MALDI TOF Mass Spectrometry Imaging of Blood Smear: Method Development and Evaluation

The aim of this study was to develop and evaluate matrix assisted LASER desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry imaging (MSI) of blood smear. Integrated light microscope and MALDI IT-TOF mass spectrometer, together with a matrix sublimation device, were used for analysis of blood smears coming from healthy male donors. Different blood plasma removal, matrix deposition, and instrumental settings were evaluated using the negative and positive ionization modes while agreement between the light microscopy images and the lateral distributions of cellular marker compounds served as the MSI quality indicator. Red and white blood cells chemical composition was analyzed using the differential m/z expression. Five seconds of exposure to ethanol followed by the 5 min of 9-aminoacridine or α-cyano-4-hydroxycinnamic acid deposition, together with two sets of instrumental settings, were selected for the MALDI TOF MSI experiments. Application of the thin and transparent matrix layers assured good correspondence between the LASER footprints and the preselected regions of interest. Cellular marker m/z signals coincided well with the appropriate cells. A metabolite databases search using the differentially expressed m/z produced hits which were consistent with the respective cell types. This study sets the foundations for application of blood smear MALDI TOF MSI in clinical diagnostics and research

Targeting Burkitt Lymphoma with a Tumor Cell–specific Heptamethine Carbocyanine-cisplatin Conjugate

BACKGROUND: Burkitt lymphoma is a fast-growing mature B cell malignancy, whose genetic hallmark is translocation and activation of the c-myc gene. Prompt multiagent immunochemotherapy regimens can have favorable outcomes, but prognosis is poor in refractory or relapsed disease. We previously identified a novel family of near-infrared heptamethine carbocyanine fluorescent dyes (HMCD or DZ) with tumor-homing properties via organic anion-transporting peptides. These membrane carriers have uptake in tumor cells but not normal cells in cell culture, mouse and dog tumor models, patient-derived xenografts, and perfused kidney cancers in human patients. METHODS: Here we report the cytotoxic effects of a synthesized conjugate of DZ with cisplatin (CIS) on B cell lymphoma CA46, Daudi, Namalwa, Raji, and Ramos cell lines in cell culture and in xenograft tumor formation. Impaired mitochondrial membrane permeability was examined as the mechanism of DZ-CIS-induced lymphoma cell death. RESULTS: The new conjugate, DZ-CIS, is cytotoxic against Burkitt lymphoma cell lines and tumor models. DZ-CIS retains tumor-homing properties to mitochondrial and lysosomal compartments, does not accumulate in normal cells and tissues, and has no nephrotoxicity in mice. DZ-CIS accumulated in Burkitt lymphoma cells and tumors induces apoptosis and retards tumor cell growth in culture and xenograft tumor growth in mice. CONCLUSION: DZ-CIS downregulated c-myc and overcame CIS resistance in myc-driven TP53-mutated aggressive B cell Burkitt lymphoma. We propose that DZ-CIS could be used to treat relapsed/refractory aggressive Burkitt lymphomas

Systemic mastocytosis in Croatia

Cilj: Ciljevi ove studije bili su identificirati bolesnike sa sistemskom mastocitozom (SM) u Republici Hrvatskoj (RH) i analizirati njihove kliničke karakteristike. Ispitanici i metode: Retrospektivno su iz osam hematoloških centara u RH identificirani bolesnici sa SM. Analizirane su kliničke karakteristike, te načini i ishodi liječenja ovih bolesnika. Rezultati: Uključeno je 20 bolesnika, medijan dobi bio je 40,5 godina (raspon 24–77), a većinu su činile žene (n=12). Dominirali su bolesnici s indolentnom SM (ISM, n=11), dok je učestalost agresivne SM (ASM, n=4), „šuljajuće“ sistemske mastocitoze (SSM, n=3) i SM s pridruženom zloćudnom hematološkom bolešću (SM-AHND, n=2) bila manja. Gotovo su svi bolesnici imali kožni osip, a značajan broj njih i dispeptične smetnje, alergijsku dijatezu, bolove u kostima i osteoporozu. Antihistaminike je primala većina bolesnika, a citoredukciju 10 bolesnika (ISM=3, SSM=2, ASM=4, SM-AHND=1). Većina bolesnika koja je zahtijevala citoreduktivno liječenje primala je interferon alfa-2a (2 ISM, 1 SSM i 3 ASM), dva steroida (1 ISM i 1 SM-AHND), te po jedan imatinib (SSM) i kladribin (ASM). Svi su bolesnici liječeni u prvoj liniji interferonom alfa-2a i kladribinom postigli parcijalnu remisiju, a dva bolesnika liječena imatinibom i steroidom bila su refraktorna na liječenje. Nije bilo prekida liječenja interferonom zbog nuspojava. Nakon medijana praćenja od 33 mjeseca preminulo je troje bolesnika, jedan s ASM i oba s SM-AHND. Medijan preživljenja bolesnika s ISM/SSM nije dostignut naspram bolesnika s ASM/SM-AHND, gdje je iznosio 105 mjeseci (p=0,009). Zaključak: Kliničke karakteristike i ishodi liječenja bolesnika sa SM u RH slični su onima iz velikih svjetskih centara. Najčešće korišten citoreduktivni lijek u RH bio je interferon alfa-2a koji se pokazao sigurnim i učinkovitim.Aim: The aims of this study were to identify patients with systemic mastocytosis (SM) in Croatia and to analyze their clinical characteristics. Patients and methods: Patients with SM treated at eight hospitals in Croatia were retrospectively identified and their clinical characteristics, treatment patterns and outcomes were analyzed. Results:Twenty patients were included, median age was 40.5years (range 24-77), and most were females (n=12) . Patients with indolent SM (ISM, n=11) predominated, followed by aggressive SM (ASM, n=4), smoldering SM (SSM, n=3) and SM with an associated hematological neoplastic disorder (SM-AHND, n=2). Only one patient (with ASM) did not have cutaneous involvement, and a significant proportion of SM patients had dyspepsia, allergic diathesis, bone pains and osteoporosis. Antihistamines were administered in the majority of the patients, whereas ten patients needed cytoreductive treatment (ISM, n=3, SSM n=2, ASM, n=4, SM-AHND, n=1). Most SM patients in need for cytoreduction received interferon alpha-2a (two ISM, one SSM and three ASM), two received steroids (one ISM and one SM-AHND), one received imatinib (SSM) and the last patient was treated with cladribine (ASM). All patients treated first-line with interferons and cladribine achieved partial remission, whereas two patients treated with imatinib and steroid were refractory. None of the patients discontinued interferon due to drug-related side-effects. After a median follow-up of 33 months, three patients died, one with ASM and two with SM-AHND.The median survival of ISM/SSM patients was higher than in ASM/SM-AHND patients in whom it was 105 months (p=0.009). Conclusion: Clinical characteristics and treatment outcomes of SM patients in Croatia are comparable to those from large international centers. The most commonly administered cytoreductive drug in Croatia was interferon alpha-2a which was shown to be safe and effective

Treatment-Related Risk Factors for Adverse Outcomes of COVID-19 in Patients Treated for Lymphoid Malignancies in the Pre-Omicron Era—A Study of KroHem, the Croatian Group for Hematologic Diseases

Patients with lymphoid malignancies are at increased risk of death or prolonged infection due to COVID-19. Data on the influence of different antineoplastic treatment modalities on outcomes are conflicting. Anti-CD20 monoclonal antibodies increase the risk of prolonged infection. It is unclear whether this risk is affected by the choice of the antibody (rituximab vs. obinutuzumab). To elucidate the role of antineoplastic therapy on COVID-19 outcomes, KroHem collected data on patients with lymphoid malignancies diagnosed with COVID-19 between October 2020 and April 2021. A total of 314 patients were identified, 75 untreated, 61 off treatment and 178 on treatment. The mortality rate in untreated and off-treatment patients was 15% and 16%; 9% and 10% had prolonged infection. In the on-treatment group, 3% were still prolonged positive at time of data collection, 62% recovered and 35% died; 42% had prolonged infection. Disease type, use of anti-CD20 monoclonal antibodies, prior autologous stem-cell transplantation (ASCT) and line of treatment did not significantly affect mortality. Mortality was higher in older patients (p = 0.0078) and those treated with purine analogues (p = 0.012). Prolonged COVID-19 was significantly more frequent in patients treated with anti-CD20 monoclonal antibodies (p = 0.012), especially obinutuzumab, and purine analogues (p = 0.012). Age, prior ASCT and treatment line did not significantly affect risk of prolonged infection. These data suggest that increased age and use of purine analogues are main risk factors for increased mortality of COVID-19 in patients with lymphoid malignancies. Obinutuzumab further increases the risk of prolonged disease, but not of death, in comparison to rituximab. Epidemiological considerations should be taken into account when choosing the appropriate antineoplastic therapy for patients with lymphoid malignancies

Clinical Dilemmas in the Treatment of Elderly Patients Suffering from Hodgkin Lymphoma: A Review

Elderly patients make up a significant number of cases of newly diagnosed Hodgkin lymphoma. However, unlike in young patients, the outcomes of elderly patients are poor, and they are under-represented in phase III trials. Prior to treatment initiation, geriatric assessment should ideally be performed to address the patient’s fitness and decide whether to pursue a curative or palliative approach. The ABVD regimen is poorly tolerated in unfit patients, with high treatment-related mortality. Alternative chemotherapy approaches have been explored, with mixed results obtained concerning their feasibility and toxicity in phase II trials. The introduction of brentuximab vedotin-based regimens led to a paradigm shift in first- and further-line treatment of elderly Hodgkin lymphoma patients, providing adequate disease control within a broader patient population. As far as checkpoint inhibitors are concerned, we are only just beginning to understand the role in the treatment of this population. In relapsed/refractory settings there are few options, ranging from autologous stem cell transplantation in selected patients to pembrolizumab, but unfortunately, palliative care is the most common modality. Importantly, published studies are frequently burdened with numerous biases (such as low numbers of patients, selection bias and lack of geriatric assessment), leading to low level of evidence. Furthermore, there are few ongoing studies on this topic. Thus, elderly Hodgkin lymphoma patients are hard to treat and represent an unmet need in hematologic oncology. In conclusion, treatment needs to be personalized and tailored on a case-by-case basis. In this article, we outline treatment options for elderly Hodgkin lymphoma patients

Keratin 13 Expression Reprograms bone and Brain Metastases of Human Prostate Cancer Cells

Lethal progression of prostate cancer metastasis can be improved by developing animal models that recapitulate the clinical conditions. We report here that cytokeratin 13 (KRT13), an intermediate filament protein, plays a directive role in prostate cancer bone, brain, and soft tissue metastases. KRT13 expression was elevated in bone, brain, and soft tissue metastatic prostate cancer cell lines and in primary and metastatic clinical prostate, lung, and breast cancer specimens. When KRT13 expression was determined at a single cell level in primary tumor tissues of 44 prostate cancer cases, KRT13 level predicted bone metastasis and the overall survival of prostate cancer patients. Genetically enforced KRT13 expression in human prostate cancer cell lines drove metastases toward mouse bone, brain and soft tissues through a RANKL-independent mechanism, as KRT13 altered the expression of genes associated with EMT, stemness, neuroendocrine/neuromimicry, osteomimicry, development, and extracellular matrices, but not receptor activator NF-κB ligand (RANKL) signaling networks in prostate cancer cells. Our results suggest new inhibitors targeting RANKL-independent pathways should be developed for the treatment of prostate cancer bone and soft tissue metastases