Multimodal Biomarkers That Predict the Presence of Gleason Pattern 4: Potential Impact for Active Surveillance.

PURPOSE: Latent grade group ≥2 prostate cancer can impact the performance of active surveillance protocols. To date, molecular biomarkers for active surveillance have relied solely on RNA or protein. We trained and independently validated multimodal (mRNA abundance, DNA methylation, and/or DNA copy number) biomarkers that more accurately separate grade group 1 from grade group ≥2 cancers. MATERIALS AND METHODS: Low- and intermediate-risk prostate cancer patients were assigned to training (n=333) and validation (n=202) cohorts. We profiled the abundance of 342 mRNAs, 100 DNA copy number alteration loci, and 14 hypermethylation sites at 2 locations per tumor. Using the training cohort with cross-validation, we evaluated methods for training classifiers of pathological grade group ≥2 in centrally reviewed radical prostatectomies. We trained 2 distinct classifiers, PRONTO-e and PRONTO-m, and validated them in an independent radical prostatectomy cohort. RESULTS: PRONTO-e comprises 353 mRNA and copy number alteration features. PRONTO-m includes 94 clinical, mRNAs, copy number alterations, and methylation features at 14 and 12 loci, respectively. In independent validation, PRONTO-e and PRONTO-m predicted grade group ≥2 with respective true-positive rates of 0.81 and 0.76, and false-positive rates of 0.43 and 0.26. Both classifiers were resistant to sampling error and identified more upgrading cases than a well-validated presurgical risk calculator, CAPRA (Cancer of the Prostate Risk Assessment; P < .001). CONCLUSIONS: Two grade group classifiers with superior accuracy were developed by incorporating RNA and DNA features and validated in an independent cohort. Upon further validation in biopsy samples, classifiers with these performance characteristics could refine selection of men for active surveillance, extending their treatment-free survival and intervals between surveillance.

Characterization of male breast cancer: results of the EORTC 10085/TBCRC/BIG/NABCG International Male Breast Cancer Program.

Background: Male breast cancer (BC) is rare, managed by extrapolation from female BC. The International Male BC Program aims to better characterize and manage this disease. We report the results of part I, a retrospective joint analysis of cases diagnosed during a 20-year period. Methods: Patients with follow-up and tumor samples, treated between 1990 and 2010, in 93 centers/9 countries. Samples were centrally analyzed in three laboratories (the United Kingdom, the Netherlands and the United States). Results: Of 1822 patients enrolled, 1483 were analyzed; 63.5% were diagnosed between 2001 and 2010, 57 (5.1%) had metastatic disease (M1). Median age at diagnosis: 68.4 years. Of 1054 M0 cases, 56.2% were node-negative (N0) and 48.5% had T1 tumors; 4% had breast conserving surgery (BCS), 18% sentinel lymph-node biopsy; half received adjuvant radiotherapy; 29.8% (neo)adjuvant chemotherapy and 76.8% adjuvant endocrine therapy (ET), mostly tamoxifen (88.4%). Per central pathology, for M0 tumors: 84.8% ductal invasive carcinomas, 51.5% grade 2; 99.3% estrogen receptor (ER)-positive; 81.9% progesterone receptor (PR)-positive; 96.9% androgen receptor (AR)-positive [ER, PR or AR Allred score ≥3]; 61.1% Ki67 expression low (<14% positive cells); using immunohistochemistry (IHC) surrogates, 41.9% were Luminal-A-like, 48.6% Luminal-B-like/HER-2-negative, 8.7% HER-2-positive, 0.3% triple negative. Median follow-up: 8.2 years (0.0-23.8) for all, 7.2 years (0.0-23.2), for M0, 2.6 years (0.0-12.7) for M1 patients. A significant improvement over time was observed in age-corrected BC mortality. BC-specific-mortality was higher for men younger than 50 years. Better overall (OS) and recurrence-free survival (RFS) were observed for highly ER+ (P = 0.001), highly PR+ (P = 0.002), highly AR+ disease (P = 0.019). There was no association between OS/RFS and HER-2 status, Ki67, IHC subtypes nor grade. Conclusions: Male BC is usually ER, PR and AR-positive, Luminal B-like/HER2-negative. Of note, 56% patients had T1 tumors but only 4% had BCS. ER was highly positive in >90% of cases but only 77% received adjuvant ET. ER, PR and AR were associated with OS and RFS, whereas grade, Ki67 and IHC surrogates were not. Significant improvement in survival over time was observed.

Integrated cytogenetic and high-resolution array CGH analysis of genomic alterations associated with MYCN amplification.

Amplification of oncogenes and closely linked flanking genes is common in some types of cancer and can be associated with complex chromosome rearrangements and/or co-amplification of non-syntenic chromosomal regions. To better understand the etiology and structural complexity of focal MYCN amplicons in human neuronal cancer, we investigated the precise chromosomal locations of high copy number genomic regions in MYCN amplified cell lines. An integrated cytogenetic map of the MYCN amplicon was created using high-resolution array CGH, spectral karyotyping (SKY), multi-color banding (mBAND), and fluorescence in situ hybridization (FISH) in 4 human neuronal tumor cell lines. The evidence of complex intra- and inter-chromosomal events, providing clues concerning the nature of the genomic mechanisms that contributed to the process of MYCN amplification, was observed. The presence of multiple co-amplified syntenic or non-syntenic sequences in the MYCN amplicon is quite intriguing. MYCN is usually centrally located in the amplicon; however, the structure and complexity of the amplicons were highly variable. It is noteworthy that clusters of unstable repetitive regions characterized by CNV sequences were present throughout the regions encompassed by MYCN gene amplification, and these sequences could provide a mechanism to destabilize this region of the genome. Complex structural rearrangements involving genomic losses and gains in the 2p24 region lead to MYCN amplification and that these rearrangements can trigger amplification events.

Genomic signatures of chromosomal instability and osteosarcoma progression detected by high resolution array CGH and interphase FISH.

Osteosarcoma (OS) is characterized by an unstable karyotype which typically has a heterogeneous pattern of complex chromosomal abnormalities. High-resolution array comparative genomic hybridization (CGH) in combination with interphase fluorescence in situ hybridization (FISH) analyses provides a complete description of genomic imbalances together with an evaluation of the contribution of cell-to-cell variation to copy number changes. There have been no analyses to date documenting genomic signatures consistent with chromosomal instability mechanisms in OS tumors using array CGH. In this study, we utilized high-resolution array CGH to identify and characterize recurrent signatures of genomic imbalances using ten OS tumors. Comparison between the genomic profiles identified tumor groups with low, intermediate and high levels of genomic imbalance. Bands 6p22-->p21, 8q24 and 17p12--> p11.2 were consistently involved in high copy gain or amplification events. Since these three locations have been consistently associated with OS oncogenesis, FISH probes from each cytoband were used to derive an index of cellular heterogeneity for copy number within each region. OS with the highest degree of genomic imbalance also exhibited the most extreme cell-to-cell copy number variation. Significantly, the three OS with the most imbalance and genomic copy number heterogeneity also had the poorest response to preoperative chemotherapy. This genome wide analysis is the first utilizing oligonucleotide array CGH in combination with FISH analysis to derive genomic signatures of chromosomal instability in OS tumors by studying genomic imbalance and intercellular heterogeneity. This comprehensive genomic screening approach provides important insights concerning the mechanisms responsible for generating complex genomes. The resulting phenotypic diversity can generate tumors with a propensity for an aggressive disease course. A better understanding of the underlying mechanisms leading to OS tumor development could result in the identification of prognostic markers and therapeutic targets.

Correlating breakage-fusion-bridge events with the overall chromosomal instability and in vitro karyotype evolution in prostate cancer.

Chromosomal instability (CIN) is thought to underlie the generation of chromosomal changes and genomic heterogeneity during prostatic tumorigenesis. The breakage-fusion-bridge (BFB) cycle is one of the CIN mechanisms responsible for characteristic mitotic abnormalities and the occurrence of specific classes of genomic rearrangements. However, there is little detailed information concerning the role of BFB and CIN in generating genomic diversity in prostate cancer. In this study we have used molecular cytogenetic methods and array comparative genomic hybridization analysis (aCGH) of DU145, PC3, LNCaP, 1532T and 1542T to investigate the in vitro role of BFB as a CIN mechanism in karyotype evolution. Analysis of mitotic structures in all five prostate cancer cell lines showed increased frequency of anaphase bridges and nuclear strings. Structurally rearranged dicentric chromosomes were observed in all of the investigated cell lines, and Spectral Karyotyping (SKY) analysis was used to identify the participating rearranged chromosomes. Multicolor banding (mBAND) and aCGH analysis of some of the more complex chromosomal rearrangements and associated amplicons identified inverted duplications, most frequently involving chromosome 8. Chromosomal breakpoint analysis showed there was a higher frequency of rearrangement at centromeric and pericentromeric genomic regions. The distribution of inverted duplications and ladder-like amplifications was mapped by mBAND and by aCGH. Adjacent spacing of focal amplifications and microdeletions were observed, and focal amplification of centromeric and end sequences was present, particularly in the most unstable line DU145. SKY analysis of this line identified chromosome segments fusing with multiple recipient chromosomes (jumping translocations) identifying potential dicentric sources. Telomere free end analysis indicated loss of DNA sequence. Moreover, the cell lines with the shortest telomeres had the most complex karyotypes, suggesting that despite the expression of telomerase, the reduced telomere length could be driving the observed BFB events and elevated levels of CIN in these lines.

Application of comparative genomic hybridization, spectral karyotyping, and microarray analysis in the identification of subtype-specific patterns of genomic changes in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) in children occurs predominantly as two major histologically defined subtypes called embryonal RMS (RMS-E) and the prognostically less favorable alveolar RMS (RMS-A). Comparative genomic hybridization (CGH) was performed on 21 RMS and identified consistent gains affecting chromosomes 2 (8/10), 5 (5/10), 6 (3/10), 7 (7/10), 8 (9/10), 11 (6/ 10), and 12 (5/10) in RMS-E. Losses/deletions involved chromosomes 19 (2/10) and chromosomes 4, 9, 10, 17, 21 (1/10 each). High copy number amplification, involving the 2p24 region (5/11) and less frequently, the 12q13-21 (2/11), 9p22 (1/11), and 17q22-25 (1/11) regions, was detected in RMS-A. Gene amplification at band 2p24 was present in 6/12 alveolar tumors, and in each case, MYCN was amplified, together with the distally placed DDX1 gene. For these patients there was a shorter disease free interval and a higher mortality than patients with tumors without amplification. Detailed spectral karyotype analysis (SKY) was performed on two RMS cell lines (one of each subtype) and identified a surprisingly high level of structural change. Gene expression studies with the Atlas Human Cancer Array (588 genes) showed that 153 genes generated a signal of similar intensity in both cell lines, and 45 genes appeared to have subtype-specific expression. The chromosomal location of differentially expressed genes was compared to the pattern of genomic alteration in RMS as determined by CGH in this study and the literature.

Use of whole genome amplification and comparative genomic hybridisation to detect chromosomal copy number alterations in cell line material and tumour tissue.

We have established that whole genome amplification (WGA), in conjunction with genomic DNA array comparative genomic hybridisation (gaCGH) allows for the identification of genome-wide copy number abnormalities (CNAs) in DNA extracted from both cell line and patient material. To determine the fidelity and reproducibility of WGA to detect copy number imbalances using gaCGH, well characterized cell line genomic DNA was analysed. The gaCGH data obtained from non-amplified DNA and amplified DNA for the neuroblastoma cell line NUB7 and a paediatric medulloblastoma patient was almost identical. In addition, laser capture microdissection (LCM) of prostate tumour cells and subsequent WGA allowed for the detection of a number of CNAs that may not have been identified if DNA had been extracted in bulk from heterogeneous tissue. The results presented here demonstrate the use of WGA for generating sufficient DNA for gaCGH analysis without the introduction of significant sequence representation bias. The combination of amplification and gaCGH using DNA extracted from archival patient material has the potential for permitting the studying of DNA from small cancerous or pre-cancerous foci, which may help to identify potential genomic markers for early diagnosis.

High-resolution cDNA microarray CGH mapping of genomic imbalances in osteosarcoma using formalin-fixed paraffin-embedded tissue.

Formalin-fixed paraffin embedded (FFPE) tumor tissue provides an opportunity to perform retrospective genomic studies of tumors in which chromosomal imbalances are strongly associated with oncogenesis. The application of comparative genomic hybridization (CGH) has led to the rapid accumulation of cytogenetic information on osteosarcoma (OS); however, the limited resolving power of metaphase CGH does not permit precise mapping of imbalances. Array CGH allows quantitative detection and more precise delineation of copy number aberrations in tumors. Unfortunately the high cost and lower density of BACs on available commercial arrays has limited the ability to comprehensively profile copy number changes in tumors such as OS that are recurrently subject to genomic imbalance. In this study a cDNA/EST microarray including 18,980 human cDNAs (which represent all 22 pairs of autosomal chromosomes and chromosome X) was used for CGH analysis of eight OS FFPE. Chromosomes 1, 12, 17, and X harbored the most imbalances. Gain/amplification of X was observed in 4/8 OS, and in keeping with other recent genomic analyses of OS, gain/amplification of 17p11.2 was often accompanied by a distal deletion in the region of the p53 gene. Gain/amplification of the X chromosome was verified using interphase FISH carried out on a subset of OS FFPE sections and OS tissue arrays.

Recurrent anomalies of 6q25 in chondromyxoid fibroma.

Chondromyxoid fibroma is a rare benign bone tumor most commonly arising in the metaphysis of long bones in young adults. Histopathologically, chondromyxoid fibroma may be difficult to distinguish from other cartilaginous neoplasms. Recently, a pericentric inversion of chromosome 6 [inv(6)(p25q13)] has been proposed as a specific genetic marker for chondromyxoid fibroma. In this study, cytogenetic and spectral karyotypic analyses of 2 chondromyxoid fibroma cases showed clonal abnormalities of chromosome 6 but at a breakpoint on the long arm (q25) distal to that described in the pericentric inversion. These findings suggest that several distinct breakpoints on chromosome 6 are nonrandomly involved in chondromyxoid fibroma.

Molecular cytogenetic analysis of non-small cell lung carcinoma by spectral karyotyping and comparative genomic hybridization.

The overall pattern of chromosomal changes detected by spectral karyotype (SKY) analysis of two cell lines of each major histological subtype of NSCLC, namely squamous cell carcinoma (SQCC) and adenocarcinoma (ADC), indicated a greater degree of chromosomal rearrangement, than was present or predicted by either comparative genomic hybridization (CGH) or G-banding analysis alone. To investigate these observations, CGH was used to screen DNA derived from 8 primary tumors and 15 cell lines. The results indicated that the most frequently gained chromosome arms were 5p (70%), 8q (65%), 15q (52%), 20q (48%), 1q (43%), 19q (39%), 3q (35%), and 11q (35%). Chromosomal losses were less frequently observed, and included 18q (39%), 9 (35%), 6q (30%), 13q (21%), 5q12-q32 (17%), and 19p (17%). Amplifications were found on 2p23-p24, 3q24-q27, 5p, 6cen-p21.1, 6q26, 7p21, 7q31, 8q, 11q13-qter, 20q12-q13.2. Comparison between CGH findings of the two major histological subtypes showed that gains at 1q22-q32.2, 15q, 20q, and losses at 6q, 13q, and 18q was common in ADCs, whereas SQCCs exhibited gains/amplifications at 3q. Distal 8q was gained by CGH in 65% of tumors of both subtypes. Low level MYCC amplification was confirmed by direct fluorescence in situ hybridization (FISH) analysis. The pattern of overall chromosomal changes detected using combinations of molecular cytogenetic analytical methods suggests that it will be easier to detect recurrent subtype-dependent aberrations in NSCLC.

Chromosomal instability in osteosarcoma and its association with centrosome abnormalities.

The mechanism that generates the extreme aneuploidy that characterizes osteosarcoma (OS) is poorly understood. In this study, interphase fluorescence in situ hybridization (FISH) analysis was used to enumerate cell-to-cell variation of several different chromosomes. We also investigated whether there was an association between TP53 mutation and centrosome aberrations in the generation of chromosomal aneuploidy in OS in four OS cell lines (HOS, SAOS2, U2OS, and MG63) and in a subset of seven tumors. Our analysis showed that there was a wide range of numerical changes affecting multiple chromosomes in OS cell lines and tumors. These data suggest that chromosomal instability (CIN) could be responsible for the extensive aneuploidy associated with this tumor. The results also showed an increased frequency of atypical mitotic figures in three OS cell lines with defective TP53, function and significantly, a more marked CIN phenotype was present in these lines. Furthermore, numerical aberrations of centrosomes were also present in these three OS cell lines with TP53 mutations. In two of three OS patients' tumors there was a large increase in the percentage of abnormal centrosome numbers. We conclude that CIN is a consistent feature of OS and that an intrinsic disturbance of the chromosomal segregation mechanisms is likely associated with centrosome aberrations.

Comparative genomic hybridization analysis identifies gains of 1p35 approximately p36 and chromosome 19 in osteosarcoma.

Osteosarcomas (OS) are aggressive tumors of the bone and often have a poor prognosis. Conventional cytogenetic analyses of OS have revealed highly complex karyotypes, with numerous abnormalities. In this study, we analyzed 18 untreated OS tumors from 17 patients of the younger incidence age group by comparative genomic hybridization (CGH), 4 tumors by spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH). Comparative genomic hybridization identified frequent copy number changes of the chromosomal region 1p (10/17) and gain of part or all of chromosome 19(8/17). In addition gains were observed at 5p(3/17), 8q(3/17), 16p(3/17), and 17p(5/17); and losses at chromosomes 2q(3/17), 10(4/17) and 13(3/17). High level gains were detected in the 8q23 approximately q24 region in two tumors as well as at 17p in one primary and a metastatic tumor. Minimal regions of gain were present at 1p35 approximately p36.3 (8/17); 5p14 approximately p15.2 (3/17), and 8q22 approximately q24.3 (3/17). SKY analysis demonstrated that OS has a complex pattern of clonal and non-clonal rearrangements and helped confirm the structural basis for the imbalances detected by CGH. Spectral karyotyping confirmed an overall pattern of chromosomal gain affecting 1p in all four tumors. Fluorescence in situ hybridization analysis from these tumors confirmed the gain of the 1p36 region in 2 tumors as determined by CGH analysis as well as the amplification of 8q.

Molecular cytogenetic analysis of medulloblastomas and supratentorial primitive neuroectodermal tumors by using conventional banding, comparative genomic hybridization, and spectral karyotyping.

OBJECT: Medulloblastomas and related primitive neuroectodermal tumors (PNETs) of the central nervous system are malignant, invasive embryonal tumors with predominantly neuronal differentiation that comprise 20% of pediatric brain tumors. Cytogenetic analysis has shown that alterations in chromosome 17, particularly the loss of 17p and the formation of isochromosome 17q, as well as the gain of chromosome 7 are the most common changes among this group of tumors. Comparative genomic hybridization (CGH) studies have largely confirmed these cytogenetic findings and have also identified novel regions of gain, loss, and amplification. The advent of more sophisticated multicolored fluorescence in situ hybridization (FISH) procedures such as spectral karyotyping (SKY) now permits complete recognition of all aberrations including extremely complex rearrangements. The authors report a retrospective analysis of 19 medulloblastoma and five PNET cases studied using combinations of classic banding analysis, FISH, CGH, and SKY to examine comprehensively the chromosomal aberrations present in this tumor group and to attempt to identify common structural rearrangement(s). METHODS: The CGH data demonstrate gains of chromosomes 17q and 7 in 60% of the tumors studied, which confirms data reported in the current literature. However, the authors have also combined the results of all three molecular cytogenetic assays (Giemsa banding, CGH, and SKY) to reveal the frequency of chromosomal rearrangement (gained, lost, or involved in structural rearrangement). CONCLUSIONS: The combined results indicate that chromosomes 7 and 17 are the most frequently rearranged chromosomes (10.1% and 8.9%, respectively, in all rearrangements detected). Furthermore, chromosomes 3 (7.8%), 14 (7%), 10 (6.7%), and 22 (6.5%) were also found to be frequently rearranged, followed by chromosomes 6 (6.5%), 13 (6.2%), and 18 (6.2%). Eight (33%) of 24 tumors exhibited high-level gains or gene amplification. Amplification of MYCN was identified in four tumors, whereas amplification of MYCC was identified in one tumor. One tumor exhibited a high-level gain of chromosome 9p. Additionally, desmoplastic medulloblastomas and large-cell medulloblastomas exhibited higher karyotype heterogeneity, amplification, and aneusomy than classic medulloblastomas.

Molecular cytogenetic analysis of glial tumors using spectral karyotyping and comparative genomic hybridization.

BACKGROUND: Glial tumors are the most common tumors of the central nervous system, affecting individuals of all ages. Conventional cytogenetics have been unable to identify a consistent chromosomal translocation or rearrangement in this group of tumors; thus, more advanced molecular cytogenetic approaches are required. METHODS AND RESULTS: In this study, 16 glial tumors, including two recurrences and six glioma cell lines, were analyzed by spectral karyotyping (SKY) and comparative genomic hybridization (CGH). From 169 rearrangements detected by SKY, chromosomes 1 and 10 were the most frequently affected by translocation (18 of 169 and 16 of 169 rearrangements, respectively). Other frequently altered chromosomes included chromosomes 3 (13 of 169 rearrangements), 5 (ten of 169 rearrangements), 7 (ten of 169 rearrangements ), and 11 (ten of 169 rearrangements). A clustering of centromeric breakpoints was detected in chromosomes 3, 5, 10, 11, 16, 17, and 20. CGH analysis identified consistent gain of part or all of chromosome 7 among the 10 astrocytic tumors (five of ten specimens) in the study group. Analysis of the three gangliogliomas and one ependymoma identified a much simpler pattern of primarily numerical change. CONCLUSION: Application of improved cytogenetic methods can increase our abilities to progress toward effective strategies of molecular diagnosis and classification of glial tumors.

Oncogene amplification in medulloblastoma: analysis of a case by comparative genomic hybridization and fluorescence in situ hybridization.

We describe amplification of the MYCC oncogene in a medulloblastoma with aggressive clinical behavior. The patient was a six year old boy who underwent gross total surgical excision of a cerebellar tumor. Despite chemotherapy and total neuraxis radiation, the clinical course was one of relentless progression, with extensive subarachnoid spread and death within eight months of presentation. The pathological features were consistent with the recently described, "large cell variant" of medulloblastoma. Tumor cells exhibited large vesicular nuclei, prominent nucleoli and strong immunoreactivity for synaptophysin. Polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) assay revealed no evidence of MYCN amplification or 1p deletion in the tumor. FISH analysis revealed evidence of MYCC amplification in the 20- to 30-fold range. Comparative genomic hybridization (CGH) revealed regions of gains and amplification in three locations, with gains of chromosome 7, amplification of 8q24 (corresponding to the MYCC locus) and gains of the long arm of chromosome 17 (suggestive of isochromosome 17q). While conventional karyotypic analysis was not successful in the present case, CGH provided invaluable information about gene amplification and losses/gains of chromosomes and chromosomal regions. Thus, CGH is a powerful technique applicable to frozen or paraffin-embedded material which helps to ascertain the presence of gene amplification even without prior knowledge of the gene to be tested.

Clinical utility of kallikrein-related peptidases (KLK) in urogenital malignancies.

Kallikrein-related peptidases (KLK), which represent a major tissue-associated proteolytic system, stand for a rich source of biomarkers that may allow molecular classification, early diagnosis and prognosis of human malignancies as well as prediction of response or failure to cancer-directed drugs. International research points to an important role of certain KLKs in female and male urogenital tract malignancies, in addition to cancers of the lung, brain, skin, head and neck, and the gastrointestinal tract. Regarding the female/male urogenital tract, remarkably, all of the KLKs are expressed in the normal prostate, testis, and kidney whereas the uterus, the ovary, and the urinary bladder are expressing a limited number of KLKs only. Most of the information regarding KLK expression in tumour-affected organs is available for ovarian cancer; all of the 12 KLKs tested so far were found to be elevated in the malignant state, depicting them as valuable biomarkers to distinguish between the normal and the cancerous phenotype. In contrast, for kidney cancer, a series of KLKs was found to be downregulated, while other KLKs were not expressed. Evidently, depending on the type of cancer or cancer stage, individual KLKs may show characteristics of a Janus-faced behaviour, by either expanding or inhibiting cancer progression and metastasis.

Expression of TEL-JAK2 in primary human hematopoietic cells drives erythropoietin-independent erythropoiesis and induces myelofibrosis in vivo.

Activation of JAK2 by chromosomal translocation or point mutation is a recurrent event in hematopoietic malignancies, including acute leukemias and myeloproliferative disorders. Although the effects of activated JAK2 signaling have been examined in cell lines and murine models, the functional consequences of deregulated JAK2 in the context of human hematopoietic cells are currently unknown. Here we report that expression of TEL-JAK2, a constitutively active variant of the JAK2 kinase, in lineage-depleted human umbilical cord blood cells results in erythropoietin-independent erythroid differentiation in vitro and induces the rapid development of myelofibrosis in an in vivo NOD/SCID xenotransplantation assay. These studies provide functional evidence that activated JAK2 signaling in primitive human hematopoietic cells is sufficient to drive key processes implicated in the pathophysiology of polycythemia vera and idiopathic myelofibrosis. Furthermore, they describe an in vivo model of myelofibrosis initiated with primary cells, highlighting the utility of the NOD/SCID xenotransplant system for the development of experimental models of human hematopoietic malignancies.

Advances in the detection of chromosomal aberrations using spectral karyotyping.

Spectral karyotyping (SKY) is a powerful 24-color, whole chromosome-painting assay allowing the visualization of each chromosome in one experiment. Subtle karyotype rearrangements can be detected easily so that small translocations lead to a transition from one color to another at the chromosomal breakpoint region. SKY has enabled the elucidation of several examples of hidden or "cryptic" structural aberrations that may otherwise have been left undetected by classical cytogenetic methods. Furthermore, the chromosomal origins of abnormalities once designated "marker chromosomes" can now be determined rather than left unidentified. SKY analysis of cancer cytogenetics samples provides a much more detailed description of the highly abnormal karyotypes that characterize advanced tumors and cancer cell lines. In addition, SKY significantly adds to the power of clinical cytogenetic analysis of constitutional chromosomal aberrations by facilitating the identification of subtle structural rearrangements that may contain aneuploidy with potential pathological consequences.

Use of multicolor spectral karyotyping in genetic analysis of pleuropulmonary blastoma.

Pleuropulmonary blastoma (PPB) is a rare, malignant intrathoracic pediatric tumor. It arises from the lung, pleura, or mediastinum and its pathogenesis and relationship to other pediatric solid tumors is not well understood. In this study, a case of PPB in a 3-year-old girl was studied using a combination of molecular genetic methods and cytogenetics. Molecular analysis of the commonly encountered fusion translocation gene products of pediatric solid tumors failed to detect a rearrangement. Cytogenetic analysis, supplemented by multicolor spectral karyotyping (SKY), identified an unbalanced translocation between chromosomes 1 and X, resulting in additional copies of 1q, an extra copy of Xq, and loss of part of Xp. In addition, trisomy 8 was detected. The identification of new chromosomal alterations and confirmation of previously reported ones in this rare neoplasm helps to improve our understanding of its pathogenesis and association with other pediatric tumors.