Targeted genomic analysis of Müllerian adenosarcoma.

Müllerian adenosarcoma (MA) is a rare mixed mesenchymal tumour of the female genital tract, composed of malignant stroma and benign-appearing epithelium. Sarcomatous overgrowth (SO) is the only established histological variable associated with higher stage and shorter survival. Specific molecular or immunohistochemistry (IHC) tools for the diagnosis of MA are lacking. Our goal was to study genomic mutations and copy number variations (CNVs) in MA to understand better its pathobiology, and develop specific diagnostic and prognostic tools. DNA was extracted from 20 samples of MA from 18 subjects (12 without SO and 6 with SO), including two in which areas of both typical MA histology and SO were independently tested. Samples were analysed using a targeted next-generation sequencing assay interrogating exonic sequences of 275 cancer genes for mutations and CNVs as well as 91 introns across 30 genes for cancer-associated rearrangements. The mean number of mutations in MA with SO (mean 9.7; range 3-14) did not differ significantly from that in MA without SO (mean 9.6; range 5-16). MA with SO had significantly higher mean numbers of gene-level CNVs (24.6) compared to MA without SO (5; p = 0.0002). The most frequent amplification involved MDM2 and CDK4 (5/18; 28%), accompanied by focal CDK4 and MDM2 and diffuse HMGA2 expression using immunohistochemistry. MYBL1 amplification was seen in 4/18 (22%), predominantly in SO. Alterations in PIK3CA/AKT/PTEN pathway members were seen in 13/18 (72%). Notably, TP53 mutations were uncommon, present in only two cases with SO. Three out of 18 (17%) had mutations in ATRX, all associated with SO. No chromosomal rearrangements were identified. We have identified a number of recurrent genomic alterations in MA, including some associated with SO. Although further investigation of these findings is needed, confirmation of one or more may lead to new mechanistic insights and novel markers for this often difficult-to-diagnose tumour.

Integrative analysis reveals 53BP1 copy loss and decreased expression in a subset of human diffuse large B-cell lymphomas.

p53-Binding protein 1 (53BP1) encodes a critical checkpoint protein that localizes to sites of DNA double-strand breaks (DSBs) and participates in DSB repair. Mice that are 53bp1 deficient or hemizygous have an increased incidence of lymphoid malignancies. However, 53BP1 abnormalities in primary human tumors have not been described. By combining high-density single nucleotide polymorphism (HD SNP) array data and gene expression profiles, we found 9 of 63 newly diagnosed human diffuse large B-cell lymphomas (DLBCLs) with single copy loss of the chromosome 15q15 region including the 53BP1 locus; these nine tumors also had significantly lower levels of 53BP1 transcripts. 53BP1 single copy loss found with the HD SNP array platform was subsequently confirmed by fluorescence in situ hybridization. These studies highlight the role of 53BP1 copy loss in primary human DLBCLs and the value of integrative analyses in detecting this genetic lesion in human tumors.

ALK expression in pseudosarcomatous myofibroblastic proliferations of the genitourinary tract.

AIMS: Pseudosarcomatous myofibroblastic proliferation of the genitourinary tract is rare and may develop after trauma or spontaneously. The aim of this study was to characterize further the clinicopathological features of these lesions and to examine their relationship to inflammatory myofibroblastic tumour (IMT). METHODS AND RESULTS: Twenty-seven cases of pseudosarcomatous myofibroblastic proliferation were analysed. There were seven males and 20 females; median age was 37 years (range 16-88). Most lesions were from the bladder (n = 21), while others were in the urethra, vulva, vagina, rectum and retrovesical space. Median tumour size was 30 mm (range 6-120 mm). Seven cases (25%) had a history of prior trauma or surgery. Three cases recurred locally but not destructively. The tumours had fasciitis-like features including bland spindle cells with evenly distributed chromatin, admixed inflammatory cells (mainly lymphocytes) and often a myxoid stroma. Immunohistochemistry showed positivity for smooth muscle actin in 14/20 cases, keratin in 8/19, desmin in 7/20 and anaplastic lymphoma kinase (ALK) in 10/21 cases. Fluorescent in situ hybridization was performed in six ALK+ cases; all were negative for ALK gene rearrangement. CONCLUSIONS: Pseudosarcomatous myofibroblastic proliferations of the genitourinary tract may show ALK immunopositivity but do not show consistent ALK rearrangement. Given subtle morphological differences and more consistently benign behaviour, their relationship to inflammatory myofibroblastic tumour at other sites remains uncertain.

Molecular cytogenetic mapping of recurrent chromosomal breakpoints in tenosynovial giant cell tumors.

Tenosynovial giant cell tumor (TGCT) is the most common benign tumor of synovium and tendon sheath. Cytogenetic data indicate that 1p11-13 is the region most frequently involved in structural rearrangements. With the aim of eventually identifying the genes associated with TGCT development, we have investigated 1p11-13 breakpoints using fluorescence in situ hybridization (FISH) analysis, with a panel of yeast artificial chromosome (YAC) probes covering 1p11-21. Twenty-six tumors were analyzed by G-banding, and 24 of these showed a breakpoint in 1p11-13. The cytogenetic findings add to previous observations that, among a variety of translocations involving 1p11-13, chromosome 2 is the most common translocation partner, with a breakpoint in 2q35-37. This aberration was found in eight cases. Other recurrent translocation partners, found in two or three cases, were 5q22-31, 11q11-12, and 8q21-22. Material from 21 tumors was available for FISH analysis, which revealed that the breakpoints clustered to one region spanned by two YAC probes, 914F6 and 885F12 located in 1p13.2, in 18 cases. Bacterial artificial chromosome probes were used to map the recurrent breakpoint on chromosome 2. In four of seven cases there was a breakpoint within the sequence covered by probe 260J21, where the RDC1 gene is located, a gene reported to fuse with HMGIC in lipomas with a 2;12 translocation.

Intravenous leiomyomatosis: molecular and cytogenetic analysis of a case.

Apart from its hormone responsiveness, little about the pathobiology of intravenous leiomyomatosis (IVL), a rare smooth muscle proliferation, is known. We investigated the cytogenetics and molecular biology of IVL in a 40-year-old female who presented with an abrupt onset of dyspnea. In addition to the intracaval tumor mass composed of histologically benign smooth muscle, four distinct retroperitoneal "fibroids" were cytogenetically investigated. An identical abnormal karyotype, 45,XX,der(14)t(12; 14)(q15;q24),-22, was observed in all five specimens. Fluorescence in situ hybridization revealed three copies of HMGIC (alias HMGA2), two on the normal chromosomes 12 at 12q15, as well as another on the der(14) in the breakpoint region, suggesting that the 12q breakpoint occurred 5' (centromeric) to HMGIC (HMGA2), as has been frequently observed in uterine leiomyoma. Such similarity in chromosomal rearrangements suggests that there may be a pathogenetic relationship between IVL and uterine leiomyomata with t(12;14). Skewed X inactivation was observed in each tumor sample, but not in the myometrium. In each tumor, the lower molecular weight allele of HUMARA was nonrandomly inactivated. This pattern of X inactivation is most consistent with origin from a single transformation event, and in this regard, IVL more closely resembles disseminated peritoneal leiomyomatosis than typical uterine leiomyomata.

BRD4 bromodomain gene rearrangement in aggressive carcinoma with translocation t(15;19).

Translocation t(15;19)(q13;p13.1) defines a lethal midline carcinoma arising adjacent to respiratory tract in young people. To characterize molecular alterations responsible for the distinctly aggressive biological behavior of this cancer, we mapped the chromosome 15 and 19 translocation breakpoints by fluorescence in situ hybridization (FISH) and Southern blotting. To evaluate preliminarily the frequency, anatomical distribution, and histological features of t(15;19) cancer, we developed a FISH assay for paraffin sections. Our findings reveal a novel oncogenic mechanism in which the chromosome 19 translocation breakpoint interrupts the coding sequence of a bromodomain gene, BRD4. These studies implicate BRD4 as a potential partner in a t(15;19)-associated fusion oncogene. In addition, we localized the chromosome 15 breakpoint to a 9-kb region in each of two cases, thereby identifying several candidate oncogenes which might represent the BRD4 fusion partner. FISH evaluation of 13 pediatric carcinomas revealed t(15;19) in one of four sinonasal carcinomas, whereas this translocation was not detected in thymic (n = 3), mucoepidermoid (n = 3), laryngeal (n = 2), or nasopharyngeal (n = 1) carcinomas. Our studies shed light on the oncogenic mechanism underlying t(15;19) and provide further evidence that this highly lethal cancer arises from respiratory mucosa.

ETV6 rearrangements in patients with infantile fibrosarcomas and congenital mesoblastic nephromas by fluorescence in situ hybridization.

Congenital mesoblastic nephroma (CMN) and infantile fibrosarcoma (IFS) are two pediatric tumors arising in the kidneys and soft tissues of infants, respectively. Recently, a t(12;15)(p13;q25) resulting in ETV6-NTRK3 gene fusion was detected in patients with IFS and in patients with the cellular type of CMN, suggesting a common pathogenetic pathway. We investigated the presence or absence of ETV6 rearrangements and numerical abnormalities of chromosome 11 by using fluorescence in situ hybridization on paraffin-embedded material from five cases of IFS, two of CMN, and one of mixed type (CMN and IFS) found in our files. In three cases of IFS, we found ETV6 gene rearrangement but a normal copy number of chromosome 11. One case each of IFS, the cellular type of CMN, and the mixed type (CMN and IFS) had both abnormalities. In a case of classic CMN, neither trisomy 11 nor gene rearrangement was found. It is possible that trisomy 11 is a later, nonessential event in the pathogenetic process or that this secondary aberration is associated with still-unrecognized clinical or biological characteristics. We confirmed that IFS and the cellular type of CMN are cytogenetically related and can occur synchronously in the same organ.

Genetics of epithelial tumors of the renal parenchyma in adults and renal cell carcinoma in children.

The classification of renal cell carcinomas has been affected by both the delineation of new histologic subtypes and the understanding that recognized histomorphologic patterns are reflective of specific sets of cytogenetic abnormalities. In fact, standard methods of clinicopathologic study and cytogenetic analysis have been cooperatively contributory in the evolution of current concepts regarding the biologic nature and classification of renal parenchymal epithelial tumors. In this review, the current classification scheme for these tumors is discussed from the perspective of both the defining histologic and cytogenetic features. Limited molecular data are described as well.

Telomere dysfunction triggers extensive DNA fragmentation and evolution of complex chromosome abnormalities in human malignant tumors.

Although mechanisms for chromosomal instability in tumors have been described in animal and in vitro models, little is known about these processes in man. To explore cytogenetic evolution in human tumors, chromosomal breakpoint profiles were constructed for 102 pancreatic carcinomas and 140 osteosarcomas, two tumor types characterized by extensive genomic instability. Cases with few chromosomal alterations showed a preferential clustering of breakpoints to the terminal bands, whereas tumors with many changes showed primarily interstitial and centromeric breakpoints. The terminal breakpoint frequency was negatively correlated to telomeric TTAGGG repeat length, and fluorescence in situ hybridization with telomeric TTAGGG probes consistently indicated shortened telomeres and >10% of chromosome ends lacking telomeric signals. Because telomeric dysfunction may lead to formation of unstable ring and dicentric chromosomes, mitotic figures were also evaluated. Anaphase bridges were found in all cases, and fluorescence in situ hybridization demonstrated extensive structural rearrangements of chromosomes, with terminal transferase detection showing fragmented DNA in 5-20% of interphase cells. Less than 2% of cells showed evidence of necrosis or apoptosis, and telomerase was expressed in the majority of cases. Telomeric dysfunction may thus trigger chromosomal fragmentation through persistent bridge-breakage events in pancreatic carcinomas and osteosarcomas, leading to a continuous reorganization of the tumor genome. Telomerase expression is not sufficient for completely stabilizing the chromosome complement but may be crucial for preventing complete genomic deterioration and maintaining cellular survival.

PLAG1 alterations in lipoblastoma: involvement in varied mesenchymal cell types and evidence for alternative oncogenic mechanisms.

Lipoblastomas are rare soft tissue tumors that occur primarily in young children. They typically contain variably differentiated adipocytes, primitive mesenchymal cells, myxoid matrix, and fibrous trabeculae. Abnormalities in chromosome 8, leading to rearrangements of the PLAG1 gene, were demonstrated recently in four lipoblastomas. In the present report, we determine the frequency of PLAG1 alterations in 16 lipoblastomas from children aged 13 years or younger, and we also evaluate the stages of lipoblastoma differentiation at which PLAG1 genomic alterations are found. Eleven lipoblastomas (69%), including those with either classic or lipoma-like histology, had rearrangements of the 8q12 PLAG1 region. Another three lipoblastomas had polysomy for chromosome 8 in the absence of PLAG1 rearrangement. Only two cases (13%) lacked a chromosome 8 abnormality. Notably, the lipoblastomas with chromosome 8 polysomy had up to five copies of chromosome 8 as an isolated cytogenetic finding in an otherwise diploid cell. We also demonstrate that PLAG1 alterations are found in a spectrum of mesenchymal cell types in lipoblastomas, including lipoblasts, mature adipocytes, primitive mesenchymal cells, and fibroblast-like cells. This finding is consistent with neoplastic origin in a primitive mesenchymal precursor and with variable differentiation to a mature adipocyte end-point. Hence, our studies provide biological validation for the clinical observation that lipoblastomas can evolve into mature, lipoma-like, lesions. They also suggest that PLAG1 dosage alterations caused by polysomy 8 might represent an alternative oncogenic mechanism in lipoblastoma.

Upper respiratory tract carcinoma with chromosomal translocation 15;19: evidence for a distinct disease entity of young patients with a rapidly fatal course.

BACKGROUND: Carcinoma of the upper respiratory tract is rare in childhood, and cytogenetic aberrations have not been characterized in this population. The chromosomal translocation 15;19 has been reported four times previously. All patients were young and had tumors arising in the thorax. The three reports that provide clinical follow-up all describe superior vena cava syndrome and death soon after presentation. All tumors were diagnosed as carcinoma (three undifferentiated, one mucoepidermoid), and the authors suggested thymus, lung, or germ cell origin. METHODS: The authors investigated the clinical and pathologic findings in two patients with poorly differentiated carcinoma showing evidence of t(15;19). This included a 13-year-old girl with a rapidly growing epiglottic mass, leading to superior vena cava syndrome and death and a 12-year-old girl with an aggressive nasopharyngeal mass showing intracranial extension. RESULTS: The laryngeal tumor was poorly differentiated, with vesicular nuclei, prominent nucleoli, extensive necrosis, and a lymphoplasmacytic infiltrate; cells were positive for cytokeratin and negative for lymphoma, melanoma, germ cell, and endocrine markers. Electron microscopy showed rare intermediate junctions and basal lamina. The nasopharyngeal tumor was poorly differentiated with areas of obvious squamous differentiation observed histologically, immunophenotypically, and ultrastructurally. Cytogenetic and fluorescent in situ hybridization studies were consistent with t(15;19)(q13;p13.1) in both cases. Both children received chemo- and radiotherapy. The first child died of disease after 36 weeks; autopsy revealed tumor in the larynx with spread to the skin/subcutis (neck and thorax) and lymph nodes (cervical, subcarinal, and pulmonary hilar). The second child developed widespread bony metastases and died of disease after 13 weeks. CONCLUSIONS: In conjunction with previous reports, the authors' findings show that t(15;19) is part of a distinct clinicopathologic entity characterized by young age, midline carcinoma of the neck or upper thorax, and a rapidly fatal course. Female gender and superior vena cava syndrome are common. The histogenesis of these distinctive tumors is unknown. The authors' findings suggest origin in the upper airway, perhaps from submucosal glands.

Growth inhibition of dermatofibrosarcoma protuberans tumors by the platelet-derived growth factor receptor antagonist STI571 through induction of apoptosis.

Dermatofibrosarcoma protuberans (DFSP) and giant cell fibroblastoma (GCF) are recurrent, infiltrative skin tumors that presently are treated with surgery. DFSP and GCF tumors are genetically characterized by chromosomal rearrangements fusing the collagen type Ialpha1 (COLIA1) gene to the platelet-derived growth factor B-chain (PDGFB) gene. It has been shown that the resulting COL1A1/PDGF-B fusion protein is processed to mature PDGF-BB. Autocrine PDGF receptor stimulation has therefore been predicted to contribute to DFSP and GCF tumor development and growth. Here we demonstrate presence of activated PDGF receptors in primary cultures derived from six different DFSP and GCF tumors. Three of the primary cultures were further characterized; their in vitro growth displayed an increased sensitivity to treatment with the PDGF receptor tyrosine kinase inhibitor STI571, as compared with normal fibroblasts. Transplantable tumors, displaying a DFSP-like histology, were established from one of the DFSP primary cultures. Treatment of tumor-bearing severe combined immunodeficient mice with STI571 reduced tumor growth. The growth-inhibitory effects in vitro and in vivo occurred predominantly through induction of tumor cell apoptosis. Our study demonstrates growth-inhibitory effects of PDGF receptor antagonists on human DFSP- and GCF-derived tumor cells and demonstrates that autocrine PDGF receptor stimulation provides antiapoptotic signals contributing to the growth of these cells. These findings suggest targeting of PDGF receptors as a novel treatment strategy for DFSP and GCF.

Molecular cytogenetic definition of three distinct chromosome arm 14q deletion intervals in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms characterized by frequent chromosome arm 14q losses. In this study, the 14q changes in a series of 39 histologically and immunohistochemically confirmed GISTs were analyzed in detail by metaphase and/or interphase fluorescence in situ hybridization (FISH) studies using 21 genetically well-characterized, region-specific 14q11-24 YAC clones. By conventional cytogenetic analysis, acquired clonal chromosome aberrations were found in 17 out of 35 tumors. Chromosome 14 was involved in 13 cases; six specimens showed complete chromosome 14 loss, while the remaining seven had structural abnormalities with the breakpoints residing within the intervals 14q11-13 or 14q22-24. Other recurrent chromosome aberrations included frequent deletions of chromosome 1p (11/17), losses of chromosome 22 (7/17), losses or deletions of chromosome arm 13 (6/17) or 15 (4/17), and gains or translocations involving chromosome 17 (4/17). Combining cytogenetic data with double-color FISH analysis, total or partial losses of 14q material were detected in 29 out of 36 tumors (81%). The 14q losses were found in all stages and histological subtypes. Two most frequent common deletion regions flanked by YACs 931B1 and 761D4, and 802E7 and 892C11 at 14q23-24 (25/30 of each; 83%) could be identified. Furthermore, 21 tumors (70%) shared a region of deletion defined by YACs 957H10 and 931E5 at 14q11-12. Our results suggest the presence of at least three distinct critical deletion regions on chromosome 14 in GISTs.

Clinical impact of molecular and cytogenetic findings in synovial sarcoma.

Synovial sarcoma is an aggressive soft-tissue tumor that accounts for up to 10% of soft-tissue sarcomas. Cytogenetically, synovial sarcoma is characterized by the t(X;18)(p11;q11), found in more than 95% of the tumors. This translocation results in rearrangements of the SYT gene in 18q11 and one of the SSX1, SSX2, or SSX4 genes in Xp11, creating a SYT/SSX1, SYT/SSX2, or SYT/SSX4 chimeric gene. It has been shown that patients with SYT/SSX1 fusion genes have a shorter metastasis-free survival than do patients with SYT/SSX2. Previous studies have also suggested that clonal evolution may be associated with disease progression. In the present study, RT-PCR analysis showed that all 64 examined synovial sarcomas from 54 patients had SYT-SSX chimeric genes. SYT/SSX1 was found in 40 tumors from 33 patients, SYT/SSX2 in 23 tumors from 20 patients, and SYT/SSX4 in one case. Two patients had variant SYT/SSX2 transcripts, with 57 bp and 141 bp inserts, respectively, between the known SYT and SSX2 sequences. Patients with tumors with SYT/SSX1 fusions had a higher risk of developing metastases compared to those with SYT/SSX2 fusions (P = 0.01). The reciprocal transcripts SSX1/SYT and SSX2/SYT were detected using nested PCR in 11 of the 40 samples with SYT/SSX1 and 5 of the 23 samples with SYT/SSX2, respectively. Among 20 blood samples, SYT/SSX1 and SYT/SSX2 were detected in one sample each. The t(X;18), or variants thereof, was found cytogenetically in all patients but three. Among 32 primary tumors, the t(X;18) or a variant translocation was the sole anomaly in 10. In contrast, of the seven metastatic lesions that were investigated prior to radiotherapy, only one had a t(X;18) as the sole anomaly; all other tumors displayed complex karyotypes. Cytogenetic complexity in primary tumors was, however, not associated with the development of metastases. Tumors with SYT/SSX2 less often (4/12 vs. 7/15) showed complex karyotypes than did tumors with SYT/SSX1, but the difference was not significant. Combining cytogenetic complexity and transcript data, we found that the subgroup of patients with tumors showing simple karyotypes and SYT/SSX2 fusion had the best clinical outcome (2/8 patients developed metastases), and those with tumors showing complex karyotypes together with SYT/SSX1 fusion the worst (6/7 patients developed metastases). This corresponded to 5-year metastasis-free survival rates of 0.58 and 0.0, respectively (P = 0.02).

Frequent fusion of the JAZF1 and JJAZ1 genes in endometrial stromal tumors.

Endometrial stromal tumors are divided into three types: benign stromal nodules, endometrial stromal sarcomas, and undifferentiated endometrial sarcomas. A variety of cytogenetic abnormalities involving chromosome 7 have been reported in endometrial stromal sarcomas, including a recurrent t(7;17)(p15;q21). We have identified two zinc finger genes, which we have termed JAZF1 and JJAZ1, at the sites of the 7p15 and 17q21 breakpoints. Analyses of tumor RNA indicate that a JAZF1/JJAZ1 fusion is present in all types of endometrial stromal tumors; however, the fusion appears to be rarer among endometrial stromal sarcomas that would be considered high-grade according to certain classification schemes. These findings suggest that the less malignant endometrial stromal tumors may evolve toward more malignant types, but that some endometrial stromal sarcomas with relatively abundant mitotic activity may compose a biologically distinct group.

The der(17)t(X;17)(p11;q25) of human alveolar soft part sarcoma fuses the TFE3 transcription factor gene to ASPL, a novel gene at 17q25.

Alveolar soft part sarcoma (ASPS) is an unusual tumor with highly characteristic histopathology and ultrastructure, controversial histogenesis, and enigmatic clinical behavior. Recent cytogenetic studies have identified a recurrent der(17) due to a non-reciprocal t(X;17)(p11.2;q25) in this sarcoma. To define the interval containing the Xp11.2 break, we first performed FISH on ASPS cases using YAC probes for OATL1 (Xp11.23) and OATL2 (Xp11.21), and cosmid probes from the intervening genomic region. This localized the breakpoint to a 160 kb interval. The prime candidate within this previously fully sequenced region was TFE3, a transcription factor gene known to be fused to translocation partners on 1 and X in some papillary renal cell carcinomas. Southern blotting using a TFE3 genomic probe identified non-germline bands in several ASPS cases, consistent with rearrangement and possible fusion of TFE3 with a gene on 17q25. Amplification of the 5' portion of cDNAs containing the 3' portion of TFE3 in two different ASPS cases identified a novel sequence, designated ASPL, fused in-frame to TFE3 exon 4 (type 1 fusion) or exon 3 (type 2 fusion). Reverse transcriptase PCR using a forward primer from ASPL and a TFE3 exon 4 reverse primer detected an ASPL-TFE3 fusion transcript in all ASPS cases (12/12: 9 type 1, 3 type 2), establishing the utility of this assay in the diagnosis of ASPS. Using appropriate primers, the reciprocal fusion transcript, TFE3-ASPL, was detected in only one of 12 cases, consistent with the non-reciprocal nature of the translocation in most cases, and supporting ASPL-TFE3 as its oncogenically significant fusion product. ASPL maps to chromosome 17, is ubiquitously expressed, and matches numerous ESTs (Unigene cluster Hs.84128) but no named genes. The ASPL cDNA open reading frame encodes a predicted protein of 476 amino acids that contains within its carboxy-terminal portion of a UBX-like domain that shows significant similarity to predicted proteins of unknown function in several model organisms. The ASPL-TFE3 fusion replaces the N-terminal portion of TFE3 by the fused ASPL sequences, while retaining the TFE3 DNA-binding domain, implicating transcriptional deregulation in the pathogenesis of this tumor, consistent with the biology of several other translocation-associated sarcomas. Oncogene (2001) 20, 48 - 57.

Amplification of AML1 in childhood acute lymphoblastic leukemias.

Amplification of AML1 has been confirmed by fluorescence in situ hybridization analysis in two cases of childhood acute lymphoblastic leukemia. It remains to be elucidated whether this amplification results in up-regulation of the normal AML1 gene product or a potentially mutant AML1 transcript.

Abnormal nuclear shape in solid tumors reflects mitotic instability.

Abnormalities in nuclear morphology are frequently observed in malignant tissues but the mechanisms behind these phenomena are still poorly understood. In this study, the relation between abnormal nuclear shape and chromosomal instability was explored in short-term tumor cell cultures. Mitotically unstable ring and dicentric chromosomes were identified by fluorescence in situ hybridization at metaphase and subsequently localized in interphase nuclei from five malignant soft tissue tumors. The vast majority (71 to 86%) of nuclear blebs, chromatin strings, and micronuclei contained material from the unstable chromosomes, whereas few (<11%) were positive for stable chromosomes. Nuclear morphology was also evaluated in fibroblasts and an osteosarcoma cell line exposed to irradiation. A linear correlation was found between the frequency of abnormalities in nuclear shape, on one hand, and cells with unstable chromosomes (r = 0.87) and anaphase bridge configurations (r = 0.98), on the other hand. The relation between nuclear shape and karyotypic pattern was investigated further in cultures from 58 tumors of bone, soft tissue, and epithelium. Blebs, strings, and micronuclei were significantly more frequent in tumors that contained rings, dicentrics, or telomeric associations than in those exhibiting only stable aberrations (P: < 0.001) and a positive correlation (r = 0.78) was found between the frequency of such nuclear abnormalities and the intratumor heterogeneity of structural chromosome aberrations. These results indicate that the formation of nuclear blebs, chromatin strings, and micronuclei in malignant tissues is closely related to the breakage-fusion-bridge type of mitotic disturbances. Abnormalities in nuclear shape may thus primarily be regarded as an indicator of genetic instability and intratumor heterogeneity, independent of cytogenetic complexity and the grade of malignancy.