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.

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.

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.

Comorbidity in patients with cancer: prevalence and severity measured by cumulative illness rating scale.

Comorbidity is defined as the presence of one or more diseases in addition to an index disease. In elderly people, the number and severity of comorbidity increase with age. We report the comorbidity data of 536 patients treated as in-patients: 231 elderly cancer patients (ECP), 172 younger cancer patients (YCP) and 133 elderly patients admitted for non-cancer reasons (EMP). Comorbidity was assessed with the cumulative illness rating scale geriatric version (CIRS-G). Data on number of affected organ systems (levels 1-4), number of affected organ systems with severe disease (levels 3-4), and sum score of levels per patient are reported. The number of comorbidities increases with age. A 76% of ECP, 51% of YCP, and 79% of EMP have severe comorbidity. Palliative treatment approach is not associated with higher levels of comorbidity in ECP. Vascular disorders were the most common comorbidity. The difficulty to rate haematological comorbidity in cancer patients is reflected. This is the first report on detailed results of assessment of comorbidity measured by CIRS-G in cancer patients. In addition, we provide a comparison to an elderly group of patients admitted for non-cancer reasons.

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.

Anaemia-related impairment in quality of life in elderly cancer patients prior to chemotherapy.

PURPOSE: Quality of life (QoL) of patients with cancer is a major area of concern for both patients and their physicians. PATIENTS AND METHODS: We investigated QoL with the EORTC-QLQ-C30 questionnaire with reference to anaemia and Karnofsky Performance Status (KPS) prior to the start of chemotherapy in 477 patients: 195 elderly cancer patients (Group A), 152 younger cancer patients (Group B), and 130 patients aged 60 years or older admitted for non-cancer related disorders (Group C). RESULTS: In univariate analysis QoL was significantly worse in 8 out of 15 scales in anaemic compared to non anaemic patients in Group A, in 2 in Group B, and in 7 in Group C. In ANOVA analysis including KPS and haemoglobin status, the influence of anaemia and KPS independently persists in most scales in Group A, in some in Group C, but not in Group B. CONCLUSIONS: Anaemia and functional impairment are independently related to QoL in elderly cancer and elderly medical patients, but not in younger cancer patients.

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.

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.

[Health services research and geriatrics: deficits and research approaches using the example of colorectal carcinoma and anaemia]. / Versorgungsforschung und Geriatrie: Defizite und Forschungsansätze am Beispiel des kolorektalen Karzinoms und der Anämie.

The number of elderly people with cancer will increase within the next decades. Cancer will surpass cardiovascular diseases as the leading cause of death. In comparison to younger patients elderly patients with cancer are less often treated within the scope of clinical trials. Data from health care research demonstrate that the treatment of elderly patients is less often guideline-directed than that of younger patients. This will be demonstrated in more detail for patients with colorectal carcinoma and for patients with anaemia. Older people are reluctant to participate in programs for the early detection of colorectal carcinoma and its precursors. They less often receive adjuvant chemotherapy in stage III disease, despite the fact that adjuvant chemotherapy is no more toxic than in younger patients and equally effective and therefore recommended in the guidelines. Compared to younger patients, elderly patients less often receive palliative chemotherapy in stage IV disease. Anaemia has a prevalence of about 10% in people aged 65 and more; the reported values are highly variable. There is a lack of data on the grade of evaluation. Also, there are no diagnostic and therapeutic guidelines in Germany. Health services research will play an important role in assessing deficits in the diagnosis and therapy of cancer diseases in the elderly and in determining the goals for future efforts in health care and research.

Novel cryptic chromosomal rearrangements detected in acute lymphoblastic leukemia detected by application of new multicolor fluorescent in situ hybridization approaches.

Routine cytogenetic analysis provides important information on diagnostic and prognostic relevance for hematological malignancies. However, it is often difficult to obtain good karyotypes, especially of cells from cases with acute lymphoblastic leukemia (ALL) because of poor morphology and spreading. Thus, detailed karyotyping can be hampered and even in case of a 'normal karyotype' according to banding cytogenetics doubts remain if the result is reliable. In order to address this problem a series of 37 ALL cases without any detectable numerical or structural chromosomal defects was selected and studied by two recently developed multicolor fluorescence in situ hybridization (FISH) approaches: 1) multitude multicolor banding (mMCB) is a FISH-banding technique, which allows the analyses of inter- and intra-chromosomal rearrangements of the whole human karyotype in one single experiment; 2) chromosome-specific subcentromere/subtelomere-specific multicolor (subCTM-)FISH applies locus-specific subtelomeric and subcentromic probes and enables the characterization of the subtelomeric and peri-centric regions of the chromosomes, not analyzable by other FISH-approaches. Thus, we detected the following recurrent cryptic chromosomal aberrations: del(12)(pter) [8 cases], del(9)(qter) [3 cases], and del(11)(pter) [2 cases]. Moreover, cryptic changes in additional nine subtelomeric and in two subcentromeric regions were observed one time, each. In summary, mMCB and subCTM were proven to be powerful methods in the screening for new cryptic chromosomal aberrations, which considerably increased the accuracy of cytogenetic diagnosis.

Impairment in functional status and survival in patients with acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is mainly affecting elderly patients. Elderly patients are increasingly affected by impairment of functional status (FS). FS is of prognostic relevance for survival in different tumours. Data for patients with AML are rare. Within a prospective trial we recruited patients with newly diagnosed AML and measured FS by two different methods: Karnofsky performance status (KPS) and instrumental activities of daily living (IADL). Sixty-three patients aged 19-85 years (median 61.1) were included. Twenty-three had prior myelodisplastic syndrome (MDS), 7 favourable, 17 unfavourable karyotype. Fifty received induction chemotherapy, 13 palliative chemotherapy. Median survival was 15.2 months (95% CI, 10.8-22.3) in all patients. Age, cytogenetic risk group, and impaired KPS and IADL significantly influenced median survival in univariate analysis. Impairment of IADL was the single most predictive variable. In multivariate analysis, impairment of IADL Score (HR:4.3, 95% CI 1.7-10.5, P = 0.001) and of KPS (HR:4.8, 95% CI 1.9-12.3, P = 0.001), and unfavourable cytogenetic risk group (HR:6.0, 95% CI 2.5-14.3, P < 0.001) significantly predicted median survival. In patients with AML, FS and not age is a major predictor of survival. The influence of FS is independent from cytogenetic risk group. IADL measurement adds information to KPS. The results have to be confirmed in a large sample of patients.

Quantification of the response of circulating epithelial cells to neodadjuvant treatment for breast cancer: a new tool for therapy monitoring.

INTRODUCTION: In adjuvant treatment for breast cancer there is no tool available with which to measure the efficacy of the therapy. In contrast, in neoadjuvant therapy reduction in tumour size is used as an indicator of the sensitivity of tumour cells to the agents applied. If circulating epithelial (tumour) cells can be shown to react to therapy in the same way as the primary tumour, then this response may be exploited to monitor the effect of therapy in the adjuvant setting. METHOD: We used MAINTRAC analysis to monitor the reduction in circulating epithelial cells during the first three to four cycles of neoadjuvant therapy in 30 breast cancer patients. RESULTS: MAINTRAC analysis revealed a patient-specific response. Comparison of this response with the decline in size of the primary tumour showed that the reduction in number of circulating epithelial cells accurately predicted final tumour reduction at surgery if the entire neoadjuvant regimen consisted of chemotherapy. However, the response of the circulating tumour cells was unable to predict the response to additional antibody therapy. CONCLUSION: The response of circulating epithelial cells faithfully reflects the response of the whole tumour to adjuvant therapy, indicating that these cells may be considered part of the tumour and can be used for therapy monitoring.

Minimal residual disease based on patient specific Flt3-ITD and -ITT mutations in acute myeloid leukemia.

We present our first experiences with determination of minimal residual disease (MRD) based on patient specific Flt3-ITD (internal tandem duplication) mutations. We analysed MRD status of 11 AML patients in a retrospective investigation and its potential impact on the follow up of these patients. In five out of six patients with a positive Flt3-ITD based MRD status a relapse of AML was observed in the follow up while one patient lacks a clinical relapse so far. In contrast, four out of five patients with a negative MRD status remain free of disease. One of these patients relapsed with a switch of FAB subtype including loss of Flt3-ITD mutation. Furthermore, in one patient we could identify a Flt3-ITT (internal tandem triplication mutation).

Bone morphogenetic protein 2 (BMP-2) induces in vitro invasion and in vivo hormone independent growth of breast carcinoma cells.

Breast cancer cell lines migrated towards a BMP-2 source depending on BMP-2 concentration. After a short exposure to BMP-2, the cells were able to migrate through matrigel. MCF-7 cells transfected with the BMP-2 gene also showed enhanced migratory properties and high expression of the metastasis-related gene BCSG1. In a xenograft model without estrogen supplementation MCF-7/BMP-2 cells formed tumors. These tumors were characterised by an enhanced vasculature and the formation of chondroid and osseous structures. In conclusion elevated levels of BMP-2 enhance the tumorigenic properties of breast carcinoma cells and drive the cells towards a more aggressive phenotype with estrogen independent growth.

Analyses of minimal residual disease based on Flt3 mutations in allogeneic peripheral blood stem cell transplantation.

PURPOSE: Activating Flt3 mutations are observed in about 30% of patients with acute myeloid leukaemia (AML) and individual Flt3 mutations are applicable for minimal residual disease (MRD) analyses. METHODS: We investigated the MRD status in four AML patients carrying different Flt3 mutations (three patients with Flt3 length mutations of the juxtamembrane domain, one patient carrying a mutation of the Flt3 tyrosine kinase domain, i.e. Flt3-TKD mutation) who underwent allogeneic peripheral blood stem cell transplantation (PBSCT). Residual leukaemia cells were retrospectively determined by real-time PCR at different time points. RESULTS: We can demonstrate a good correlation between the course of MRD status and clinical events in all four investigated patients. CONCLUSION: These examples demonstrate the potential impact of Flt3 based MRD status not only after but also prior to allogeneic PBSCT.

Increase in number of circulating disseminated epithelial cells after surgery for non-small cell lung cancer monitored by MAINTRAC(R) is a predictor for relapse: A preliminary report.

BACKGROUND: Lung cancer still remains one of the most commonly occurring solid tumors and even in stage Ia, surgery fails in 30% of patients who develop distant metastases. It is hypothesized that these must have developed from occult circulating tumor cells present at the time of surgery, or before. The aim of the present study was to detect such cells in the peripheral blood and to monitor these cells following surgery. METHODS: 30 patients treated for lung cancer with surgery were monitored for circulating epithelial cells (CEC) by taking peripheral blood samples before, 2 weeks and 5 months after surgery and/or radiotherapy (RT) chemotherapy (CT) or combined RT/CT using magnetic bead enrichment and laser scanning cytometry (MAINTRAC(R)) for quantification of these cells. RESULTS: In 86% of the patients CEC were detected before surgery and in 100% at 2 weeks and 5 months after surgery. In the control group, which consisted of 100 normal donors without cancer, 97 % were negative for CEC. A significantly higher number of CEC was found preoperatively in patients with squamous cell carcinoma than in those with adenocarcinoma. In correlation to the extent of parenchymal manipulation 2 weeks after surgery, an increase in numbers of CEC was observed with limited resections (18/21) whereas pneumonectomy led to a decrease (5/8) of CEC, 2 weeks after surgery. The third analysis done 5 months after surgery identified 3 groups of patients. In the group of 5 patients who received neo- or adjuvant chemo/radiotherapy there was evidence that monitoring of CEC can evaluate the effects of therapy. Another group of 7 patients who underwent surgery only showed a decrease of CEC and no signs of relapse. A third group of 11 patients who had surgery only, showed an increase of CEC (4 with an initial decrease after surgery and 7 with continuous increase). In the group with a continuous increase during the following 24 months, 2 early relapses in patients with stage Ia adenocarcinoma were observed. The increase of CEC preceded clinical detection by six months. CONCLUSION: We consider, therefore, that patients with adenocarcinoma and a continuous increase of CEC after complete resection for lung cancer are at an increased risk of early relapse.