Preferred co-localization of chromosome 8 and 21 in myeloid bone marrow cells detected by three dimensional molecular cytogenetics.

The impact of chromosome architecture in the formation of chromosome aberrations is a recent finding of interphase directed molecular cytogenetic studies. Also positive correlation of translocation frequencies and spatial proximity of chromosomes was described. Thus, disease specific chromosomal translocations could be due to tissue specific genomic organization. However, no three-dimensional interphase fluorescence in situ hybridization (FISH) studies for the nuclear architecture of bone marrow (BM) cells have previously been done. In this study, BM of three secondary acute myelogenous leukemia (AML) cases with trisomy 8 and otherwise normal karyotype were evaluated. Bone marrow cells of one AML and one ALL (acute lymphoblastic leukemia) case, peripheral blood lymphocytes and human sperm, all of them with normal karyotype, served as controls. Multicolor banding (MCB) probes for chromosomes 8 and 21 were applied in suspension-FISH (S-FISH). Interestingly, in myeloid bone marrow cells chromosomes 8 (di- and trisomic) and 21 tended to co-localize with their homologue chromosome(s), rather than to be separated. Thus, the co-localization of chromosomes 8 and 21 might promote a translocation providing a selective advantage of t(8;21) cells in AML-M2. In summary, the concept that tissue specific spatial proximity of chromosomes leads to enhanced translocation frequencies was further supported.

Delineation of yet unknown cryptic subtelomere aberrations in 50% of acute myeloid leukemia with normal GTG-banding karyotype.

Acute myeloid leukemia (AML) is a heterogeneous disease with respect to clinical prognosis and acquired chromosomal aberrations. After routine banding cytogenetic analysis 45% of AML patients show a normal karyotype (NK-AML). For a better understanding of development and progression in AML, it is important to find markers which could be primary genetic aberrations. Therefore, in this study 31 patients with NK-AML were analyzed by new high resolution molecular cytogenetic approaches. A combination of multitude multicolor banding and metaphase microdissection-based comparative genomic hybridization revealed deletions of the subtelomeric regions in 6% of the studied cases. According to these results, locus-specific probes for the subtelomeric regions of chromosomes 5, 9, 11, 12 and 13 were applied on 22 of the studied 31 NK-AML cases. Surprisingly, 50% of them showed deletions or duplications. These aberrations occurred in the in vitro proliferating as well as in the non-proliferating cells. Meta-analysis of the aberrant regions revealed that they often include genes known to be associated with tumors, e.g. RASA3 on chromosome 13. These results implicate that aberrations in the subtelomeric regions of NK-AML occur quite often and may be considered as primary genetic changes, and should not be neglected in future diagnostic approaches.

Clinical implications of molecular genetic aberrations in acute myeloid leukemia.

The role of different cytogenetic changes has been extensively evaluated in patients with acute myeloid leukemia (AML), and cytogenetic analysis of AML blasts is essential to form prognostic subgroups in order to stratify for the extent of therapy. Nevertheless, 40-45% of AML patients lack such cytogenetic markers, i.e., cytogenetically normal AML (CN-AML). In the past decade, different molecular aberrations were identified in AML and especially CN-AML can now be discriminated into certain prognostic subgroups. This review considers the latest advances to define the prognostic impact of molecular aberrations in AML and gives insights how such molecular markers can be applied for analysis of minimal residual disease. Furthermore, therapeutic implications as well as the potential role of new methodological techniques in analyzing expression patterns of AML blasts are discussed.

Differential expression of cancer-related genes by single and permanent exposure to bone morphogenetic protein 2.

PURPOSE: Bone morphogenetic proteins (BMPs) are multifunctional regulators of various cell functions. The BMP-signalling network plays a pivotal role during embryogenesis and tumorigenesis. BMPs, e.g. BMP-2 exert their biological function in a time and concentration-dependent manner but also modulated by the context of the cellular microenvironment. In this study, we investigated the effect of a steady high level of BMP-2 versus a single application of BMP-2 on the breast cancer cell line MCF-7. METHODS: The effect of the incubation regimes was analysed by DNA microarray expression profiling. Data were verified by real-time PCR. The protein expression of apoptosis-related genes was studied by western blot analysis. RESULTS: We found a clear difference in the altered gene expression between the constant high level and the single application of BMP-2. After grouping the genes of interest into the biological processes of Gene Ontology, the group of apoptosis-related genes like BAX, BAG5 or PKR, was predominantly affected under the single-application regime of BMP-2. Among these protein kinase R was the most prominently regulated. Further studies on the protein level showed activation of PKR after 4 h with a subsequent enhanced phosphorylation of the PKR substrate eIF2alpha for several hours. CONCLUSIONS: The duration of treatment and the concentration of BMP-2 affect the global expression pattern of MCF-7 cells. Among the regulated cancer-related genes, the cohort of the apoptosis-related genes showed the pronounced alterations. Our data point to a novel role of BMP-2 in the regulation of the PKR pathway in tumorigenesis.

Monitoring the response of circulating epithelial tumor cells to adjuvant chemotherapy in breast cancer allows detection of patients at risk of early relapse.

PURPOSE: To demonstrate that it is possible to monitor the response to adjuvant therapy by repeated analysis of circulating epithelial tumor cells (CETCs) and to detect patients early who are at risk of relapse. PATIENTS AND METHODS: In 91 nonmetastatic primary breast cancer patients, CETCs were quantified using laser scanning cytometry of anti-epithelial cell adhesion molecule-stained epithelial cells from whole unseparated blood before and during adjuvant chemotherapy. RESULTS: Numbers of CETCs were analyzed before therapy, before each new cycle, and at the end of chemotherapy. The following three typical patterns of response were observed: (1) decrease in cell numbers (> 10-fold); (2) marginal changes in cell numbers (< 10-fold); and (3) an (sometimes saw-toothed) increase or an initial decrease with subsequent reincrease (> 10-fold) in numbers of CETCs. Twenty relapses (22%) were observed within the accrual time of 40 months, including one of 28 patients from response group 1, five of 30 patients from response group 2, and 14 of 33 patients from response group 3. The difference in relapse-free survival was highly significant for CETC (hazard ratio = 4.407; 95% CI, 1.739 to 9.418; P < .001) between patients with decreasing cell numbers and those with marginal changes and between patients with marginal changes and those with an increase of more than 10-fold (linear Cox regression model). CONCLUSION: These results show that peripherally circulating tumor cells are influenced by systemic chemotherapy and that an increase (even after initial response to therapy) of 10-fold or more at the end of therapy is a strong predictor of relapse and a surrogate marker for the aggressiveness of the tumor cells.

Additive effects of PI3-kinase and MAPK activities on NB4 cell granulocyte differentiation: potential role of phosphatidylinositol 3-kinase gamma.

PURPOSE: In acute promyelocytic leukemia (APL) the chromosome translocation t(15;17) resulting in the PML-RAR alpha fusion protein is responsible for a blockage of myeloid differentiation. In this study we investigated the expression of different Phosphatidylinositol 3-kinase (PI3K) isoforms during granulocyte differentiation of NB4 cells induced by all-trans-retinoic acid (ATRA), 9-cis-retinoic acid (9cisRA) or retinoic acid receptor (RAR) agonists. METHODS: NB4 cells were analysed for their ability to differentiate into granulocytic lineage by the use of ATRA, 9cisRA or RAR agonists. Expression of signalling proteins was investigated by western blot and real-time PCR. PI3K activity was determined by in vitro kinase assays. RESULTS: Co-treatment of NB4 cells with either LY294002 to inhibit PI3Ks or PD98059 in order to suppress MEK activity led to significant reduction of CD11b surface expression during ATRA, 9cisRA or the RAR alpha agonist Ro40-6055 dependent NB4 cells granulocyte differentiation. We also show that only the G-protein coupled receptor activated PI3Kgamma isoform demonstrates up-regulated protein and mRNA expression during myeloid differentiation of NB4 cells via RAR alpha and RAR beta-dependent mechanism. Furthermore, activation of MAPK cascade including phosphorylation of MEK increases during retinoid induced differentiation of NB4 cells. Interestingly, protein kinase assays of immunoprecipitated PI3Kgamma revealed a protein of about 50 kDa that is phosphorylated when NB4 cells were treated with the RAR alpha agonist Ro40-6055. CONCLUSION: Collectively, our data suggest additive effects of PI3K and MAPK activity on ATRA-dependent NB4 cells granulocyte differentiation.

Clinical impact of nucleophosmin mutations and Flt3 internal tandem duplications in patients older than 60 yr with acute myeloid leukaemia.

BACKGROUND: Nucleophosmin (NPM1) and Flt3 internal tandem duplications (Flt3-ITD mutations) represent the most frequent molecular aberrations in patients with acute myeloid leukemia (AML). While NPM1 mutations are associated with favourable prognosis in younger AML patients, Flt3-ITD mutations reflect an unfavourable prognostic factor in these patients. So far, especially NPM1 mutations have not yet been evaluated exclusively in older patients. PATIENTS AND METHODS: We retrospectively analysed the prevalence of NPM1 and Flt3-ITD mutations and its association with complete remission (CR), and survival in 99 elderly patients (median age 71 yr, range 60-85 yr) newly diagnosed for AML. Primary treatment approach was curative in 54, and palliative in 38 patients, while seven patients received best supportive care only. The mean follow-up of surviving patients was 600 d. RESULTS: Sixty-seven patients were tested negative for NPM1 and Flt3-ITD mutations (group 1), 16 patients carried only a NPM1 mutation (group 2) and nine patients had only a Flt3-ITD mutation (group 3) while additional seven patients were positive for both aberrations (group 4). We can demonstrate a significant higher rate of CR comparing wildtype vs. NPM1 positive patients (40.5% for group 1 vs. 80.0% for group 2, P = 0.03) for patients receiving curative therapy. Interestingly, there is no significant difference in overall survival between group 1 and group 2 (Log-rank test P = 0.22, median 440 d vs. 1125 d). In contrast, patients carrying a Flt3-ITD mutation had a significant worse overall survival compared to wildtype patients (P = 0.03, median 210 d for group 3 + 4 vs. 634 d for group 1 + 2) while no difference of CR rate could be observed (42.8% vs. 48.9%, P = 0.91). CONCLUSION: As elderly but medically fit patients with AML carrying a NPM1 mutation have a high CR rate, age itself should not be a barrier for induction treatment. However, new therapeutic concepts of postremission therapy (e.g. allogeneic stem cell transplantation after dose-reduced conditioning) should be considered for these patients in first CR.

Depression and functional impairment independently contribute to decreased quality of life in cancer patients prior to chemotherapy.

BACKGROUND: An inverse association either between depression or impaired functional status and quality of life (QoL) has been reported for cancer patients, but the independent effect of depression or depressive symptoms and of functional impairment on QoL is unclear. PATIENTS AND METHODS: We investigated the prevalence of depression or depressive symptoms with the Beck Depression Inventory (BDI), the functional impairment with the ECOG-Performance-Status (ECOG-PS) and the QoL with the EORTC-QLQ-C30 questionnaire in a sample of 175 hospitalised cancer patients prior to the start of chemotherapy. RESULTS: Sixteen of 175 patients (9.1%) screened positive for major depression, 29 (16.6%) had mild to moderate depressive symptoms. In 11 of 15 scales of the EORTC-QLQ-C30 questionnaire depression or depressive symptoms were significantly associated with worse QoL in univariate analysis and in 12 of 15 scales poor ECOG-PS was significantly associated with worse QoL. In multivariate analysis including ECOG-PS and BDI, the effect of depression and/ or depressive symptoms on QoL was persistent in seven scales: global QoL, physical- and role functioning, fatigue, nausea & vomiting, pain, and constipation, that of ECOG-PS in five scales: global QoL, emotional functioning, nausea & vomiting, pain, and appetite loss. CONCLUSIONS: Signs of major depression or depressive symptoms and impaired functional status contribute independently to poorer QoL in cancer patients prior to chemotherapy.

Specific pattern of protein expression in acute myeloid leukemia harboring FLT3-ITD mutations.

FLT3 activating mutations can be detected in about 35% of acute myeloid leukemia (AML). FLT3 internal tandem duplications (FLT3-ITD) represent the majority of FLT3 mutations (25 - 30%) while FLT3-TKD (tyrosine kinase domain) mutations can be found in about 7% of AML patients. In this study, we addressed the question whether especially primary AML cells carrying FLT3-ITD mutations show differences in terms of their protein expression pattern compared to FLT3 wild-type blasts. We investigated bone marrow samples that were isolated at diagnosis from 36 AML patients expressing either FLT3 wild-type (n = 16) or an activating FLT3 mutation (FLT3-ITD, n = 15; FLT3-TKD, n = 5). Proteomic analysis was performed by means of surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry which has shown its high efficiency in finding biomarkers in solid tumors. Here, we demonstrate that a large series of proteins is differently expressed in primary AML blasts harboring FLT3-ITD mutations. Furthermore, there are also significant differences of the protein expression profile between FLT3-ITD and FLT3-TKD mutations. Interestingly, further analysis of FLT3-ITD positive AML according to its response to the induction chemotherapy demonstrates putative prognostic markers for this subgroup of AML. We suggest that SELDI-TOF mass spectrometry represents a promising tool of proteomic analysis of AML that might help to establish new prognostic markers in AML.

Quality-of-life in elderly patients with cancer: a short review.

BACKGROUND: Prolongation of survival and maintenance or improvement of health-related quality-of-life (HRQoL) are the two important goals within the treatment of individual patients. Due to the severity of symptoms and the toxicity of treatment, HRQoL has become a major area of concern when treating cancer patients in general and elderly patients in particular. PATIENTS AND METHODS: We present a literature review of HRQoL aspects in elderly patients with cancer and especially address the topic whether impairments in the different tools of a comprehensive geriatric assessment (CGA) are associated with decreased HRQoL in elderly cancer patients. RESULTS: Elderly cancer patients tend to weight their HRQoL as more important than gain in survival, when compared to younger patients. An age-dependent decrease in different scales of HRQoL is reported in patients and normative samples. HRQoL is also a predictor of survival. The variation of HRQoL can be used in trials comparing different treatment options. In individual patients, regular measurement of HRQoL aims to improve patients-centred care. Age related impairments of different areas of CGA are associated with decreased HRQoL in elderly cancer patients. CONCLUSIONS: HRQoL is an important outcome with elderly cancer patients and should be assessed regularly and thoroughly.

Age, severe comorbidity and functional impairment independently contribute to poor survival in cancer patients.

PURPOSE: With the increasing number of elderly patients suffering from cancer, comorbidity and functional impairment become common problems in patients with cancer. Both comorbidity and functional impairment are associated with a shorter survival time in cancer patients, but their independent role has rarely been addressed before. METHODS: Within a prospective trial we recruited 427 cancer patients, irrespective of age and type of cancer, admitted as inpatients prior to the start of chemotherapy. Comorbidity was assessed with the cumulative illness rating scale (CIRS-G), functional impairment with WHO performance status (WHO-PS), basal (ADL) and instrumental (IADL) activities of daily living. RESULTS: Median follow-up was 34.2 months. A total, 61.4%. of patients died. Median survival time was 21.0 months. Age, kind of tumour (solid vs. haematological), treatment approach (non-curative vs. curative), WHO-PS (2-4 vs. 0-1), IADL (<8 vs. 8), and severe comorbidity (CIRS-level 3-4 vs. none) were significantly associated with shorter survival time in univariate analysis. In a multivariate Cox-regression-analysis, age (HR 1.019; 95%-CI 1.007-1.032; P=0.003), kind of tumour (HR 1.832; 95%-CI 1.314-2.554; P<0.001), WHO-PS (HR 1.455; 95%-CI 1.059-2.000; P=0.021), and comorbidity level 3-4 (HR 1.424; 95%-CI 1.012-2.003; P=0.043) maintained their significant association. CONCLUSIONS: Age, severe comorbidity, functional impairment, and kind of tumour are independently related to shorter survival time in cancer patients.

Rituximab added to first-line mitoxantrone, chlorambucil, and prednisolone chemotherapy followed by interferon maintenance prolongs survival in patients with advanced follicular lymphoma: an East German Study Group Hematology and Oncology Study.

PURPOSE: Rituximab has been shown to be active in follicular lymphoma (FL), both as monotherapy and in combination with chemotherapy. We conducted a randomized trial comparing mitoxantrone, chlorambucil, and prednisolone (MCP) chemotherapy plus rituximab with MCP alone. PATIENTS AND METHODS: Previously untreated patients with stage III or IV CD20+ indolent or mantle cell lymphoma were randomly assigned to either eight 28-day cycles of MCP plus rituximab (R-MCP; n = 181) or eight cycles of MCP alone (n = 177). All patients who achieved a complete or partial remission were treated with interferon maintenance until relapse. Herein, we report the results from the primary analysis population of patients with FL, who constituted the majority of patients (56%) recruited to the trial (n = 201; R-MCP, n = 105; MCP, n = 96). RESULTS: Rates of overall and complete response were significantly higher in the R-MCP arm than the MCP arm (overall response, 92% v 75%, respectively; P = .0009; complete response, 50% v 25%, respectively; P = .004). With a median follow-up time of 47 months, median event-free survival (EFS) and progression-free survival (PFS) times were significantly prolonged with R-MCP compared with MCP (EFS, not reached v 26 months, respectively; P < .0001; PFS, not reached v 28.8 months, respectively; P < .0001), and overall survival (OS) was significantly improved with R-MCP compared with MCP (4-year OS rate, 87% v 74%, respectively; P = .0096). CONCLUSION: The R-MCP regimen significantly improves complete and overall response rates, EFS, PFS, and OS in patients with previously untreated advanced FL, without a clinically significant increase in toxicity.

Co-morbidity and functional deficits independently contribute to quality of life before chemotherapy in elderly cancer patients.

UNLABELLED: GOAL OF THE WORK: The quality of life (QoL) of patients with cancer is a major area of concern for both patients and their physicians. The independent contribution of functional impairment and co-morbidity to QoL is unclear. MATERIALS AND METHODS: We investigated initial global QoL in 477 patients: 195 cancer patients aged 60 years or older (group A), 152 cancer patients below the age of 60 years (group B), admitted as inpatients for chemotherapy initiation and 130 patients aged 60 years or older admitted for non-cancer-related disorders (group C). Global QoL was assessed by the EORTC-QLQ-C30 subscale, functional status by the Karnofsky Performance Scale (KPS) and the Instrumental Activities of Daily Living (IADL) scale, and co-morbidity by the Cumulative Illness Rating Scale (CIRS). RESULTS: In multivariate analyses, global QoL is significantly associated with KPS, IADL and co-morbidity in group A (r (2) = 0.27), with KPS and IADL in group B (r (2) = 0.23), and with age, KPS and IADL in group C (r (2) = 0.38). CONCLUSIONS: IADL contributes to global QoL in addition to the known effect of KPS. In addition, co-morbidity independently influences global QoL in elderly cancer patients.

Tolerance to chemotherapy in elderly patients with cancer.

BACKGROUND: Due to demographic changes, the number of elderly people with cancer will increase in the next decades. In the past, elderly patients with cancer were often excluded from clinical trials. Chronological age has been considered a risk factor for increased toxicity and reduced tolerance to chemotherapy. METHODS: We present a review on toxicity of chemotherapy and factors associated with toxicity in elderly patients with cancer, and we discuss chemotherapeutic agents and treatment options in treating this patient population. RESULTS: Age is a risk factor for increased toxicity to chemotherapy and decreased tolerance. However, few trials have been reported with adjustment for age-associated changes such as impairment of functional status and increased comorbidity, which also show an independent association with increased toxicity. Published data may include several biases, such as referral and publication bias. CONCLUSIONS: Decision making in elderly cancer patients should be based on the results of a geriatric assessment. Patients with few or no limitations should be treated as younger patients are treated. Data with a high level of evidence are unavailable for patients showing moderate or severe limitations in a geriatric assessment.