De novo‐generated small palindromes are characteristic of amplicon boundary junction of double minutes

Double minutes (DMs) are hallmarks of gene amplification. However, their molecular structure and the mechanisms of formation are largely unknown. To elucidate the structure and underlying molecular mechanism of DMs, we obtained and cloned DMs using microdissection; and degenerated oligonucleotide primed polymerase chain reaction (DOP‐PCR) from the ovarian cancer cell line UACC‐1598. Two large amplicons, the 284 kb AmpMYCN, originating from locus 2p24.3 and the 391 kb AmpEIF5A2, from locus 3q26.2, were found co‐amplified on the same DMs. The two amplicons are joined through a complex 7 kb junction DNA sequence. Analysis of the junction has revealed three de novo created small palindromes surrounding the six breakpoints. Consistent with these observations, we further found that 70% of the 57 reported DM junction sequences have de novo creation of small palindromic sequences surrounding the breakpoints. Together, our findings indicate that de novo‐generated small palindromic sequences are characteristic of amplicon boundary junctions on DMs. It is possible that the de novo‐generated small palindromic sequences, which may be generated through non‐homologous end joining in concert with a novel DNA repair machinery, play a common role in amplicon rejoining and gene amplification.     

Plasmids with a mammalian replication origin and a matrix attachment region initiate the event similar to gene amplification

Gene amplification plays a crucial role in the development of many human malignancies. Amplified genes are frequently localized on double minutes (DMs). We show here that plasmids bearing both a mammalian replication origin and a nuclear matrix attachment region were able to integrate into DMs if transfected to cells having DMs (COLO 320DM). Furthermore, these plasmids triggered the events leading to the de novo formation of the structure similar to DMs if transfected to the cells without DMs (COLO 320HSR or HeLa). Autonomous replication of these plasmids was suggested to be a prerequisite for these events to occur, because the presence of the origin sequences in the plasmids was required. The presence of matrix attachment region in the plasmids is also required for these events to occur, suggesting that matrix attachment plays an indispensable role in extrachromosomal replication. This model system will allow us to investigate the mechanism of gene amplification as well as to analyze the autonomous replication of the plasmid with mammalian replication origins.     

双微体与食管癌放疗临床疗效的关系

目的研究食管癌患者外周血白细胞染色体双微体畸变与放疗疗效的关系。方法检查食管癌患者外周血３８例，观察白细胞染色体畸变的情况及肿瘤的放疗效果。结果３８例患者中的７例患者发现双微体，占１８．４％，含双微体患者放疗有效率７１．４％，无双微体患者放疗有效率９６．８％，１年生存率分别为１４．３％和５４．８％。结论食管癌患者外周血发现双微体者多为晚期病人，这类病人对放射治疗者一定抗拒性，生存期短，病程发展快。     

Tumour karyotype discriminates between good and bad prognostic outcome in neuroblastoma

In 28 patients with neuroblastoma of different stages the karyotype was determined in the primary tumour and/or in the metastases by direct chromosome preparation or short term cell culture. In addition, DNA analysis for the proto-oncogene N-myc was performed for comparison in 10 cases. Abnormalities (deletions, translocations, derivations) of the short arm of chromosome 1 with the most frequent breakpoint at 1p32 (besides rarer aberrations in other chromosomes) were found in the tumour karyotype of 15 of 18 (83%) patients with metastatic disease (stage IV) and in 2 of 3 patients with stage III, but in none of the 7 patients with stages I, II, IV-S who are all alive with no evidence of disease. These 7 surviving patients with good prognosis had a hyperploid tumour karyotype, mainly in the triploid range. Eleven of the 18 (61%) patients with stage IV and 1 of 3 patients with stage III also contained double minutes (DMs) and/or homogeneously staining regions (HSRs) in their tumour karyotypes. N-myc amplification (30 to 60 copies) in the tumour DNA was detected in 2 of 6 (33%) examined cases with stage IV, in 1 out of 2 examined cases with stage III, and correlated with the presence of DMs/HSRs. Life table analysis showed a 90% probability of surviving in patients lacking the 1p abnormality as compared to less than 10% in patients with an aberrant 1p chromosome in the tumour cells. We conclude that tumour karyotype, in particular the structure of the short arm of chromosome 1, is the most important factor in determining the different outcome in children with neuroblastoma.     

Biology of desmoplastic melanoma: a case-control comparison with other melanomas.

PURPOSE: Previous studies have established that patients with desmoplastic melanoma (DM) have thicker primary tumors. Consequently, comparisons with other forms of melanoma have been strongly biased by differences in Breslow stage. This is the first case-matched control study comparing DM with other forms of melanoma. PATIENTS AND METHODS: From a database of 3,202 melanoma patients treated at one institution, 89 patients with DM and 178 case-matched control patients (2:1) were identified by matching for tumor thickness, age, sex, and year of diagnosis. Clinical, pathologic, and outcome information was obtained from chart review. RESULTS: Controls were matched successfully to patients for tumor thickness, age, sex, and year of diagnosis. Presentation with American Joint Committee on Cancer stage III or IV disease is less common in patients with DM compared to case-matched control patients (5% v 21%; P < .001). Re-excisions to obtain clear surgical margins are required more often in patients with DM compared to case-matched control patients (21% v 6%; P < .001). Risk of positive sentinel nodes is lower in patients with DM compared to case-matched control patients (8% v 34%; P = .013). Despite these differences, survival rates of patients with DM are the same as case-matched control patients. CONCLUSION: Use of case-matched control patients matched for tumor thickness avoids biases introduced by the advanced Breslow stage of DMs. DMs are more locally aggressive than thickness-matched controls, and positive sentinel nodes are limited to patients with thick primary tumors. Importantly, patients with DM have survival rates similar to patients with other melanomas of similar thickness.     

Gender and ploidy in cancer survival

Background

Females carry a better prognosis than men for many cancer types. We hypothesized that chromosomal changes, in particular numerical alterations of the sex chromosomes or the presence of near-triploidy may contribute to these gender differences.

Methods

To characterize the influence of gender a literature search was performed for survival data of 27 tumor types. All entities were categorized by the strength of evidence for differences in survival between females and males. To test our hypothesis the Mitelman database of chromosomal alterations was evaluated for the major tumor types occurring in both women and men. Numerical gonosome alterations were documented and mean chromosome numbers were converted into histograms to provide insight into the ploidy level of 37 cancer types.

Results

In general, a survival advantage of women could be shown for most, but not all cancer types. In addition, 36.859 karyograms were analyzed. Numerical gonosome alterations were more frequent in males than females indicating a potential link with gender differences in survival. Neartriploidy was a common phenomenon in many cancer types suggesting that it represents a metastable condition of the cancer genome. It was not related to gender differences in survival. However, the extent of triploidy and aneuploidy was associated with poor prognosis in carcinomas. There was no single case in the Mitelman database with normal chromosome number (n?=?46) that did not carry at least one structural or numerical aberration.

Conclusions

Our study highlights the importance of chromosomal changes in tumor formation and progression. In addition, it suggests potential associations with gender specific differences in survival.     

MYCN amplification in neuroblastoma: A paradigm for the clinical use of an oncogene

Increase of the dosage of cellular oncogenes by DNA amplification is a frequent genetic alteration of cancer cells. The presence of amplified cellular oncogenes is usually signalled by conspicuous chromosomal abnormalities, “double minutes” (DMs) or “homogeneously staining chromsomal regions” (HSRs). Some human cancers carry a specific amplified oncogene at high incidence. In neuroblastomas the amplification of MYCN has been found associated with aggressively growing cancers and is an indicator for poor prognosis. MYCN amplification is of predictive value for identifying neuroblastoma patiens that require specific therapeutic regimens and for identifying patients that will not benefit from chemotherapy. This has been a lecture presented on a Tempus-course (S-JEP 11198–96) “Harmonization of Ph. D. degree to EU standards”     

A new myeloblastic leukemia cell line with double minute chromosomes. Induction of methotrexate resistance and dihydrofolate reductase gene amplification.

To test the relationship between DMs and drug resistance in newly established AML cell lines, KY821, and its clone KY821A3, the latter had lost DMs during cloning, were cultured in increasing concentrations of MTX. KY821 became resistant against 2 x 10(-4) M MTX, whereas KY821A3 did against 2 x 10(-5) M MTX in a same period. Enhanced enzyme activities of DHFR were correspondent to the increased DMs numbers and DHFR gene amplification in both resistant clones. The amplified DHFR gene was located on DMs by in situ hybridization. These data indicated that the presence of DMs in KY821 would facilitate the acquisition of drug resistance.     

Increased level of amplification of the c-myc oncogene in tumors induced in nude mice by a human breast carcinoma cell line

Cell line SW 613-S, derived from a human breast carcinoma, contained double minute chromosomes (DMs) but lost them progressively upon in vitro cultivation. These cells were tumorigenic in nude mice. Cell lines were derived from the tumors and were found to have a high DM content. In three such cell lines, DMs were stably maintained upon in vitro cultivation, whereas in another they were progressively lost. We found that the c-myc oncogene is amplified 5- to 10-fold in SW 613-S and 20- to 90-fold in the different cell lines derived from the tumors. At least part of the additional c-myc copies were found associated with a purified DM fraction. In cell lines which lost the DMs during in vitro passages, the level of amplification was maintained. In situ hybridization experiments indicated that this loss was compensated by the acquisition of copies of the c-myc gene integrated into a chromosome. No major rearrangement of the amplified c-myc gene was detected. The amount of c-myc messenger RNAs is roughly proportional to the level of amplification. Our results indicate that growth of SW 613-S cells as tumors in nude mice selected cells with an increased level of amplification and expression of the c-myc oncogene.     

Chromosome findings in human neuroblastomas xenografted in nude mice

Chromosomes were successfully studied in 8 of 9 human neuroblastomas (NBs) xenografted in nude mice. Structural abnormalities in the short arm of chromosome #1 were found in 6 of the 8 tumors; these included nonreciprocal translocations and simple deletions. The breakpoints were distributed between 1p11 and 1p34, and all of them had lost the terminal portion of 1p (1pter----1p34). Double minutes (DMs) and homogeneously staining regions (HSRs) were observed in 7 tumors; 6 had either DMs or HSRs, and one had some cells with DMs and other cells with HSR. Only one tumor had neither DMs, nor HSRs. Our study revealed that structural abnormalities in the NB xenografts were essentially the same as those in the NB cell lines and fresh tumors reported previously, and that DMs and HSRs were seen in most NB xenografts as frequently as in NB cell lines.     

Frontline extended surgery is associated with improved survival in retroperitoneal low- to intermediate-grade soft tissue sarcomas

BackgroundThe purpose of the study was to retrospectively reassess in our institutional series at a longer follow-up the value of a systematic attempt to carry out wide resections in retroperitoneal soft tissue sarcoma.Patients and methodsThree hundred and thirty-one consecutive patients surgically treated were analyzed. Since a shift toward a systematic more extended surgical approach took place starting from 2002, patients were divided in two groups according to the time of surgery. Overall survival (OS), crude cumulative incidence of local recurrence (LR) and distant metastases (DMs) were estimated. Cox model multivariate analysis was carried out.ResultsFive-year OS of patients operated in the recent period was 66%, compared with 48% for those operated in the previous period. This was associated with less LR (28% versus 49%), while the number of DMs was higher in the recent group (25% versus 12%). Beside the treatment period, the only independent determinant for survival was histological grade.ConclusionsThe adoption of a policy of more liberal visceral en bloc resections was associated with a higher local control and OS. This benefit was evident in patients with grade I–II tumors, while DMs were a limiting factor in high-grade ones. New therapies are needed to control systemic disease as local surgery may improve local control.     

Molecular/cytogenetic alterations accompanying the development of multidrug resistance in the J774.2 murine cell line

Mouse macrophage-like J774.2 cells were selected for resistance to colchicine and examined by molecular/cytogenetic analysis to determine whether the acquisition of the multidrug resistant (mdr) phenotype was associated with specific chromosomal rearrangements. Cytogenetic studies of the J774.2 parental and two colchicine-resistant (CLCR) sublines--J7.Cl-30 (770-fold CLCR) and J7.Cl-100 (2500-fold CLCR)--demonstrated specific numeric and structural karyotypic alterations accompanying the emergence of mdr. The parental cells demonstrated a modal chromosome number of 63, while the modal number of the J7.Cl-30 subline was 53. The most striking difference between the parental and J7.Cl-30 subline was the presence of an average of 60 double minutes (DMs) per cell in the CLCR cells. The 2500-fold resistant J7.Cl-100 subline displayed a modal number of 50, which included structural rearrangements involving chromosomes 2 and 7 and concomitant replacement of DMs by a homogeneously staining region (HSR). Southern blotting analysis demonstrated a approximately 35-fold amplification of P-glycoprotein homologous sequences in the J7.Cl-30 subline and approximately 70-fold amplification in the J7.Cl-100 subline. Chromosomal in situ hybridization localized the amplified P-glycoprotein sequences to DMs (J7.Cl-30) and the HSR (J7.Cl-100) in these CLCR sublines. Our results suggest that CLCR in J774.2 cells results from overexpression of P-glycoprotein via gene amplification which was accompanied by chromosomal evolution from DMs to an HSR.     

Molecular evidence demonstrating local treatment failure is the source of distant metastases in some patients treated for breast cancer

PURPOSE: To examine the clonality relationships among initial invasive breast carcinoma (IBC), ipsilateral breast failure (IBF), and distant metastasis (DM) to determine the effect of local tumor recurrence on the development of DMs. METHODS AND MATERIALS: A total of 18 patients treated with breast-conserving therapy who developed an IBF followed by DMs were studied using a 20 informative-marker, polymerase chain reaction-based allelic imbalance clonality assay. RESULTS: Four relationships were identified. First, in 7 cases, the IBF and DMs were clonally related to the initial IBC as one progressively genetic unstable process. Second, in 3 cases, the IBF and DMs were each clonally related to the IBC but clonally distinct from each other. Third, in 3 cases, the IBC and the IBF were clonally related and the DMs were clonally related to the IBFs, with a weak relationship to the initial IBC. Finally, in 5 cases, the IBF was clonally distinct from the initial IBC (new second primary) and the DMs were clonally related to the IBF and clonally distinct from the initial IBC. CONCLUSION: These findings provide molecular evidence demonstrating that some DMs can directly develop from IBFs and support the importance of local tumor control in the overall treatment of breast cancer patients.     

Evidence for selection of homogeneously staining regions in a human melanoma cell line

Cytogenetic analysis of tumor cells from a human malignant melanoma was performed on both the primary tumor colony-forming cells and a cell line (HA-A) established subsequently from the clonogenic population. Chromosome-banding analysis demonstrated identical karyotypic alterations in both the tumor colony-forming cells and the HA-A cell line, documenting their origin from a common precursor. The most distinctive chromosome alterations shared between tumor colony-forming cells and the HA-A cell line were double-minute bodies (DMs) and a homogeneously staining region (HSR) on chromosome 7 at band p22. This represents the first observation of DMs or HSRs in cells from a human malignant melanoma. The frequency of HSR-bearing cells observed in the original tumor was less than 1%, while DM-containing cells were present in greater than 90% of all cells examined. In contrast, serial chromosome harvests at early passage of the HA-A cell line revealed positive selection for HSR-bearing cells with concomitant loss of DM-containing cells after increasing time in vitro. Following the ninth serial passage in vitro, HSRs were observed in 100% of cells with DMs no longer observed in the HA-A cell line. The finding of an HSR-bearing marker in the original tumor sample supports the view that HSRs are not an artifact of in vitro monolayer growth. However, our results demonstrate that the frequency of HSR-bearing cells within established cell lines may reflect in vitro selection and therefore not accurately reflect the frequency of this population in vivo.     

Glycoconjugate profiling of primary melanoma and its sentinel node and distant metastases: implications for diagnosis and pathophysiology of metastases

Aiming at more precise detection of melanoma cells in sentinel lymph nodes and better understanding of the mechanisms underlying metastatic spread, expression of L1, CEACAM1, and binding of the lectins HPA, ML-I and PNA, was assessed in benign nevi (n=12), primary melanomas (PTs: n=67), their corresponding sentinel lymph nodes (SLNs: n=40), and distant metastases (DMs: n=35). Sensitivity and specificity of CEACAM1 (95-97%; 66%) and L1 (90-93%; 100%) exceeded that of the standard markers MelanA, S100, and HMB45 in single marker use. Lectin binding was found in PTs and DMs (HPA: 69% and 77%; ML-I: 82% and 77%, respectively), but rarely in SLNMs (HPA: 20%, ML-I: 20%, PNA: 5%, respectively). The highly specific and sensitive L1-11A against L1 and 4D1/C2 against CEACAM1 antibodies are a worthy completion to standard antibody panels for diagnosis of melanoma cells. Both CAMs seem to be functionally involved in lymphatic and haematogenous spread, and are thus promising target molecules for immunotoxins.     

Expulsion of micronuclei containing amplified genes contributes to a decrease in double minute chromosomes from malignant tumor cells

Double minute chromosomes (DMs) are a hallmark of gene amplification. The relationship between the formation of DMs and the amplification of DM‐carried genes remains to be clarified. The human colorectal cancer cell line NCI‐H716 and human malignant primitive neuroectodermal tumor cell line SK‐PN‐DW are known to contain many DMs. To examine the amplification of DM‐carried genes in tumor cells, we performed Affymetrix SNP Array 6.0 analyses and verified the regions of amplification in NCI‐H716 and SK‐PN‐DW tumor cells. We identified the amplification regions and the DM‐carried genes that were amplified and overexpressed in tumor cells. Using RNA interference, we downregulated seven DM‐carried genes, (NDUFB9, MTSS1, NSMCE2, TRIB1, FAM84B, MYC and FGFR2) individually and then investigated the formation of DMs, the amplification of the DM‐carried genes, DNA damage and the physiological function of these genes. We found that suppressing the expression of DM‐carried genes led to a decrease in the number of DMs and reduced the amplification of the DM‐carried genes through the micronuclei expulsion of DMs from the tumor cells. We further detected an increase in the number of γH2AX foci in the knockdown cells, which provides a strong link between DNA damage and the loss of DMs. In addition, the loss of DMs and the reduced amplification and expression of the DM‐carried genes resulted in a decrease in cell proliferation and invasion ability.     

Evidence for a new tumour suppressor gene locus for γ-radiation-induced thymic lymphoma located on the proximal part of mouse chromosome 18

Introduction

Five distinct thymic lymphoma suppressor regions have been identified on chromosome 4 (TLSR 1–5), and one on chromosome 19 (TLSR 8) through detection of loss of heterozygosity (LOH). Additionally, the involvement ofp16/INK4a, p15/INK4b, p73, Pten andFas had been reported in the most advanced stages of thymic lymphomagenesis.

Material and methods

Advanced thymic lymphomas (n=110) induced by gamma-irradiation in F1 hybrids from a BALB/c and C57BL/6J cross were analysed using micro-satellite markers on chromosomes 13, 14 and 18.

Results

Of the 110 tumours, 15 (14.5%) exhibited allelic losses on chromosome 18 atD18Mit21. Comparative analyses revealed significant differences (p<0.0001) for this marker and led us to define a new critical region of LOH on the proximal part of mouse chromosome 18, and named as TLSR9 according to the Mouse Nomenclature Committee.

Conclusions

The proposed TLSR9 region is orthologous with 18q11 region, which is affected in various types of human cancers (Mitelman Database of Chromosome Aberrations in cancer (2002) Mitelman F, Johansson B and Mertens F (eds), http://cgap.nci.nih.gov/Chromosomes/Mitelman), and containsN-cadherin as a possible candidate gene.     

Endoscopic Resection of Sinonasal Malignancies

Malignant tumors of the sinonasal tract are rare, accounting for only 1% of all malignancies. Although they are associated with substantial histological heterogeneity, surgery plays a key role in their management. This review addresses the evolution of current treatments in view of the introduction of endoscopic resection techniques. The absence of facial incisions and osteotomies, decreased hospitalization time, better control of bleeding, improved visualization of tumor borders, and reduced morbidity and mortality rate are the major advantages of endoscopic techniques in comparison to traditional external approaches. The major criticisms focus on oncologic results in view of the short/intermediate follow-up of large series, which have commonly grouped together several histologies that may be associated with different prognoses. Since prospective studies contrasting the results of endoscopic and craniofacial resections are difficult to carry out given the rarity of the disease together with ethical issues, the creation of a large database would favor the analysis of several variables related to the patient, tumor, and treatment on survival performed on a large number of patients.     

Predictors of tumour involvement in remaining axillary lymph nodes of breast cancer patients with positive sentinel lymph node.

AIMS: To characterize the various clinicopathologic features in cases of breast cancer with positive sentinel lymph nodes (SLNs), in order to determine factors that might help in predicting the involvement of the non-SLNs. METHODS: A retrospective database review was performed of 726 breast cancer patients with stage 0-II, in whom SLNs were successfully identified. One hundred eighty-five of these patients showed positive SLNs, and subsequently underwent axillary lymph node dissection (ALND). These cases were divided into two groups based on the presence or absence of metastases in the non-SLNs, i.e. positive non-SLNs (NSLN+; 81 cases) and negative non-SLNs (NSLN-; 104 cases). RESULTS: Multivariate analysis revealed that a larger size of the primary tumour (>2.0cm), presence of lymphatic invasion, larger size of the largest SLN metastasis (>2mm), and a 100% metastatic rate in the SLNs (number of positive SLNs/number of harvested SLNs) were significantly associated with NSLN+. Among the cases in which all the four factors were present, 73% (30/41) were found to have NSLN+. CONCLUSION: We found four independent predictors in relation to non-SLN metastasis. Although these factors might be useful for determining the need of additional ALND, it would seem that even the presence of all of these four factors in combination may be insufficient to safely omit ALND. Thus, until further evidence is accumulated from the results of large clinical trials, ALND would still be recommended for patients with SLN metastasis.     

Codeletions at 1p and 19q predict a lower risk of pseudoprogression in oligodendrogliomas and mixed oligoastrocytomas

Background

Pseudoprogression (PsP) occurs at a higher rate in glioblastoma multiforme with a methylated MGMT promoter—a subset with increased sensitivity to chemoradiotherapy and better overall prognosis. In oligodendroglioma (OG) and oligoastrocytoma (OA), presence of 1p/19q codeletions is highly predictive of response to treatment and is often associated with the methylated MGMT promoter; hence, this study queries whether the presence of 1p/19q codeletions in OG/OA correlates with a higher rate of PsP following therapy.

Methods

A retrospective analysis was performed on all OG/OA in a database of patients with brain tumors who underwent resection of their tumor since 1998. Eighty-eight cases (37 with and 51 without 1p/19q codeletions) met inclusion criteria, and their patient data were analyzed to determine whether the presence of 1p/19q codeletions was associated with PsP and survival.

Results

OG/OA (World Health Organization grades II and III) with 1p/19q codeletions had a significantly improved survival (P = .041). Multivariate analysis found that PsP occurred less frequently in OG/OA with 1p/19q codeletions compared with tumors without codeletions (odds ratio, 0.047; 95% confidence interval, 0.005–0.426; P = .0066). The rate of PsP was 19% for the entire cohort, 31% for tumors without codeletions, and 3% for tumors with codeletions. When early posttreatment contrast enhancement developed in tumors with 1p/19q codeletions, it occurred exclusively in tumors that were histologically OA and not OG.

Conclusion

Codeletions of 1p/19q are a marker of good prognosis but are unexpectedly associated with a lower likelihood of PsP. PsP does not correlate with sensitivity to treatment and improved survival in OG/OA.