Abnormal biomarkers predict complex FAS or FADD defects missed by exome sequencing.

BACKGROUND: Elevated TCRαß+CD4-CD8- double-negative T cells (DNT) and serum biomarkers help identify FAS mutant patients with autoimmune lymphoproliferative syndrome (ALPS). However, in some patients with clinical features and biomarkers consistent with ALPS, germline or somatic FAS mutations cannot be identified on standard exon sequencing (ALPS-undetermined: ALPS-U). OBJECTIVE: We sought to explore whether complex genetic alterations in the FAS gene escaping standard sequencing or mutations in other FAS pathway-related genes could explain these cases. METHODS: Genetic analysis included whole FAS gene sequencing, copy number variation analysis, and sequencing of FAS cDNA and other FAS pathway-related genes. It was guided by FAS expression analysis on CD57+DNT, which can predict somatic loss of heterozygosity (sLOH). RESULTS: Nine of 16 patients with ALPS-U lacked FAS expression on CD57+DNT predicting heterozygous "loss-of-expression" FAS mutations plus acquired somatic second hits in the FAS gene, enriched in DNT. Indeed, 7 of 9 analyzed patients carried deep intronic mutations or large deletions in the FAS gene combined with sLOH detectable in DNT; 1 patient showed a FAS exon duplication. Three patients had reduced FAS expression, and 2 of them harbored mutations in the FAS promoter, which reduced FAS expression in reporter assays. Three of the 4 ALPS-U patients with normal FAS expression carried heterozygous FADD mutations with sLOH. CONCLUSION: A combination of serum biomarkers and DNT phenotyping is an accurate means to identify patients with ALPS who are missed by routine exome sequencing.

In-depth molecular analysis of lymphomas with lymphoplasmacytic differentiation may provide more precise diagnosis and rational treatment allocation.

We performed a molecular analysis of formalin-fixed paraffin embedded and decalcified bone marrow trephine biopsies of 41 patients with a B-cell disorder with lymphoplasmacytic differentiation to enable a more precise diagnosis and to describe potentially prognostic and therapeutic relevant mutations. Analysis was performed with a commercially available next-generation sequencing (NGS) lymphoma panel (Lymphoma Solution, SophiaGenetics). Results were correlated with clinical and pathological parameters. Our group covered a spectrum of B-cell disorders with plasmacytic differentiation ranging from Waldenstroem's macroglobulinemia (WM), to small-B-cell lymphomas with plasmacytic differentiation (SBCL-PC) to IgM myeloma (MM). The most helpful diagnostic criteria included morphology and immuno-phenotype as a prerequisite for the interpretation of molecular analysis. MYD88 mutation was present in nearly all WM, but also in 50% of SBCL-PCs, while MM were consistently negative. Driver mutations, such as TP53, were already detectable early in the course of the respective diseases indicating a higher risk of progression, transformation, and reduced progression-free survival. In addition, we report on a novel BIRC3 frameshift mutation in one case of a progressive WM. Our data indicate that patients with LPL/WM might benefit from thorough pathological work-up and detailed molecular analysis in terms of precise diagnosis and targeted treatment allocation.

Adherence to Patient-Reported Symptom Monitoring and Subsequent Clinical Interventions for Patients With Multiple Myeloma in Outpatient Care: Longitudinal Observational Study.

BACKGROUND: The use of software to monitor patient-reported outcome measures (PROMs) can improve outcomes for patients with cancer receiving anticancer therapy; however, evidence from applications used in routine clinical practice is lacking. OBJECTIVE: We aimed to investigate adherence to and patient perceptions of a weekly, web-based PROM symptom monitoring program in routine clinical practice for patients with Multiple Myeloma. Moreover, we aimed to capture how clinical alerts prompted by the system influenced clinical care. METHODS: We conducted a single-center longitudinal observational study to evaluate patient adherence to and perceptions of the PROM monitoring software in routine practice. Patients with Multiple Myeloma remotely completed weekly treatment-specific PROMs to monitor key symptoms via a dedicated web-based platform. Alarming symptoms triggered clinical alerts in the application for the treatment team, which could initiate clinical interventions. The primary outcomes were the web-based assessment completion rate and patients' perceptions of the monitoring program, as assessed by an evaluation questionnaire. Moreover, clinical alerts prompted by the system and consequential clinical interventions were analyzed. RESULTS: Between July 2021 and June 2022, a total of 55 patients were approached for participation; 39 patients participated (24, 61% male, mean age 63.2, SD 9.2 years). The median assessment completion rate out of all weekly scheduled assessments was 70.3% (IQR 41.2%-89.6%). Most patients (77%) felt that the health care team was better informed about their health status due to the web-based assessments. Clinical alerts were triggered for 1758 of 14,639 (12%) reported symptoms. For 548 of 1758 (31.2%) alerts, the symptom had been registered before and no further action was required; for 348 of 1758 (19.9%) alerts, telephone consultation and self-management advice sufficed. Higher-level interventions were seldom needed in response to alerts: referral to a doctor or specialist (88/1758, 5% alerts), medication changes (22/1758, 1.3%), scheduling additional diagnostics (9/1758, 0.5%), or unplanned emergency visits (7/1758, 0.4%). Most patients (55%) reported the calls in response to alerts gave them "quite a bit" or "very much" of an added feeling of security during therapy. CONCLUSIONS: Our study shows that high adherence to regular and tailored PROM monitoring can be achieved in routine clinical care. The findings provide valuable insight into how the PROM monitoring program and the clinical alerts and resulting interventions shaped clinical practice. TRIAL REGISTRATION: ClinicalTrials.gov NCT05036863; https://clinicaltrials.gov/study/NCT05036863.

Attrition Rates in Multiple Myeloma Treatment under Real World Conditions-An Analysis from the Austrian Myeloma Registry (AMR).

Multiple myeloma (MM) is characterized by serial relapses, necessitating the application of sequential lines of therapy (LoT). Reports on attrition rates (ARs) vary widely. The present study analysed ARs from the Austrian Myeloma Registry. Attrition was defined as being either deceased, progressive without having received another LoT, or lack of follow-up for ≥5 years. A total of 571 patients diagnosed between January 2009 and August 2021 were included (median age: 72 years; median follow-up: 50.8 months). Some 507 patients received at least one LoT. Of the total, 43.6% underwent autologous stem cell transplantation (SCT, transplant eligible = TE)) with primarily VRd (Bortezomib/Lenalidomide/Dexamethasone) given as induction (26.5%), followed by lenalidomide maintenance in 55.7% of cases. Transplant-ineligible (NTE) patients were predominantly treated with Vd (Bortezomib/Dexamethasone, 21.6%), receiving maintenance in 27.1%. A total of 37.5% received a second LoT. ARs across one to five LoTs were 16.7-27%. Frontline induction/ SCT followed by maintenance reduced ARs associated with age and achievement of deep remission in the frontline. Deep remission prolongs follow-up and time-to-next-treatment (TTNT), while high-risk-cyctogenetics negatively affected these outcomes. Our results demonstrate considerably lower ARs for MM patients within the AMR data versus other healthcare systems. Young age and the achievement of significant remissions after optimal frontline therapy resulted in particularly low ARs. These promising results support a key role for the ease of drug access and reimbursement policies in governing long-term MM patient outcomes.

Application of mid-infrared microscopic imaging for the diagnosis and classification of human lymphomas.

Mid-infrared (MIR) microscopic imaging of indolent and aggressive lymphomas was performed including formalin-fixed and paraffin-embedded samples of six follicular lymphomas and 12 diffuse large B-cell-lymphomas as well as reactive lymph nodes to investigate benefits and challenges for lymphoma diagnosis. MIR images were compared to defined pathological characteristics such as indolent versus aggressive versus reactive, germinal centre versus activated cell-of-origin (COO) subtypes, or a low versus a high proliferative index and level of PD-L1 expression. We demonstrated that MIR microscopic imaging can differentiate between reactive lymph nodes, indolent and aggressive lymphoma samples. Also, it has potential to be used in the subtyping of lymphomas, as shown with the differentiation between COO subtypes, the level of proliferation and PD-L1 expression. MIR microscopic imaging is a promising tool for diagnosis and subtyping of lymphoma and further evaluation is needed to fully explore the advantages and disadvantages of this method for pathological diagnosis.

Visible and near-infrared hyperspectral imaging techniques allow the reliable quantification of prognostic markers in lymphomas: A pilot study using the Ki67 proliferation index as an example.

In this study, we examined the suitability of visible and infrared (Vis-NIR) hyperspectral imaging (HSI) for the quantification of prognostic markers in non-Hodgkin lymphoma on the example of the Ki67 proliferation index. Ki67 quantification was done on six follicular lymphomas (FLs) and 12 diffuse large B-cell lymphomas (DLBCLs) by applying classic immunohistochemistry. The Ki67 index was comparatively assessed visually, using HSI-based quantification and a digital imaging analysis (DIA) platform. There was no significant difference between visual assessment (VA), DIA, and HSI in FLs. For DLBCLs, VA resulted in significantly higher Ki67 values than HSI (p = 0.023) and DIA (p = 0.006). No such difference was seen comparing analysis by HSI and DIA (p = 0.724). Cohen's κ revealed a "substantial correlation" of Ki67 values for HSI and DIA in FLs and DLBCLs (κ = 0.667 and 0.657). Here we provide the first evidence that, comparably to traditional DIA, HSI can be used reliably to quantify protein expression, as exemplified by the Ki67 proliferation index. By covering the near-infrared spectrum, HSI might offer additional information on the biochemical composition of pathological specimens, although our study could not show that HSI is clearly superior to conventional DIA. However, the analysis of multiplex immunohistochemistry might benefit from such an approach, especially if overlapping immunohistochemical reactions were possible. Further studies are needed to explore the impact of this method on the analysis and quantification of multiple marker expression in pathological specimens.

Erratum: Willenbacher, E., et al. Curcumin: New Insights into an Ancient Ingredient against Cancer. Int. J. Mol. Sci. 2019, 20, 1808.

We would like to change the authors' names and affiliations on Page 1 of paper [...].

Patients with double/triple copy number gains on C-MYC, BCL2, and/or BCL6 treated with standard chemotherapy have a similarly poor prognosis than those with high-grade B cell lymphoma with C-MYC and BCL2 and/or BCL6 rearrangements: a single-center experience on a consecutive cohort of large B cell lymphomas.

High-grade B cell lymphomas with rearrangements on C-MYC and BCL2 and/or BCL6 (HGBL with MYC and BCL2 and/or Bcl6 rearrangement) are associated with worse clinical outcomes and thus were introduced as a separate new category in the recently updated WHO classification. From 2012 to 2016, we analyzed a consecutive cohort of large B cell lymphomas (LBCLs) for C-MYC, BCL2, and BCL6 rearrangements and correlated our results with clinical-pathological parameters. Ten of 78 (13%) cases had a C-MYC and BCL2 and/or BCL6 rearrangement, so-called double or triple hit (DH), while double/triple copy number gains (CNGs) were found in eight (10%) patients. Patients with a high-grade lymphoma with DH or CNG progressed significantly more often after first-line chemotherapy (p = 0.005). When treated with standard chemotherapy, patients with a DH or CNG had a significantly worse overall (OS) and recurrence free survival (RFS) compared with all other patients (p = 0.033 and p < 0.001, respectively). Thus, patients with a diffuse large B cell lymphoma, harboring a double/triple CNG, seem to have a similar poor prognosis than those with a DH. Though our data can only be regarded as preliminary, our results warrant further investigations to fully elucidate the role of CNGs as well as underlying molecular mechanisms resulting in aggressive behavior in LBCL.

Pixantrone demonstrates significant in vitro activity against multiple myeloma and plasma cell leukemia.

Treatment results for multiple myeloma and plasma cell leukemia have considerably improved, but cure remains elusive and establishing new therapeutic approaches constitutes a major unmet clinical need. We analyzed the anti-myeloma properties of the aza-anthracenedione pixantrone which has been successfully used in a phase III study for the treatment of patients with aggressive non-Hodgkin's lymphoma as monotherapy as well as in combination regimes in vitro and in an adapted in vivo model (ex ovo chicken chorioallantoic membrane (CAM) assay). Pixantrone significantly inhibited proliferation and metabolic activity of all investigated myeloma cell lines. Importantly, anti-myeloma effects were more pronounced in tumor cell lines than in stromal cells, mesenchymal stem cells, and peripheral blood mononuclear cells of healthy controls. Apoptosis of myeloma cell lines was observed only after a 7-day incubation period, indicating a fast cytostatic and a slower cytotoxic effect of this drug. Pixantrone reduced the viability of primary plasma cells of patients and induced downregulation of myeloma-cell growth in the CAM assay. Additionally, we demonstrate in vitro synergism between pixantrone and the histone deacetylase inhibitor panobinostat with respect to its anti-proliferative features. From these data, we conclude that systematic investigations of the clinical usefulness of pixantrone in the framework of controlled clinical trials are clearly indicated (e.g., in penta-refractory patients).

Curcumin: New Insights into an Ancient Ingredient against Cancer.

Cancer patients frequently use complementary medicine. Curcumin (CUR) and its derivates (from the extract of Curcuma longa L.) represent some of the most frequently used ones, having a long history in traditional Asian medicine. CUR was demonstrated, both in vitro and in vivo, to have significant anti-inflammatory effects, thus potentially counteracting cancer-promoting inflammation, which is a hallmark of cancer. CUR modulate a plethora of signaling pathways in cancer cells, comprising the NF-κB (nuclear factor k-light-chain-enhancer of activated B cells), the JAK/STAT (Janus-Kinase/Signal Transducers and Activators of Transcription), and the TGF-ß (transforming growth factor-ß) pathways. Furthermore, CUR confers properties of electron receptors, which destabilize radical oxygen species (ROS), explaining its antioxidant and anti-apopototic effects. Although CUR has a low bioavailability, its role in advanced cancer treatment and supportive care was addressed in numerous clinical trials. After promising results in phase I⁻II trials, multiple phase III trials in different indications are currently under way to test for direct anti-cancer effects. In addition, CUR exerts beneficial effects on cancer treatment-related neurotoxcity, cardiotoxicity, nephrotoxicity, hemato-toxicity, and others. More efficient galenic formulations are tested to optimze CUR's usability in cancer treatment. This review should provide a comprehensive overview of basic science, and pre-clinical and clinical data on CUR in the field of oncology.

Impact of renal impairment on outcomes after autologous stem cell transplantation in multiple myeloma: a multi-center, retrospective cohort study.

BACKGROUND: Renal impairment (RI) is a negative prognostic factor in Multiple Myeloma (MM) and affected patients are often excluded from autologous stem cell transplantation (ASCT). However, it remains unclear whether historically inferior outcome data still hold true. METHODS: From a total of 475 eligible MM patients who had undergone ASCT between 1998 and 2016, 374 were included in this multi-centric retrospective cohort study. Renal function was determined both at the time of MM diagnosis and ASCT by estimated glomerular filtration rate (eGFR according to the MDRD formula, RI defined as eGFR < 60 ml/min/1.73m2). Patients were categorized into 3 groups: A) no RI diagnosis and ASCT, B) RI at diagnosis with normalization before ASCT and C) RI both at the time of diagnosis and ASCT. Log-rank testing was used for overall and progression-free survival (OS, PFS) analysis. CONCLUSION: While severe RI at MM diagnosis confers a risk of shorter OS, MM progression after ASCT is not affected by any stage of renal failure. It can be concluded that ASCT can be safely carried out in MM patients with mild to moderate RI and should be pro-actively considered in those with severe RI. RESULTS: When comparing all groups, no difference in OS and PFS was found (p = 0.319 and p = 0.904). After further stratification according to the degree of RI at the time of diagnosis, an OS disadvantage was detected for patients with an eGFR < 45 ml/min/m2. PFS was not affected by any RI stage.

Towards Molecular Profiling in Multiple Myeloma: A Literature Review and Early Indications of Its Efficacy for Informing Treatment Strategies.

Multiple myeloma (MM), the second most common hematologic malignancy, is characterized by the clonal expansion of plasma cells. Despite dramatic improvements in patients' survival over the past decade due to advances in therapy exploiting novel molecular targets (immunomodulatory drugs, proteasome inhibitors and monoclonal antibodies), the treatment of relapsed and refractory disease remains challenging. Recent studies confirmed complex, dynamic, and heterogeneous genomic alterations without unifying gene mutations in MM patients. In the current review, we survey recent therapeutic strategies, as well as molecular profiling data on MM, with emphasis on relapsed and refractory cases. A critical appraisal of novel findings and of their potential therapeutic implications will be discussed in detail, along with the author's own experiences/views.

Short overview on the current standard of treatment in newly diagnosed multiple myeloma.

The treatment of newly diagnosed multiple myeloma has changed dramatically over the past 20 years, from near uniform application of chemotherapy to a patient performance status- and risk-based approach. Furthermore, initiation of treatment criteria have evolved from a pure end-organ damage-based definition to include risk factors of transformation to frank myeloma. Besides, the mainly cytogenetically defined Multiple Myeloma (MM) risk status, transplant eligibility of patients still serves primarily to allocate patients within a rational treatment algorithm. While all transplant-eligible MM patients should receive a triplet induction therapy followed by autologous transplantation and, in most cases, lenalidomide maintenance, other therapeutic elements (e. g., other maintenance strategies, consolidation, tandem transplantation,..) have to be decided on an individualized appraisal of risk and toxicities. Standard-risk patients should never be undertreated, as they derive the highest relative benefit from using the best available registered therapies. However, high-risk patients should be preferentially treated inside clinical trials testing additive innovative treatments, as the improvement in the prognosis of this group of patients by standard therapies has been underwhelming. Furthermore, the evaluation process of non-transplant-eligible patients should always comprise an evaluation of performance status, frailty, and comorbidities (e. g., a comprehensive geriatric assessment) to facilitate the allocation of individualized therapies.

T cells in multiple myeloma display features of exhaustion and senescence at the tumor site.

BACKGROUND: Multiple myeloma is an incurable plasma cell malignancy that is mostly restricted to the bone marrow. Cancer-induced dysfunction of cytotoxic T cells at the tumor site may be responsible for immune evasion and therapeutical failure of immunotherapies. Therefore, enhanced knowledge about the actual status of T cells in myeloma bone marrow is urgently needed. Here, we assessed the expression of inhibitory molecules PD-1, CTLA-4, 2B4, CD160, senescence marker CD57, and CD28 on T cells of naive and treated myeloma patients in the bone marrow and peripheral blood and collected data on T cell subset distribution in both compartments. In addition, T cell function concerning proliferation and expression of T-bet, IL-2, IFNγ, and CD107a was investigated after in vitro stimulation by CD3/CD28. Finally, data was compared to healthy, age-matched donor T cells from both compartments. METHODS: Multicolor flow cytometry was utilized for the analyses of surface molecules, intracellular staining of cytokines was also performed by flow cytometry, and proliferation was assessed by 3H-thymidine incorporation. Statistical analyses were performed utilizing unpaired T test and Mann-Whitney U test. RESULTS: We observed enhanced T cell exhaustion and senescence especially at the tumor site. CD8+ T cells expressed several molecules associated with T cell exhaustion (PD-1, CTLA-4, 2B4, CD160) and T cell senescence (CD57, lack of CD28). This phenotype was associated with lower proliferative capacity and impaired function. Despite a high expression of the transcription factor T-bet, CD8+ T cells from the tumor site failed to produce IFNγ after CD3/CD28 in vitro restimulation and displayed a reduced ability to degranulate in response to T cell stimuli. Notably, the percentage of senescent CD57+CD28- CD8+ T cells was significantly lower in treated myeloma patients when compared to untreated patients. CONCLUSIONS: T cells from the bone marrow of myeloma patients were more severely impaired than peripheral T cells. While our data suggest that terminally differentiated cells are preferentially deleted by therapy, immune-checkpoint molecules were still present on T cells supporting the potential of checkpoint inhibitors to reactivate T cells in myeloma patients in combination therapies. However, additional avenues to restore anti-myeloma T cell responses are urgently needed.

Real-World Use of 3rd Line Therapy for Multiple Myeloma in Austria: An Austrian Myeloma Registry (AMR) Analysis of the Therapeutic Landscape and Clinical Outcomes prior to the Use of Next Generation Myeloma Therapeutics.

OBJECTIVE: Clinical trials demonstrate improving survival in patients with multiple myeloma (MM) after treatment. However, it is unclear whether increased survival translates to a similar benefit in a real world setting. METHODS: We analyzed the overall survival of 347 multiple myeloma patients in Austria by means of a national registry (AMR), focused on results from 3rd and later lines of therapy. This benchmark was chosen to define a baseline prior to the broad application of upcoming 2nd generation drugs (carfilzomib, pomalidomide). RESULTS: Projected 10 years survival for patients with MM in Austria is estimated to be 56% in patients diagnosed in between the years 2011-2014, 21% in patients with a diagnosis made between 2000-2005, and 39% in those with a diagnosis made between 2006-2010). For the same intervals a significant increase in the use of both bortezomib, lenalidomide and thalidomide-so called IMiDs (from 2005 onwards) and their simultaneous use in combination therapies (from 2010 onwards) could be shown. The use of autologous transplantation (ASCT) remained more or less constant at ~ 35% of patients in the 1st line setting over the whole period, comparing well to international practice patterns, while the use of 2nd line ASCT increased from 5.5% to 18.7% of patients. Patients in 3rd or later line treatment (n = 105), showed that even in relapsed and refractory disease median survival was 27 months with a considerable proportion of long-term survivors (~20%). CONCLUSION & PERSPECTIVE: With the expected emergence of additional active anti-myeloma compounds, we aim to assess survival in patients with relapsed and refractory MM.

Low Beclin-1 expression predicts improved overall survival in patients treated with immunomodulatory drugs for multiple myeloma and identifies autophagy inhibition as a promising potentially druggable new therapeutic target: an analysis from The Austrian Myeloma Registry (AMR).

Beclin-1 is a key regulator of autophagy and has been suggested to be involved in the development of drug resistance in multiple myeloma (MM). We analyzed the expression of Beclin-1 in a retrospective cohort of 70 MMs. Beclin-1 expression did not influence overall survival (OS) and progression-free survival (PFS) in patients with therapy-naïve MM. In patients treated with immunomodulatory drugs (IMiDs) lack of or low Beclin-1 expression resulted in a significantly improved OS and PFS compared to those treated with bortezomib or nonnovel agents. Beclin-1 expression was more frequently detected in relapsed MM than in therapy-naïve MM probably being a hallmark of tumor progression and therapy resistance. If validated prospectively, Beclin-1 expression might identify patients prone to profit above average from IMiDs and enable a more rational allocation of antimyeloma therapies. Furthermore, the inhibition of autophagy could be a new promising target to improve response to treatment in the relapsed/refractory setting.