Martin Granzow

From latent disseminated cells to overt metastasis: Genetic analysis of systemic breast cancer progression

According to the present view, metastasis marks the end in a sequence of genomic changes underlying the progression of an epithelial cell to a lethal cancer. Here, we aimed to find out at what stage of tumor development transformed cells leave the primary tumor and whether a defined genotype corresponds to metastatic disease. To this end, we isolated single disseminated cancer cells from bone marrow of breast cancer patients and performed single-cell comparative genomic hybridization. We analyzed disseminated tumor cells from patients after curative resection of the primary tumor (stage M0), as presumptive progenitors of manifest metastasis, and from patients with manifest metastasis (stage M1). Their genomic data were compared with those from microdissected areas of matched primary tumors. Disseminated cells from M0-stage patients displayed significantly fewer chromosomal aberrations than primary tumors or cells from M1-stage patients (P < 0.008 and P < 0.0001, respectively), and their aberrations appeared to be randomly generated. In contrast, primary tumors and M1 cells harbored different and characteristic chromosomal imbalances. Moreover, applying machine-learning methods for the classification of the genotypes, we could correctly identify the presence or absence of metastatic disease in a patient on the basis of a single-cell genome. We suggest that in breast cancer, tumor cells may disseminate in a far less progressed genomic state than previously thought, and that they acquire genomic aberrations typical of metastatic cells thereafter. Thus, our data challenge the widely held view that the precursors of metastasis are derived from the most advanced clone within the primary tumor.

A Practical Approach to Microarray Data Analysis

A Practical Approach to Microarray Data Analysis is for all life scientists, statisticians, computer experts, technology developers, managers, and other professionals tasked with developing, deploying, and using microarray technology including the necessary computational infrastructure and analytical tools. The book addresses the requirement of scientists and researchers to gain a basic understanding of microarray analysis methodologies and tools. It is intended for students, teachers, researchers, and research managers who want to understand the state of the art and of the presented methodologies and the areas in which gaps in our knowledge demand further research and development. The book is designed to be used by the practicing professional tasked with the design and analysis of microarray experiments or as a text for a senior undergraduate- or graduate level course in analytical genetics, biology, bioinformatics, computational biology, statistics and data mining, or applied computer science. Key topics covered include: -Format of result from data analysis, analytical modeling/experimentation; -Validation of analytical results; -Data analysis/Modeling task; -Analysis/modeling tools; -Scientific questions, goals, and tasks; -Application; -Data analysis methods; -Criteria for assessing analysis methodologies, models, and tools.

Hodgkin's lymphoma cell lines are characterized by frequent aberrations on chromosomes 2p and 9p including REL and JAK2

Four Hodgkins lymphoma cell lines (KM‐H2, HDLM‐2, L428, L1236) were analyzed for cytogenetic aberrations, applying multiplex fluorescence in situ hybridization, chromosome banding and comparative genomic hybridization. Each line was characterized by a highly heterogeneous pattern of karyotypic changes with a large spectrum of different translocated chromosomes (range 22–57). A recurrent finding in all cell lines was the presence of chromosomal rearrangements of the short arm of chromosome 2 involving the REL oncogene locus. Furthermore, multiple translocated copies of telomeric chromosomal segments were frequently detected. This resulted in a copy number increase of putative oncogenes, e.g., JAK2 (9p24) in 3 cell lines, FGFR3 (4p16) and CCND2 (12p13) in 2 cell lines as well as MYC (8q24) in 1 cell line. Our data confirm previous cytogenetic results from primary Hodgkins tumors suggesting an important pathogenic role of REL and JAK2 in this disease. In addition, they provide evidence for a novel cytogenetic pathomechanism leading to increased copy numbers of putative oncogenes from terminal chromosomal regions, most probably in the course of chromosomal stabilization by telomeric capture.

Progression in Smoldering Myeloma Is Independently Determined by the Chromosomal Abnormalities del(17p), t(4;14), Gain 1q, Hyperdiploidy, and Tumor Load

PURPOSE The aim of this study was to analyze chromosomal aberrations in terms of frequency and impact on time to progression in patients with smoldering multiple myeloma (SMM) on the background of clinical prognostic factors. PATIENTS AND METHODS The chromosomal abnormalities 1q21, 5p15/5q35, 9q34, 13q14.3, 15q22, 17p13, t(11;14)(q13;q32), and t(4;14)(p16.3;q32) were assessed in CD138-purified myeloma cells by interphase fluorescent in situ hybridization (iFISH) alongside clinical parameters in a consecutive series of 248 patients with SMM. RESULTS The high-risk aberrations in active myeloma (ie, del(17p13), t(4;14), and +1q21) present in 6.1%, 8.9%, and 29.8% of patients significantly confer adverse prognosis in SMM with hazard ratios (HRs) of 2.90 (95% CI, 1.56 to 5.40), 2.28 (95% CI, 1.33 to 3.91), and 1.66 (95% CI, 1.08 to 2.54), respectively. Contrary to the conditions in active myeloma, hyperdiploidy, present in 43.3% of patients, is an adverse prognostic factor (HR, 1.67; 95% CI, 1.10 to 2.54). Percentage of malignant bone marrow plasma cells assessed by iFISH and combination of M-protein and plasma cell infiltration as surrogates of tumor load significantly confer adverse prognosis with HRs of 4.37 (95% CI, 2.79 to 6.85) and 4.27 (95% CI, 2.77 to 6.56), respectively. In multivariate analysis, high-risk aberrations, hyperdiploidy, and surrogates of tumor load are independently prognostic. CONCLUSION The high-risk chromosomal aberrations del(17p13), t(4;14), and +1q21 are adverse prognostic factors in SMM just as they are in active myeloma, independent of tumor mass. Hyperdiploidy is the first example for an adverse prognostic factor in SMM of opposite predictiveness in active myeloma. Risk association of chromosomal aberrations is not only a priori treatment dependent (predictive) but is also an intrinsic property of myeloma cells (prognostic).

Translocation t(11;14) Is Associated With Adverse Outcome in Patients With Newly Diagnosed AL Amyloidosis When Treated With Bortezomib-Based Regimens

PURPOSE Bortezomib has become a cornerstone in the treatment of AL amyloidosis. In this study, we addressed the prognostic impact of cytogenetic aberrations for bortezomib-treated patients. PATIENTS AND METHODS We analyzed a consecutive series of 101 patients with AL amyloidosis treated with bortezomib-dexamethasone as first-line treatment by interphase fluorescence in situ hybridization (iFISH). Patients were ineligible for high-dose chemotherapy, which would put them at risk for cardiac or renal failure, and thus represented a poor-risk group. RESULTS Presence of t(11;14), versus its absence, was associated with inferior hematologic event-free survival (median, 3.4 v 8.8 months, respectively; P = .002), overall survival (median, 8.7 v 40.7 months, respectively; P = .05), and remission rate (≥ very good partial remission; 23% v 47%, respectively; P = .02). In multivariable Cox regression models incorporating established hematologic and clinical risk factors, t(11;14) was an independent adverse prognostic marker for hematologic event-free survival (hazard ratio, 2.94; 95% CI, 1.37 to 6.25; P = .006) and overall survival (hazard ratio, 3.13; 95% CI, 1.16 to 8.33; P = .03), but not for remission (≥ very good partial remission). Markedly, the multiple myeloma high-risk iFISH aberrations t(4;14), t(14;16), del(17p), and gain of 1q21 conferred no adverse prognosis in this bortezomib-dexamethasone-treated group. After backward variable selection, the final multivariable model was validated in a consecutive series of 32 patients treated with bortezomib, dexamethasone, and cyclophosphamide. CONCLUSION iFISH results are important independent prognostic factors in AL amyloidosis. In contrast to our recently published results with melphalan and dexamethasone standard therapy, bortezomib is less beneficial to patients harboring t(11;14), whereas it effectively alleviates the poor prognosis inherent to high-risk aberrations. Given the discrepant response to different treatment modalities, iFISH may help to guide therapeutic choices in these poor-risk patients requiring rapid hematologic response.

Gain of chromosome 1q21 is an independent adverse prognostic factor in light chain amyloidosis patients treated with melphalan/dexamethasone.

Abstract Chromosomal aberrations of plasma cells are well established pathogenetic and prognostic factors in multiple myeloma, but their prognostic implication in systemic light chain (AL) amyloidosis is unclear. Therefore, the aim of this study was to identify prognostic cytogenetic risk factors by interphase FISH in a series of 103 consecutive AL amyloidosis patients treated uniformly with melphalan/dexamethasone as first-line therapy. Detection of gain of 1q21 was predictive for a poor overall survival (OS) (median 12.5 versus 38.2 months, p = 0.002). Hematologic event free survival (hem EFS) for gain of 1q21 was 5.0 versus 8.5 months in median (p = 0.08) and haematologic remission rates (≥VGPR) after three cycles were 5% versus 25% (p = 0.06). Most important, in multivariate concordance analyses the adverse prognosis carried by gain of 1q21 was retained as an independent prognostic factor (OS: p = 0.003, average hazard ratio (AHR) = 3.64, hemEFS: p = 0.008, AHR = 2.35), along with the well established Mayo cardiac staging. Patients with t(11;14) had a longer median OS with 38.2 months versus 17.5 months, though no statistical significance was reached. Deletion 13q14 and hyperdiploidy turned out to be prognostically neutral. In conclusion, we have identified gain of 1q21 as an independent adverse prognostic factor in AL amyloidosis patients treated with standard chemotherapy.

Recurrent CDKN1B (p27) mutations in hairy cell leukemia.

Hairy cell leukemia (HCL) is marked by near 100% mutational frequency of BRAFV600E mutations. Recurrent cooperating genetic events that may contribute to HCL pathogenesis or affect the clinical course of HCL are currently not described. Therefore, we performed whole exome sequencing to explore the mutational landscape of purine analog refractory HCL. In addition to the disease-defining BRAFV600E mutations, we identified mutations in EZH2, ARID1A, and recurrent inactivating mutations of the cell cycle inhibitor CDKN1B (p27). Targeted deep sequencing of CDKN1B in a larger cohort of HCL patients identify deleterious CDKN1B mutations in 16% of patients with HCL (n = 13 of 81). In 11 of 13 patients the CDKN1B mutation was clonal, implying an early role of CDKN1B mutations in the pathogenesis of HCL. CDKN1B mutations were not found to impact clinical characteristics or outcome in this cohort. These data identify HCL as having the highest frequency of CDKN1B mutations among cancers and identify CDNK1B as the second most common mutated gene in HCL. Moreover, given the known function of CDNK1B, these data suggest a novel role for alterations in regulation of cell cycle and senescence in HCL with CDKN1B mutations.

Study of 30 patients with unexplained developmental delay and dysmorphic features or congenital abnormalities using conventional cytogenetics and multiplex FISH telomere (M-TEL) integrity assay

Abstract. Cryptic subtelomeric chromosome rearrangements are a major cause of mild to severe mental retardation pointing out the necessity of sensitive screening techniques to detect such aberrations among affected patients. In this prospective study a group of 30 patients with unexplained developmental retardation and dysmorphic features or congenital abnormalities were analysed using the recently published multiplex FISH telomere (M-TEL) integrity assay in combination with conventional G-banding analysis. The patients were selected by one or more of the following criteria defined by de Vries et al.: (a) family history with two or more affected individuals, (b) prenatal onset growth retardation, (c) postnatal growth abnormalities, (d) facial dysmorphic features, (e) non-facial dysmorphism and congenital abnormalities. In addition, we included two patients who met these criteria and revealed questionable chromosome regions requiring further clarification. In four patients (13.3%) cryptic chromosome aberrations were successfully determined by the M-TEL integrity assay and in two patients with abnormal chromosome regions intrachromosomal aberrations were characterized by targetted FISH experiments. Our results accentuate the requirement of strict selection criteria prior to patient testing with the M-TEL integrity assay. Another essential precondition is high-quality banding analysis to identify structural abnormal chromosomes. The detection of familial balanced translocation carriers in 50% of the cases emphasizes the significance of such an integrated approach for genetic counselling and prenatal diagnosis.

Multiplex FISH telomere integrity assay identifies an unbalanced cryptic translocation der(5)t(3;5)(q27;p15.3) in a family with three mentally retarded individuals

Abstract. Cryptic rearrangements involving the terminal regions of chromosomes are suspected to be the cause of idiopathic mental retardation in a significant number of cases. This finding highlights the necessity of a primary screening test for such chromosome aberrations. Here we present a multiplex fluorescence in situ hybridization telomere integrity assay which allows the detection of submicroscopic aberrations in the telomeric regions of all chromosomes. This novel approach identified an unbalanced cryptic translocation der(5)t(3;5)(q27;p15.3) in a family with three cases of unexplained mental retardation and dysmorphic features. The symptoms of the patients represent neither the classical dup(3q)- nor cri du chat syndrome, although all affected individuals demonstrate several features of both syndromes. The identification of two balanced translocation carriers emphasizes the significance of the telomere integrity assay for genetic counseling and prenatal diagnosis

Prognostic impact of cytogenetic aberrations in AL amyloidosis patients after high-dose melphalan: a long-term follow-up study

Cytogenetic aberrations detected by interphase fluorescence in situ hybridization (iFISH) of plasma cells are routinely evaluated as prognostic markers in multiple myeloma. This long-term follow-up study aimed to assess the prognosis of systemic light chain amyloidosis (AL) patients treated with high-dose melphalan (HDM) chemotherapy and autologous stem cell transplantation, depending on iFISH results. Therefore, we analyzed a consecutive cohort of 123 AL patients recruited from 2003 to 2014. HDM was safe, with only 1 of 123 patients dying as a result of treatment-related mortality, and effective, with a complete remission (CR) rate of 34%. Translocation t(11;14) as the most prevalent aberration (59%) led to an improved CR rate after high-dose therapy (41.2% vs 20.0%; P = .02), translating into a prolonged hematologic event-free survival (hemEFS; median, 46.1 vs 28.1 months; P = .05) and a trend for better overall survival (median, not reached vs 93.7 months; P = .07). In multivariate analysis, t(11;14) was confirmed as a favorable prognostic factor regarding hemEFS along with lower values for the difference between involved and uninvolved free light chains. Conversely, deletion 13q14, gain of 1q21, and hyperdiploidy had no significant prognostic impact. The high-risk cytogenetic aberrations t(4;14), t(14;16), and del(17p13) conferred an unfavorable prognosis, although statistical significance was reached only for univariate CR analysis in this small group of 9 patients. Thus, t(11;14) positivity in HDM-treated AL patients conferred superior CR rates and hemEFS. In view of the reduced response of t(11;14) to bortezomib, this highlights the impact of therapy on the prognostic role of cytogenetic aberrations.