) is a precancerosis comprising two different kinds of cancer: lymphoid/lymphoplasmocytoid MGUS and plasma cell MGUS that represents about 85% of all MGUS cases. This type of MGUS has low but persistent tendency to transform to malignant disease, mainly multiple myeloma (MM), with frequency of about 1% per year. Using known risk stratification models based on clinical parameters, it is possible to identify patients' groups with average rates of progression as low as 0.26% and as high as 12% per year. However, due to the lack of clear genetic and/or phenotypic markers distinguishing MGUS from MM, we are not able to predict if and when MGUS will progress to MM in individual patients. There are partially overlapping molecular pathogenic events shared by MGUS and MM. Better understanding of pathogenesis of MGUS and MM using molecular-genetic approaches will help disclose the mechanisms of myeloma genesis; it can be also useful for identification of novel molecular targets. The ultimate goal for the near future is to develop better markers for definition of high-risk MGUS patients who will be candidates for early treatment intervention.

[Monoclonal gammopathies of undetermined significance]. / Monoklonálne gamapatie nejasného významu.

Monoclonal gammopathy of undetermined significance (MGUS) is the most common type of monoclonal gammopathies. Genetic changes, various cytokines and bone marrow angiogenesis play an important role in the pathogenesis. As far as the malignant transformation of MGUS is concerned, size and type of the serum M-protein, serum kappa and lambda free light chain ratio and number of plasma cells in peripheral blood seem to play a predictive role. A new possible risk-stratification model predicting progression of MGUS to multiple myeloma or other related disorders was presented in 2006. The model takes three parametres in consideration, type and initial size of the serum M-protein and serum kappa and lambda ratio. Patients are divided into four risk groups with different risk of progression, from 5% at 20 years in low risk group to 58% in high risk group. The interval from MGUS diagnosis to the evolution of multiple myeloma or other related malignancies ranges from 1 to 30 years. Nevertheless, the risk of progression persists even after more than 30 years after MGUS diagnosis.

High-dose chemotherapy followed by autologous stem cell transplantation changes prognosis of IgD multiple myeloma.

Immunoglobulin D (IgD) multiple myeloma (MM) is a rare plasma cell disorder constituting less than 2% of all MM cases. Survival of patients with IgD MM is generally shorter than that of patients with other types of monoclonal (M-) protein. We have retrospectively analyzed patients with IgD MM participating in clinical trials of the Czech Myeloma Group. Twenty-six IgD MM patients treated between 1996 and 2006 were identified, 14 (54%) men and 12 (46%) women. The median age was 61 years (range: 37-79 years). Ten of 26 patients (39%) were treated with first-line high-dose chemotherapy (HDCT) using melphalan 200 mg/m(2) followed by autologous stem cell transplantation (ASCT). Thirteen of 26 patients (50%) received conventional chemotherapy (CHT), mostly melphalan and prednisone or a vincristine/doxorubicin/dexamethasone (VAD) regimen. Treatment responses were evaluable for 23 of 26 (89%) patients. All HDCT patients had treatment responses, including seven patients (70%) with complete responses and three patients (30%) with partial responses. The median progression-free survival was 18 months for HDCT patients and 20 months for CHT patients. The median overall survival (OS) for all patients was 34 months. The median OS for the HDCT group has not yet been reached (70% of the patients are still alive). In contrast, the median OS for CHT patients was only 16 months. The difference in OS between the two groups was statistically significant (P=0.005). In conclusion, the overall response rate for patients with IgD MM aged 65 years or less treated with HDCT and ASCT is similar to that seen in other MM types.

[Low-dose thalidomide in refractory and relapsing multiple myeloma]. / Nízkodávkovaný talidomid u refrakterního a relabujícího mnohocetného myelomu.

INTRODUCTION: Thalidomide is one of the drugs which are newly used in the therapy of multiple myeloma. Its immunomodulating action and a number of additional effects have been proven in the treatment of advanced and refractory stage of the disease. However, the best dosing scheme has not yet been discovered and is the subject of research in a number of clinical studies today. PATIENTS AND METHODS: On a retrospective basis, we evaluated results for 59 patients with multiple myeloma who were treated with thalidomide in our facility (median dose of 100 mg), in monotherapy or in combination with corticosteroids, between 2000 and 2005. The objective was to determine the percentage of responses to treatment in patients at different stages of the disease. Response to treatment was evaluated in accordance with EBMT standards. RESULTS: Thalidomide was used as 2nd line therapy (1st relapse or primarily resistant disease) in 59% of cases (35 patients), and as 3rd line therapy (2nd relapse) in 37 % of cases (22 patients). 2 patients were receiving thalidomide as 4th line therapy. None of the patients had taken thalidomide as part of previous treatment. The response rate at 1st relapse (CR - complete remission, PR - partial remission, MR - minimum response) was 60% (21 patients), of which CR was recorded in 2 patients (6%), PR was recorded in 12 patients (35%) and MR in 6 patients (17%). The response rate at 2nd relapse was 45% (10 patients), of which CR was recorded in 3 patients (14%), PR in 1 patient (5%) and MR in 5 patients (23%). Even though we did not record any statistically significant difference in the response of the evaluated group of patients to the treatment with thalidomide at 1st and 2nd relapse, a higher percentage or progression during treatment (32% vs. 14%) was observed in patients at 2nd relapse. 2 patients treated with thalidomide at 3rd relapse did not have a satisfactory response to the treatment (progression or short stabilisation of the disease with subsequent progression). Only 3 patients (5%) of the evaluated group had to discontinue the treatment due to severe adverse events (neuropathy, allergic reaction, leukopenia). The follow up time for Thalidomide therapy ranged between 3 and 62 months for both groups (with a median of 10 months) and spanned from 3 to 60 months at 1st relapse (median of 12 months) and from 3 to 57 months at 2nd relapse (median of 6 months). No statistically significant differences were observed between the 1st and 2nd relapse groups of patients in terms of response rates or length of effect. CONCLUSION: Thalidomide is highly efficient in the treatment of multiple myeloma. The results of study document effectiveness of thalidomide regardless of the disease stage. Comparison of study data with the results of other studies shows that the effectiveness of lower doses we used is comparable with that of higher doses. The fact that lower doses of thalidomide reduce the incidence of adverse events is a clear advantage.

Isotype class switching after transplantation in multiple myeloma.

Switching of the paraprotein isotype or transient presence of oligoclonal bands detectable by serum immunofixation electrophoresis has been reported following not only transplantations, but also after intensive chemotherapy for leukemia. Retrospective analysis of 72 transplanted myeloma patients was carried out to determine the frequency and clinical significance of the appearance of abnormal proteins bands (APB) distinct from the original paraprotein. APB presence was observed in 31 patients (43%) already after the first autotransplant, the median interval from transplant was 2 months (range, 1 to 6 months). The most frequent occurrence of APB was observed after allogeneic transplantation. In the group of patients with APB presence more patients achieved complete remission (32.2% versus 17.1%), statistically significant differences were also established when we compared the percentage of surviving patients and overall survival, to the present date, among both groups of patients (p=0.03). All relapsed patients with previous isotype class switching had disease characterized by the same type of paraprotein as that detected at diagnosis. The development of APB is likely related to the recovery of impaired immunoglobulin production after transplantation. We confirmed favourable prognostic significance of this finding in transplanted myeloma patients.

Combined effect of sodium selenite and campthotecin on cervical carcinoma cells.

The effects of selenite, campthotecin and their combination were investigated in cervical carcinoma cell line Hep-2 HeLa during 24h. The measured parameters included morphological changes, proliferation, oxidative stress, mitochondrial status, caspase-3 activation and nuclear fragmentation. Selenite at all but lowest concentrations inhibited cell growth and proliferation and induced cell death characterized by membrane blebbing, oxidative stress and mitochondrial damage, occurring in the absence of caspase-3 activation and nuclear fragmentation. Campthotecin at all concentrations induced gradual apoptosis including all measured morphological and molecular parameters with exception of oxidative stress. A combination of selenite and campthotecin induced both antagonistic and synergistic effects on cervical carcinoma cells. While low selenium concentration slightly reduced cytotoxicity and proapoptotic effects of campthotecin, moderate and higher concentrations of selenium enhanced them, changing simultaneously apoptosis into more necrosis-like death. These results show importance of selenium as a potential modulator and enhancer of campthotecin-based anticancer therapy in nonovarian malignancies.

[Zinc and its role in the regulation of cell death]. / Zinek a jeho role pri regulaci bunecné smrti.

Zinc is a key element for maintenance of the structural and functional integrity of eukaryotic cells and tissues. In living systems, it forms stable complexes with macromolecules as well as so called labile pools called zincosomes, which are nowadays considered crucial for the regulation of apoptosis and cell proliferation. Zinc may block apoptosis induced by many external factors by inhibiting caspases and endonucleases, through interactions with transcription factors and kinases or due to its antioxidant activities. On the other hand, depletion of zinc may lead to rapid activation of apoptotic cascade and consequent cell death in many types of cells. Imbalances in intracellular zinc pools lead to improper regulation of cell death and proliferation, which is often causing or accompanying diseases. Therefore, detailed elucidation of the role of zinc in these regulations presents a solution for various pathophysiological conditions.