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

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

The role of BAALC (brain and acute leukemia, cytoplasmic) and associated genes in acute leukemia

Akute Leukämien stellen klinisch und molekulargenetisch sehr heterogene Erkrankungen dar. In den vergangenen Jahren konnte durch die Identifizierung molekulargenetischer Veränderungen eine Zuordnung in spezifische Risikogruppen vorgenommen und somit eine Therapieoptimierung erzielt werden. Molekulare Marker erlauben zudem Einblicke in die der Leukämogenese zugrunde liegenden pathogenetischen Mechanismen und bilden potentielle Angriffspunkte für die Entwicklung von neuen zielgerichteten Substanzen. Vor diesem Hintergrund wurde sowohl die prognostische Bedeutung als auch die biologische Funktion von BAALC und dessen assoziierten Genen in der vorliegenden Arbeit untersucht. Für BAALC wurde eine prognostische Bedeutung bereits für die CN-AML und T-ALL beschrieben, die einen therapieresistenten Leukämiesubtyp charakterisiert. Im Rahmen dieser Arbeit konnte die prognostische Wertigkeit von BAALC in der B-ALL dargestellt werden und somit eine linienunabhängige pathophysiologische Bedeutung des Gens postuliert werden. Darüber hinaus wurde einer Identifizierung von Prognosemarkern in der T-ALL nachgegangen. In vorangegangenen Studien konnte eine prognostische Wertigkeit von WT1-Mutationen bei AML-Patienten gezeigt werden, die mit einer hohen BAALC- Expression assoziiert waren. Aufgrund der linienübergreifenden prognostischen Bedeutung von BAALC wurde die Rolle von WT1 in der T-ALL untersucht. WT1-Mutationen wurden in 8% der T-ALL-Patienten identifiziert, zeigten jedoch eine geringere prognostische Wertigkeit als bei der AML. Im Gegensatz dazu konnte eine aberrante WT1-Expression mit einem schlechten Gesamtüberleben der Patienten assoziiert werden. Die Bestimmung der BAALC-Expression in der B-ALL sowie der WT1-Expression in der T-ALL stellt insbesondere für schlecht charakterisierte Subgruppen einen interessanten Ansatzpunkt zur Risikostratifizierung dar. Neben der Bestimmung der prognostischen Bedeutung von BAALC und assoziierter Gene wurden deren pathophysiologische Mechanismen untersucht. Funktionelle Analysen konnten einer aberranten BAALC-Expression jedoch keine aktive Rolle in der Hämatopoese und Leukämogenese zuweisen. Daher ist anzunehmen, dass BAALC einen Surrogatmarker darstellt und andere ko- regulierte Gene für die Chemotherapieresistenz verantwortlich sind. Hier konnte IGFBP7 als interessantes Target-Gen identifiziert werden. Ähnlich wie BAALC konnte eine erhöhte Expression von IGFBP7 mit einem ungünstigen primären Therapieansprechen und einem verkürzten Überleben assoziiert werden. Im Gegensatz zu BAALC konnten funktionelle Analysen IGFBP7 eine aktive Rolle in akuten Leukämien zuschreiben. So konnte für IGFBP7 eine Hemmung der Zellproliferation, insbesondere der S-Phase, gezeigt werden. Diese Hemmung der Proliferation könnte zu einer geringeren Sensitivität der Zellen gegenüber zellzyklusaktiven Zytostatika führen. Darüber hinaus konnte eine hohe Expression von BAALC und IGFBP7 mit Stammzelleigenschaften assoziiert werden. Vor diesem Hintergrund wurde ein Modell entwickelt, welches eine mögliche Rolle von BAALC und IGFBP7 in der Interaktion zwischen der HSC bzw. LSC und der Stammzellnische beschreibt. Die Identifizierung von IGFBP7 als eine neue molekulare Zielstruktur könnte zur Klärung der zugrunde liegenden Regulationsmechanismen der BAALC-assoziierten Chemotherapieresistenz beitragen und für mögliche neue therapeutische Konzepte herangezogen werden.Acute leukemias are a heterogeneous group of malignancies with varying clinical, morphological, immunological, and molecular characteristics. Over the past decades identification of molecular abnormalities allowed the assignment of patients into specific risk groups, which contributed to optimized leukemia treatment. In addition to the prognostic value of the molecular risk factors, characterization of these abnormalities provided insights into the pathomechanisms of acute leukemias. In this context, the prognostic and biological role of BAALC and related genes was evaluated. BAALC is a known prognostic marker in CN-AML and T-ALL. Aberrantly high BAALC expression in adult acute leukemia patients has been associated with chemotherapy resistance. In the first study, we provided evidence of the prognostic value of BAALC in B-precursor ALL, supporting a lineage independent role of BAALC in leukemogenesis. In order to define a molecularly-based treatment stratification in T-ALL, the role of prognostic markers were investigated in this entity. In recent studies, WT1 mutations were of prognostic significance in AML patients and intrestingly, mutations were associated with high expression of BAALC. As BAALC was shown to share prognostic significance in AML and T-ALL, we reasoned that WT1 mutations might also be of prognostic significance in T-ALL. WT1 gene mutations were identified in similar frequency as previously shown in AML but of less prognostic significance in T-ALL. However, aberrant WT1 expression was of independent prognostic significance associated with inferior outcome, thus suggesting a role of WT1 in T-ALL. Determination of WT1 and BAALC expression might in the future facilitate treatment stratification for molecularly undefined subgroups of T-ALL and adult B-precursor ALL, respectively. In order to gain insights into the biological function of prognostic markers, we focused on the role of BAALC in hematopoiesis and leukemogenesis. However, functional analyses did not provide a molecular role of BAALC on drug resistance, proliferation and differentiation. Consequently, we concluded that BAALC is rather a surrogate marker for treatment failure in acute leukemia and co-regulated genes might contribute to the molecular basis of drug resistance. Hence, we identified IGFBP7 as a new candidate gene involved in acute leukemia. Similar to BAALC, elevated IGFBP7 expression was associated with a lower response rate to induction therapy and an inferior overall survival in T-ALL patients. However, in contrast to BAALC, IGFBP7 may have a functional role in leukemogenesis and might contribute to the molecular mechanisms of drug resistance. In particular we showed that IGFBP7 suppressed proliferation, suggesting that IGFBP7 might promote resistance to cell cycle specific cytotoxic agents via a decrease in replicative activity in these cells. Moreover, high expression of BAALC and IGFBP7 in acute leukemias was associated with stem cell features. In this context, a model was developed based on experimental data, suggesting a role for BAALC and IGFBP7 in the interaction of the HSC and LSC within the bone marrow niche. Identification of IGFBP7 as new candidate gene involved in leukemia might help to gain further insights into the mechanism of BAALC-associated chemotherapyresistance and may result in the design of new targeted therapies

How people construct their experience of living with secondary lymphoedema in the context of their everyday lives in Australia

Purpose: The purpose of this work was to explore how men and women construct their experiences living with lymphoedema following treatment for any cancer in the context of everyday life. Methods: The design and conduct of this qualitative study was guided by Charmaz' social constructivist grounded theory. To collect data, focus groups and telephone interviews were conducted. Audiotapes were transcribed verbatim and imported into NVivo8 to organize data and codes. Data were analyzed using key grounded theory principles of constant comparison, data saturation, and initial, focused, and theoretical coding. Results: Participants were 3 men and 26 women who had developed upper- or lower-limb lymphoedema following cancer treatment. Three conceptual categories were developed during data analysis and were labeled "accidental journey," "altered normalcy," and "ebb and flow of control." "Altered normalcy" reflects the physical and psychosocial consequences of lymphoedema and its relationship to everyday life. "Accidental journey" explains the participants' experiences with the health care system, including the prevention, treatment, and management of their lymphoedema. "Ebb and flow of control" draws upon a range of individual and social elements that influenced the participants' perceived control over lymphoedema. These conceptual categories were interrelated and contributed to the core category of "sense of self," which describes their perceptions of their identity and roles. Conclusions: Results highlight the need for greater clinical and public awareness of lymphoedema as a chronic condition requiring prevention and treatment, and one that has far-reaching effects on physical and psychosocial well-being as well as overall quality of life

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

Additional file 1: Table S1. of Inhibition of IGF1-R overcomes IGFBP7-induced chemotherapy resistance in T-ALL

Probe sets in the IGF1-R signatures that are over-expressed in the high IGF1-R group. Table S2. Probe sets in the IGF1-R signatures that are under-expressed in the high IGF1-R group. (PDF 209 kb

Molecular characteristics of <i>FLT3</i>mut ETP-ALL versus <i>FLT3</i>wt ETP-ALL patients.

<p>A: P-values were calculated by Mann-Whitney-U-test.</p><p>B: P-values were calculated by Pearson's Chi-square test and Fisher's exact test, respectively.</p