The Hierarchy-of-Hypotheses Approach: A Synthesis Method for Enhancing Theory Development in Ecology and Evolution.

In the current era of Big Data, existing synthesis tools such as formal meta-analyses are critical means to handle the deluge of information. However, there is a need for complementary tools that help to (a) organize evidence, (b) organize theory, and (c) closely connect evidence to theory. We present the hierarchy-of-hypotheses (HoH) approach to address these issues. In an HoH, hypotheses are conceptually and visually structured in a hierarchically nested way where the lower branches can be directly connected to empirical results. Used for organizing evidence, this tool allows researchers to conceptually connect empirical results derived through diverse approaches and to reveal under which circumstances hypotheses are applicable. Used for organizing theory, it allows researchers to uncover mechanistic components of hypotheses and previously neglected conceptual connections. In the present article, we offer guidance on how to build an HoH, provide examples from population and evolutionary biology and propose terminological clarifications.

Inhibition of IGF1-R overcomes IGFBP7-induced chemotherapy resistance in T-ALL.

BACKGROUND: T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease with the need for treatment optimization. Previously, high expression of Insulin-like growth factor binding protein 7 (IGFBP7), a member of the IGF system, was identified as negative prognostic factor in adult T-ALL patients. Since aberrant IGFBP7 expression was observed in a variety of neoplasia and was relevant for prognosis in T-ALL, we investigated the functional role of IGFBP7 in Jurkat and Molt-4 cells as in vitro models for T-ALL. METHODS: Jurkat and Molt-4 cells were stably transfected with an IGFBP7 over-expression vector or the empty vector as control. Proliferation of the cells was assessed by WST-1 assays and cell cycle status was measured by flow-cytometry after BrDU/7-AAD staining. The effect of IGFBP7 over-expression on sensitivity to cytostatic drugs was determined in AnnexinV/7-AAD assays. IGF1-R protein expression was measured by Western Blot and flow-cytometric analysis. IGF1-R associated gene expression profiles were generated from microarray gene expression data of 86 T-ALL patients from the Microarrays Innovations in Leukemia (MILE) multicenter study. RESULTS: IGFBP7-transfected Jurkat cells proliferated less, leading to a longer survival in a nutrient-limited environment. Both IGFBP7-transfected Jurkat and Molt-4 cells showed an arrest in the G0/G1 cell cycle phase. Furthermore, Jurkat IGFBP7-transfected cells were resistant to vincristine and asparaginase treatment. Surface expression and whole protein measurement of IGF1-R protein expression showed a reduced abundance of the receptor after IGFBP7 transfection in Jurkat cells. Interestingly, combination of the IGF1-R inhibitor NPV-AEW541 restored sensitivity to vincristine in IGFBP7-transfected cells. Additionally, IGF1-R associated GEP revealed an up-regulation of important drivers of T-ALL pathogenesis and regulators of chemo-resistance and apoptosis such as NOTCH1, BCL-2, PRKCI, and TP53. CONCLUSION: This study revealed a proliferation inhibiting effect of IGFBP7 by G0/G1 arrest and a drug resistance-inducing effect of IGFBP7 against vincristine and asparaginase in T-ALL. These results provide a model for the previously observed association between high IGFBP7 expression and chemotherapy failure in T-ALL patients. Since the resistance against vincristine was abolished by IGF1-R inhibition, IGFBP7 could serve as biomarker for patients who may benefit from therapies including IGF1-R inhibitors in combination with chemotherapy.

Acute leukemias of ambiguous lineage in adults: molecular and clinical characterization.

Acute leukemias of ambiguous lineage represent a heterogeneous group of rare, poorly characterized leukemias with adverse outcome. No larger studies have yet performed a combined approach of molecular and clinical characterization of acute undifferentiated leukemia (AUL) and biphenotypic acute leukemia (BAL) in adults. Here we describe 16 adults with AUL and 26 with BAL and performed mutational as well as expression studies of genes with prognostic impact in acute leukemia (BAALC, ERG, MN1, WT1, and IGFBP7). AUL showed overexpression of these genes compared to T-lymphoblastic leukemia (T-ALL), B-precursor ALL, and to acute myeloid leukemia (AML). Genotype alterations were not detectable in AUL. BAL samples were characterized by frequent WT1 mutations (18 %) and BCR-ABL translocations (30 %). ALL-based treatment protocols induced complete remissions in 40 % and AML-like therapies in 22 % of AUL/BAL patients. The outcome in both groups was very poor; a long-term survival was only observed in patients undergoing allogeneic stem cell transplantation (SCT). Our findings indicate that AUL and BAL share important molecular and high-risk features of both myeloid and lymphoid leukemias. BAL patients exhibited genetic alterations, which can be targeted therapeutically. Importantly, ALL therapy might be more effective than AML protocols and AUL/BAL patients should be considered for allogeneic SCT.

Differences in mortality in Switzerland by citizenship during the first and second COVID-19 waves: Analysis of death statistics.

Background: Early in the COVID-19 pandemic, it became apparent that members of marginalized populations and immigrants were also at risk of being hospitalized and dying more frequently from COVID-19. To examine how the pandemic affected underserved and marginalized populations, we analyzed data on changes in the number of deaths among people with and without Swiss citizenship during the first and second SARS-CoV-2 waves. Method: We analyzed the annual number of deaths from the Swiss Federal Statistical Office from 2015 to 2020, and weekly data from January 2020 to May 2021 on deaths of permanent residents with and without Swiss citizenship, and we differentiated the data through subdivision into age groups. Results: People without Swiss citizenship show a higher increase in the number of deaths in 2020 than those who were Swiss citizens. The increase in deaths compared to the previous year was almost twice as high for people without Swiss citizenship (21.8%) as for those with it (11.4%). The breakdown by age group indicates that among people between the ages of 64 and 75, those without Swiss citizenship exhibited an increase in mortality (21.6%) that was four times higher than that for people with Swiss citizenship (4.7%). Conclusion: This study confirms that a highly specialized health care system, as is found in Switzerland, does not sufficiently guarantee that all parts of the population will be equally protected in a health crisis such as COVID-19.

Expression of IGFBP7 in acute leukemia is regulated by DNA methylation.

The important role of insulin-like growth factor binding protein 7 (IGFBP7) as a tumor suppressor in solid tumors has been revealed in several studies. Interestingly, in a recent study IGFBP7 was also shown to be aberrantly expressed in acute leukemia. Moreover, in acute T-lymphoblastic leukemia (T-ALL), high IGFBP7 expression predicts primary therapy resistance. In order to elucidate the mechanisms underlying aberrant IGFBP7 expression, we used pyrosequencing technology to investigate the DNA methylation of IGFBP7 in 109 T-ALL patient samples. Aberrant methylation was shown and hypomethylation was associated with an early immunophenotype and co-expression of the stem cell markers CD117 (P < 0.001) and CD34 (P < 0.001). In concordance, gene expression profiles of 86 T-ALL patients revealed upregulation of stem cell markers (CD34 and CD133) as well as genes associated with poor outcome and pathogenesis of leukemia (MN1, BAALC, FLT3) in the high IGFBP7 expression group. In conclusion, aberrant IGFBP7 expression is regulated by DNA methylation in acute leukemia. Hypomethylation of the gene is likely to characterize an immature and a more malignant subtype of the disease.

Do cancer stem cells exist? A pilot study combining a systematic review with the hierarchy-of-hypotheses approach.

The phenomenon of cancer cell heterogeneity has been explained by different hypotheses, each entailing different therapy strategies. The most recent is the cancer stem cell model, which says that tumourigenicity and self-renewal are restricted to rare stem cell-like cancer cells. Since its conception, conflicting evidence has been published. In this study, we tested the applicability of a new approach developed in the field of ecology, the hierarchy-of-hypotheses approach, for the Cancer Stem Cell hypothesis. This approach allows to structure a broad concept into more specific sub-hypotheses, which in turn can be connected to available empirical studies. To generate a dataset with empirical studies, we conducted a systematic literature review in the Web of Science limited to the first 1000 publications returned by the search. From this pool, 51 publications were identified that tested whether a cell sub-population had cancer stem cell properties. By classifying the studies according to: (1) assessed indicators, (2) experimental assays and (3) model cancer cells used, we built a hierarchical structure of sub-hypotheses. The empirical tests from the selected studies were subsequently assigned to this hierarchy of hypotheses, and the percentage of supporting, undecided and questioning evidence was calculated for each sub-hypothesis, as well as additional experimental characteristics. Our approach successfully allowed us to determine that within our dataset, the empirical support for the CSC hypothesis was only 49.0%. The support of different sub-hypotheses was highly variable. Most noticeable, the conception that putative cancer stem cells are a rare subset of cells could not be confirmed by most studies (13.5% support). The empirical support varied also between types of cancer, animal models and cell isolation method used. For the first time, this study showed the applicability of the hierarchy-of-hypotheses approach for synthesizing and evaluating empirical evidence for a broad hypothesis in the field of bio-medical research.

Mutational spectrum of adult T-ALL.

Novel target discovery is warranted to improve treatment in adult T-cell acute lymphoblastic leukemia (T-ALL) patients. We provide a comprehensive study on mutations to enhance the understanding of therapeutic targets and studied 81 adult T-ALL patients. NOTCH1 exhibitedthe highest mutation rate (53%). Mutation frequencies of FBXW7 (10%), WT1 (10%), JAK3 (12%), PHF6 (11%), and BCL11B (10%) were in line with previous reports. We identified recurrent alterations in transcription factors DNM2, and RELN, the WNT pathway associated cadherin FAT1, and in epigenetic regulators (MLL2, EZH2). Interestingly, we discovered novel recurrent mutations in the DNA repair complex member HERC1, in NOTCH2, and in the splicing factor ZRSR2. A frequently affected pathway was the JAK/STAT pathway (18%) and a significant proportion of T-ALL patients harboured mutations in epigenetic regulators (33%), both predominantly found in the unfavourable subgroup of early T-ALL. Importantly, adult T-ALL patients not only showed a highly heterogeneous mutational spectrum, but also variable subclonal allele frequencies implicated in therapy resistance and evolution of relapse. In conclusion, we provide novel insights in genetic alterations of signalling pathways (e.g. druggable by γ-secretase inhibitors, JAK inhibitors or EZH2 inhibitors), present in over 80% of all adult T-ALL patients, that could guide novel therapeutic approaches.

Low expression of T-cell transcription factor BCL11b predicts inferior survival in adult standard risk T-cell acute lymphoblastic leukemia patients.

BACKGROUND: Risk stratification, detection of minimal residual disease (MRD), and implementation of novel therapeutic agents have improved outcome in acute lymphoblastic leukemia (ALL), but survival of adult patients with T-cell acute lymphoblastic leukemia (T-ALL) remains unsatisfactory. Thus, novel molecular insights and therapeutic approaches are urgently needed. METHODS: We studied the impact of B-cell CLL/lymphoma 11b (BCL11b), a key regulator in normal T-cell development, in T-ALL patients enrolled into the German Multicenter Acute Lymphoblastic Leukemia Study Group trials (GMALL; n = 169). The mutational status (exon 4) of BCL11b was analyzed by Sanger sequencing and mRNA expression levels were determined by quantitative real-time PCR. In addition gene expression profiles generated on the Human Genome U133 Plus 2.0 Array (affymetrix) were used to investigate BCL11b low and high expressing T-ALL patients. RESULTS: We demonstrate that BCL11b is aberrantly expressed in T-ALL and gene expression profiles reveal an association of low BCL11b expression with up-regulation of immature markers. T-ALL patients characterized by low BCL11b expression exhibit an adverse prognosis [5-year overall survival (OS): low 35% (n = 40) vs. high 53% (n = 129), P = 0.02]. Within the standard risk group of thymic T-ALL (n = 102), low BCL11b expression identified patients with an unexpected poor outcome compared to those with high expression (5-year OS: 20%, n = 18 versus 62%, n = 84, P < 0.01). In addition, sequencing of exon 4 revealed a high mutation rate (14%) of BCL11b. CONCLUSIONS: In summary, our data of a large adult T-ALL patient cohort show that low BCL11b expression was associated with poor prognosis; particularly in the standard risk group of thymic T-ALL. These findings can be utilized for improved risk prediction in a significant proportion of adult T-ALL patients, which carry a high risk of standard therapy failure despite a favorable immunophenotype.

FLT3 mutations in early T-cell precursor ALL characterize a stem cell like leukemia and imply the clinical use of tyrosine kinase inhibitors.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.