Conditional survival to assess prognosis in patients with chronic lymphocytic leukemia.

Biomarkers in chronic lymphocytic leukemia (CLL) allow assessment of prognosis. However, the validity of current prognostic biomarkers based on a single assessment point remains unclear for patients who have survived one or more years. Conditional survival (CS) studies that address how prognosis may change over time, especially in prognostic subgroups, are still rare. We performed CS analyses to estimate 5-year survival in 1-year increments, stratified by baseline disease characteristics and known risk factors in two community-based cohorts of CLL patients (Freiburg University Hospital (n = 316) and Augsburg University Hospital (n = 564)) diagnosed between 1984 and 2021. We demonstrate that 5-year CS probability is stable (app. 75%) for the entire CLL patient cohort over 10 years. While age, sex, and stage have no significant impact on CS, patients with high-risk disease features such as non-mutated IGHV, deletion 17p, and high-risk CLL-IPI have a significantly worse prognosis at diagnosis, and 5-year CS steadily decreases with each additional year survived. Our results confirm that CLL patients have a stable survival probability with excess mortality and that the prognosis of high-risk CLL patients declines over time. We infer that CS-based prognostic information is relevant for disease management and counseling of CLL patients.

Individualized targeted treatment in a case of a rare TFG::ROS1 fusion positive inflammatory myofibroblastic tumor (IMT).

BACKGROUND: Inflammatory myofibroblastic tumor (IMTs) are rare mesenchymal neoplasms with slow growth. Resection is considered as therapeutic standard, with chemotherapy being insufficiently effective in advanced disease. ALK translocations are present in 50% of cases, ROS1 fusions (YWHAE::ROS1, TFG::ROS1) are extremely rare. Here, we present a case with TFG::ROS1 fusion and highlight the significance of molecular tumor boards (MTBs) in clinical precision oncology for post-last-line therapy. CASE PRESENTATION: A 32-year-old woman presented with IMT diagnosed at age 27 for biopsy and treatment evaluation. Previous treatments included multiple resections and systemic therapy with vinblastine, cyclophosphamide, and methotrexate. A computed tomography scan showed extensive tumor infiltration of the psoas muscles and the posterior abdomen. Next generation sequencing revealed an actionable ROS1 fusion (TFG::ROS1) with breakpoints at exon 4/35 including the kinase domain and activating the RAS-pathway. TFG, the Trk-fused gene, exerts functions such as intracellular trafficking and exhibits high sequence homology between species. Based on single reports about efficacy of ROS1-targeting in ROS1 translocation positive IMTs the patient was started on crizotinib, an ATP-competitive small molecule c-MET, ALK and ROS1-inhibitor. With a follow-up of more than 9 months, the patient continues to show a profound response with major tumor regression, improved quality of life and no evidence for severe adverse events. CONCLUSION: This case underscores the importance of the availability of modern molecular diagnostics and interdisciplinarity in precision oncology to identify rare, disease-defining genotypes that make an otherwise difficult-to-treat disease targetable.

[NGS-based molecular genetics of leukemia-a powerful and decentralized approach]. / NGS-basierte Molekulargenetik der Leukämie ­ ein leistungsfähiger und dezentraler Lösungsansatz.

The diagnosis of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), originally based on morphological assessment alone, has to bring together more and more disciplines. Today, modern AML/MDS diagnostics rely on cytomorphology, cytochemistry, immunophenotyping, cytogenetics, and molecular genetics. Only the integration of all these methods allows a comprehensive and complementary characterization of each case, which is a prerequisite for optimal AML/MDS diagnosis and treatment. In the following, we present why multidisciplinary and local diagnosis is essential today and will become even more important in the future, especially in the context of precision medicine. We present our idea and strategy implemented at Augsburg University Hospital, which has realized multidisciplinary diagnostics in AML/MDS in an interdisciplinary and decentralized approach. In particular, this includes the recent technical advances that molecular genetics provides with modern methods. The enormous amount of data generated by these techniques represents a major challenge, but also a unique opportunity. We will reflect on how this increase in knowledge can be integrated into routine practice to lead the way for personalized medicine in AML/MDS to improve patient care.

Predictive value of DNA methylation patterns in AML patients treated with an azacytidine containing induction regimen.

BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease with a poor prognosis. Dysregulation of the epigenetic machinery is a significant contributor to disease development. Some AML patients benefit from treatment with hypomethylating agents (HMAs), but no predictive biomarkers for therapy response exist. Here, we investigated whether unbiased genome-wide assessment of pre-treatment DNA-methylation profiles in AML bone marrow blasts can help to identify patients who will achieve a remission after an azacytidine-containing induction regimen. RESULTS: A total of n = 155 patients with newly diagnosed AML treated in the AMLSG 12-09 trial were randomly assigned to a screening and a refinement and validation cohort. The cohorts were divided according to azacytidine-containing induction regimens and response status. Methylation status was assessed for 664,227 500-bp-regions using methyl-CpG immunoprecipitation-seq, resulting in 1755 differentially methylated regions (DMRs). Top regions were distilled and included genes such as WNT10A and GATA3. 80% of regions identified as a hit were represented on HumanMethlyation 450k Bead Chips. Quantitative methylation analysis confirmed 90% of these regions (36 of 40 DMRs). A classifier was trained using penalized logistic regression and fivefold cross validation containing 17 CpGs. Validation based on mass spectra generated by MALDI-TOF failed (AUC 0.59). However, discriminative ability was maintained by adding neighboring CpGs. A recomposed classifier with 12 CpGs resulted in an AUC of 0.77. When evaluated in the non-azacytidine containing group, the AUC was 0.76. CONCLUSIONS: Our analysis evaluated the value of a whole genome methyl-CpG screening assay for the identification of informative methylation changes. We also compared the informative content and discriminatory power of regions and single CpGs for predicting response to therapy. The relevance of the identified DMRs is supported by their association with key regulatory processes of oncogenic transformation and support the idea of relevant DMRs being enriched at distinct loci rather than evenly distribution across the genome. Predictive response to therapy could be established but lacked specificity for treatment with azacytidine. Our results suggest that a predictive epigenotype carries its methylation information at a complex, genome-wide level, that is confined to regions, rather than to single CpGs. With increasing application of combinatorial regimens, response prediction may become even more complicated.

Embryonic stem cell factor FOXD3 (Genesis) defects in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms, believed to originate from the interstitial cells of Cajal (ICC), often caused by overexpression of tyrosine kinase receptors (TKR) KIT or PDGFRA. Here, we present evidence that the embryonic stem cell factor FOXD3, first identified as 'Genesis' and involved in both gastrointestinal and neural crest cell development, is implicated in GIST pathogenesis; its involvement is investigated both in vitro and in zebrafish and a mouse model of FOXD3 deficiency. Samples from a total of 58 patients with wild-type GISTs were used for molecular analyses, including Sanger sequencing, comparative genomic hybridization, and methylation analysis. Immunohistochemistry and western blot evaluation were used to assess FOXD3 expression. Additionally, we conducted in vitro functional studies in tissue samples and in transfected cells to confirm the pathogenicity of the identified genetic variants. Germline partially inactivating FOXD3 sequence variants (p.R54H and p.Ala88_Gly91del) were found in patients with isolated GISTs. Chromosome 1p loss was the most frequent chromosomal abnormality identified in tumors. In vitro experiments demonstrate the impairment of FOXD3 in the presence of those variants. Animal studies showed disruption of the GI neural network and changes in the number and distribution in the ICC. FOXD3 suppresses KIT expression in human cells; its inactivation led to an increase in ICC in zebrafish, as well as mice, providing evidence for a functional link between FOXD3 defects and KIT overexpression leading to GIST formation.

Quantitative multiorgan proteomics of fatal COVID-19 uncovers tissue-specific effects beyond inflammation.

SARS-CoV-2 may directly and indirectly damage lung tissue and other host organs, but there are few system-wide, untargeted studies of these effects on the human body. Here, we developed a parallelized mass spectrometry (MS) proteomics workflow enabling the rapid, quantitative analysis of hundreds of virus-infected FFPE tissues. The first layer of response to SARS-CoV-2 in all tissues was dominated by circulating inflammatory molecules. Beyond systemic inflammation, we differentiated between systemic and true tissue-specific effects to reflect distinct COVID-19-associated damage patterns. Proteomic changes in the lungs resembled those of diffuse alveolar damage (DAD) in non-COVID-19 patients. Extensive organ-specific changes were also evident in the kidneys, liver, and lymphatic and vascular systems. Secondary inflammatory effects in the brain were related to rearrangements in neurotransmitter receptors and myelin degradation. These MS-proteomics-derived results contribute substantially to our understanding of COVID-19 pathomechanisms and suggest strategies for organ-specific therapeutic interventions.

No association of malignant B-cell non-Hodgkin lymphomas with ipsilateral SARS-CoV-2 vaccination.

PURPOSE: SARS-CoV-2 vaccines cause acute ipsilateral lymph node swelling in an important proportion of vaccines. Thus far, no malignant lymphadenopathies have been reported in temporal context to vaccination in the ipsilateral draining lymph node areas. EXPERIMENTAL DESIGN: Prompted by two cases with unilateral axillary lymphomas that occurred ipsilaterally to prior SARS-CoV-2 vaccination, we systematically retrieved all B-cell non-Hodgkin lymphomas at two German University Medical Centers diagnosed before and after introduction of SARS-CoV-2 vaccines in Germany. Available lymphoma tissue (n=19) was subjected to next-generation immunosequencing of the IGH locus. Malignant clonotypes were mined in the CoVabDab database and published data sets from 342 uninfected individuals, 55 individuals 28 days after anti-SARS-CoV-2 vaccination and 139 individuals with acute COVID-19 together encompassing over 1 million CDR3 sequences in total. RESULTS: Of 313 newly diagnosed cases in the two centers and observation periods, 27 unilateral manifestations in the defined deltoid draining regions were identified. The majority thereof were diffuse large B-cell lymphomas (18 of 27 cases). Eleven unilateral cases were diagnosed in the era of SARS-CoV-2 vaccination and 16 in the control period before introduction of such vaccines. Of the 11 unilateral lymphomas that occurred during the vaccination period, ten had received a SARS-CoV-2 vaccine prior to lymphoma diagnosis. These cases were further evaluated. While left-sided were more frequent than right-sided lymphomas (19 vs 8 cases), no statistically significant association of vaccination site and laterality of the lymphoma manifestation was found. The unilateral lymphomas showed a normal range of B-cell receptors typically found in these lymphoma subtypes with no evidence for anti-SARS-CoV-2 sequences in the malignant clonotype. CONCLUSIONS: Together, we found no evidence that the current SARS-CoV-2 vaccines could serve as a trigger for lymphomagenesis in the draining lymph node areas of the deltoid region used for vaccination.

Impact of age and gender on lymphocyte subset counts in patients with COVID-19.

In symptomatic patients with acute Coronavirus disease 2019 (COVID-19), lymphocytopenia is one of the most prominent laboratory findings. However, to date age and gender have not been considered in assessment of COVID-19-related cell count alterations. In this study, the impact of COVID-19 as well as age and gender on a large variety of lymphocyte subsets was analyzed in 33 COVID-19 patients and compared with cell counts in 50 healthy humans. We confirm that cell counts of total lymphocytes, B, NK, cytotoxic and helper T cells are reduced in patients with severe COVID-19, and this tendency was observed in patients with moderate COVID-19. Decreased cell counts were also found in all subsets of these cell types, except for CD4+ and CD8+ effector memory RA+ (EMRA) and terminal effector CD8+ cells. In multivariate analysis however, we show that in addition to COVID-19, there is an age-dependent reduction of total, central memory (CM), and early CD8+ cell subsets, as well as naïve, CM, and regulatory CD4+ cell subsets. Remarkably, reduced naïve CD8+ cell counts could be attributed to age alone, and not to COVID-19. By contrast, decreases in other subsets could be largely attributed to COVID-19, and only partly to age. In addition to COVID-19, male gender was a major factor influencing lower counts of CD3+ and CD4+ lymphocyte numbers. Our study confirms that cell counts of lymphocytes and their subsets are reduced in patients with COVID-19, but that age and gender must be considered when interpreting the altered cell counts.

Identification of Disparities in Personalized Cancer Care-A Joint Approach of the German WERA Consortium.

(1) Background: molecular tumor boards (MTBs) are crucial instruments for discussing and allocating targeted therapies to suitable cancer patients based on genetic findings. Currently, limited evidence is available regarding the regional impact and the outreach component of MTBs; (2) Methods: we analyzed MTB patient data from four neighboring Bavarian tertiary care oncology centers in Würzburg, Erlangen, Regensburg, and Augsburg, together constituting the WERA Alliance. Absolute patient numbers and regional distribution across the WERA-wide catchment area were weighted with local population densities; (3) Results: the highest MTB patient numbers were found close to the four cancer centers. However, peaks in absolute patient numbers were also detected in more distant and rural areas. Moreover, weighting absolute numbers with local population density allowed for identifying so-called white spots-regions within our catchment that were relatively underrepresented in WERA MTBs; (4) Conclusions: investigating patient data from four neighboring cancer centers, we comprehensively assessed the regional impact of our MTBs. The results confirmed the success of existing collaborative structures with our regional partners. Additionally, our results help identifying potential white spots in providing precision oncology and help establishing a joint WERA-wide outreach strategy.

Progressive multifocal leukoencephalopathy associated with chemotherapy induced lymphocytopenia in solid tumors - case report of an underestimated complication.

Background: JC virus reactivation causing progressive multifocal leukoencephalopathy (PML) occurs preferentially in human immunodeficiency virus (HIV) positive individuals or patients suffering from hematologic neoplasms due to impaired viral control. Reactivation in patients suffering from solid malignancies is rarely described in published literature. Case Presentation: Here we describe a case of PML in a male patient suffering from esophageal cancer who underwent neoadjuvant radiochemotherapy and surgical resection in curative intent resulting in complete tumor remission. The radiochemotherapy regimen contained carboplatin and paclitaxel (CROSS protocol). Since therapy onset, the patient presented with persistent and progredient leukopenia and lymphopenia in absence of otherwise known risk factors for PML. Symptom onset, which comprised aphasia, word finding disorder, and paresis, was apparent 7 months after therapy initiation. There was no relief in symptoms despite standard of care PML directed supportive therapy. The patient died two months after therapy onset. Conclusion: PML is a very rare event in solid tumors without obvious states of immununosuppression and thus harbors the risk of unawareness. The reported patient suffered from lymphopenia, associated with systemic therapy, but was an otherwise immunocompetent individual. In case of neurologic impairment in patients suffering from leukopenia, PML must be considered - even in the absence of hematologic neoplasia or HIV infection.

Designing and Implementing the MySusCof App-A Mobile App to Support Food Waste Reduction.

Consumers are responsible for almost 50 percent of food waste. Consumer-focused interventions are crucial to achieve many Sustainable Development Goals (SDGs), especially SDG 12.3. There are many factors that cause food waste, and these can be prevented by changing the consumption behavior of adults. Mobile apps are seen as promising tools to change consumer behavior for ensuring more sustainable food consumption. This study describes the development process and examines the perceived quality of MySusCof, an app intended to reduce the food waste of consumers. The uMARS scale was used for collecting data from consumers. Within the scope of the study, two studies were conducted to examine the development process of the application and to determine the user reactions to the mobile application. Results show that gamification elements with hedonic and social components, as well as functional aspects, are important features for user engagement and perceived impact. The qualitative results also supported the user experience in both hedonic and functional value and role of mobile apps to lead behavior change. This study serves as a guideline for future developers of mobile apps intended to lead consumers to a more sustainable food consumption.

High viral loads: what drives fatal cases of COVID-19 in vaccinees? - an autopsy study.

The rate of SARS-CoV-2 infections in vaccinees has become a relevant serious issue. This study aimed to determine the causes of death, histological organ alteration, and viral spread in relation to demographic, clinical-pathological, viral variants, and vaccine types for deceased individuals with proven SARS-CoV-2 infection after vaccination who died between January and November 2021. Twenty-nine consecutively collected cases were analyzed and compared to 141 nonvaccinated control cases. Autopsies were performed on 16 partially and 13 fully vaccinated individuals. Most patients were elderly and suffered from several relevant comorbidities. Real-time RT-PCR (RT-qPCR) identified a significantly increased rate of generalized viral dissemination within organ systems in vaccinated cases versus nonvaccinated cases (45% vs. 16%, respectively; P = 0.008) mainly with Ct-values of higher than 25 in non-respiratory samples. However, vaccinated cases also showed high viral loads, reaching Ct-values below 10, especially in the upper airways and lungs. This was accompanied by high rates of pulmonal bacterial or mycotic superinfections and the occurrence of immunocompromising factors, such as malignancies, immunosuppressive drug intake, or decreased immunoglobulin levels. All these findings were particularly accentuated in partially vaccinated patients compared to fully vaccinated individuals. The virus dissemination observed in our case study may indicate that patients with an impaired immune system have a decreased ability to eliminate the virus. However, the potential role of antibody-dependent enhancement must also be ruled out in future studies. Fatal cases of COVID-19 in vaccinees were rare and often associated with severe comorbidities or other immunosuppressive conditions.

Restoring Immune Mediated Disease Control by Ipilimumab Re-exposition in a Heavily pretreated Patient With MSI-H mCRC.

BACKGROUND: Immune-Checkpoint-inhibitors (ICIs) are approved in first line therapy of microsatellite-instable, deficient miss-match-repair (MSI-H-dMMR) metastatic colorectal cancer (mCRC), and in second line after standard chemotherapy. Evidence supporting immunotherapy after immunotherapy is scarce. CASE REPORT: This case report highlights the course of a heavily pretreated patient with MSI-H mCRC with progression after multiple local therapies, standard chemotherapies and pembrolizumab. After 4 cycles of ipilimumab and nivolumab followed by nivolumab-maintenance he achieved a long-lasting disease control of 22 months. After further subsequent progression he regained immune mediated disease control by a second "boost" of ipilimumab. CONCLUSION: Re-exposition with ipilimumab is a potential option to restore immune-mediated-disease-control in patients with preceding long-lasting response to ipilimumab/nivolumab and with dMMR-tumors. The clinical situation of progress after long-lasting disease control on ICIs becomes more common and is an opportunity to investigate potential strategies for restoring immune mediated disease control.

Alterations of circulating lymphocyte subsets in patients with colorectal carcinoma.

INTRODUCTION: Cellular immune response to cancer is known to be of great importance for tumor control. Moreover, solid tumors influence circulating lymphocytes, which has been shown for several types of cancer. In our prospective study we elucidate changes in lymphocyte subsets in patients with colorectal carcinoma compared to healthy volunteers. METHODS: Flow cytometry was performed at diagnosis of colon carcinoma to analyze B cells, T cells and NK cells including various subtypes of each group. Univariate and multivariate analyses including age, gender, tumor stage, sidedness and microsatellite instability status (MSI) were performed. RESULTS: Forty-seven patients and 50 healthy volunteers were included. Median age was 65 years in patients and 43 years in the control group. Univariate analysis revealed lower total lymphocyte counts, lower CD4 + cells, CD8 + cells, B cells and NKs including various of their subsets in patients. In multivariate analysis patients had inferior values of B cells, CD4 + cells and NK cells and various subsets, regardless of age and gender. Naïve, central memory and HLADR + CD8 + cells showed an increase in patients whereas all other altered subsets declined. MSI status had no influence on circulating lymphocytes except for higher effector memory CD8 + cells in MSI-high patients. Localization in the left hemicolon led to higher values of total cytotoxic T cells and various T cell subsets. CONCLUSION: We found significant changes in circulating lymphocyte subsets in colon carcinoma patients, independent of physiological alterations due to gender or age. For some lymphocyte subsets significant differences according to tumor localization or MSI-status could be seen.

Impaired Dendritic Cell Homing in COVID-19.

The high mortality of COVID-19 is mostly attributed to acute respiratory distress syndrome (ARDS), whose histopathological correlate is diffuse alveolar damage (DAD). Furthermore, severe COVID-19 is often accompanied by a cytokine storm and a disrupted response of the adaptive immune system. Studies aiming to depict this dysregulation have mostly investigated the peripheral cell count as well as the functionality of immune cells. We investigated the impact of SARS-CoV-2 on antigen-presenting cells using multiplexed immunofluorescence. Similar to MERS-CoV and SARS-CoV, SARS-CoV-2 appears to be impairing the maturation of dendritic cells (DCs). DC maturation involves a switch in surface antigen expression, which enables the cells' homing to lymph nodes and the subsequent activation of T-cells. As quantitative descriptions of the local inflammatory infiltrate are still scarce, we compared the cell population of professional antigen-presenting cells (APC) in the lungs of COVID-19 autopsy cases in different stages of DAD. We found an increased count of myeloid dendritic cells (mDCs) in later stages. Interestingly, mDCs also showed no significant upregulation of maturation markers in DAD-specimens with high viral load. Accumulation of immature mDCs, which are unable to home to lymph nodes, ultimately results in an inadequate T-cell response.

Whole-genome analysis of SARS-CoV-2 samples indicate no tissue specific genetic adaptation of the virus in COVID-19 patients' upper and lower respiratory tract.

Sample panels of SARS-CoV-2 cases were retrospectively whole-genome sequenced. In three individuals, samples of upper and lower respiratory tract resulted in identical sequences suggesting virus stability including the spike protein cleavage site. In a fourth case, low-level intra-host genomic evolution and a unique 5-nucleotide deletion was observed.

One Year after Mild COVID-19: The Majority of Patients Maintain Specific Immunity, But One in Four Still Suffer from Long-Term Symptoms.

After COVID-19, some patients develop long-term symptoms. Whether such symptoms correlate with immune responses, and how long immunity persists, is not yet clear. This study focused on mild COVID-19 and investigated correlations of immunity with persistent symptoms and immune longevity. Persistent complications, including headache, concentration difficulties and loss of smell/taste, were reported by 51 of 83 (61%) participants and decreased over time to 28% one year after COVID-19. Specific IgA and IgG antibodies were detectable in 78% and 66% of participants, respectively, at a 12-month follow-up. Median antibody levels decreased by approximately 50% within the first 6 months but remained stable up to 12 months. Neutralizing antibodies could be found in 50% of participants; specific INFgamma-producing T-cells were present in two thirds one year after COVID-19. Activation-induced marker assays identified specific T-helper cells and central memory T-cells in 80% of participants at a 12-month follow-up. In correlative analyses, older age and a longer duration of the acute phase of COVID-19 were associated with higher humoral and T-cell responses. A weak correlation between long-term loss of taste/smell and low IgA levels was found at early time points. These data indicate a long-lasting immunological memory against SARS-CoV-2 after mild COVID-19.