Array-CGH and multipoint FISH to decode complex chromosomal rearrangements

BACKGROUND: Recently, several high-resolution methods of chromosome analysis have been developed. It is important to compare these methods and to select reliable combinations of techniques to analyze complex chromosomal rearrangements in tumours. In this study we have compared array-CGH (comparative genomic hybridization) and multipoint FISH (mpFISH) for their ability to characterize complex rearrangements on human chromosome 3 (chr3) in tumour cell lines. We have used 179 BAC/PAC clones covering chr3 with an approximately 1 Mb resolution to analyze nine carcinoma lines. Chr3 was chosen for analysis, because of its frequent rearrangements in human solid tumours. RESULTS: The ploidy of the tumour cell lines ranged from near-diploid to near-pentaploid. Chr3 locus copy number was assessed by interphase and metaphase mpFISH. Totally 53 chr3 fragments were identified having copy numbers from 0 to 14. MpFISH results from the BAC/PAC clones and array-CGH gave mainly corresponding results. Each copy number change on the array profile could be related to a specific chromosome aberration detected by metaphase mpFISH. The analysis of the correlation between real copy number from mpFISH and the average normalized inter-locus fluorescence ratio (ANILFR) value detected by array-CGH demonstrated that copy number is a linear function of parameters that include the variable, ANILFR, and two constants, ploidy and background normalized fluorescence ratio. CONCLUSION: In most cases, the changes in copy number seen on array-CGH profiles reflected cumulative chromosome rearrangements. Most of them stemmed from unbalanced translocations. Although our chr3 BAC/PAC array could identify single copy number changes even in pentaploid cells, mpFISH provided a more accurate analysis in the dissection of complex karyotypes at high ploidy levels

Mandatory chromosomal segment balance in aneuploid tumor cells

Copyright: Copyright 2013 Elsevier B.V., All rights reserved.Background: Euploid chromosome balance is vitally important for normal development, but is profoundly changed in many tumors. Is each tumor dependent on its own structurally and numerically changed chromosome complement that has evolved during its development and progression? We have previously shown that normal chromosome 3 transfer into the KH39 renal cell carcinoma line and into the Hone1 nasopharyngeal carcinoma line inhibited their tumorigenicity. The aim of the present study was to distinguish between a qualitative and a quantitative model of this suppression. According to the former, a damaged or deleted tumor suppressor gene would be restored by the transfer of a normal chromosome. If so, suppression would be released only when the corresponding sequences of the exogenous normal chromosome are lost or inactivated. According to the alternative quantitative model, the tumor cell would not tolerate an increased dosage of the relevant gene or segment. If so, either a normal cell derived, or, a tumor derived endogenous segment could be lost. Methods: Fluorescence in Situ Hybridization based methods, as well as analysis of polymorphic microsatellite markers were used to follow chromosome 3 constitution changes in monochromosomal hybrids. Results: In both tumor lines with introduced supernumerary chromosomes 3, the copy number of 3p21 or the entire 3p tended to fall back to the original level during both in vitro and in vivo growth. An exogenous, normal cell derived, or an endogenous, tumor derived, chromosome segment was lost with similar probability. Identification of the lost versus retained segments showed that the intolerance for increased copy number was particularly strong for 3p14-p21, and weaker for other 3p regions. Gains in copy number were, on the other hand, well tolerated in the long arm and particularly the 3q26-q27 region. Conclusion: The inability of the cell to tolerate an experimentally imposed gain in 3p14-p21 in contrast to the well tolerated gain in 3q26-q27 is consistent with the fact that the former is often deleted in human tumors, whereas the latter is frequently amplified. The findings emphasize the importance of even minor changes in copy number in seemingly unbalanced aneuploid tumors.publishersversionPeer reviewe

Involvement of evolutionarily plastic regions in cancer associated CHR3 aberrations

A functional test to identify tumor antagonizing regions on chromosome 3 (chr3), called the Elimination Test, was developed in our group. It is based on microcell mediated transfer of human chr3 into mouse or human tumor cells and analysis of the monochromosomal hybrids after their growth in vivo. We identified three regions on 3p14-p22, which were frequently lost in derived tumors (common or frequently eliminated regions). In order to understand the role of these regions in tumor development, we continued the study following two leads: analysis of breakpoint clusters to identify and characterize possible instability features and search for tumor suppressor genes within the deleted regions. First, we identified and characterized a common eliminated region 1 (CER1) homologous sequence in mouse, where it was divided into two syntenic blocks on chromosome 9. In these blocks, the gene order and content was maintained with exception of two mouse gene duplications. Comparative analysis helped us to characterize five previously not identified mouse genes (Kiss et al. 2002). A more extensive comparative study of CER1 showed that its border regions are characterized by evolutionary plasticity: synteny breaks in several species, recent tandem gene duplications, retroposed pseudogene insertions, and horizontal evolution of the genes. Thus we showed that the cancer associated breakpoint regions have features of evolutionary plasticity. These results and other publications from our group suggested structural instability at the borders of eliminated regions identified by the Elimination Test (Darai et al. 2005) (Kost-Alimova et al. 2003; Kost-Alimova et al. 2004). As a next step, in order to analyze rearrangements of entire chr3 in human tumor cells, we developed and compared two high resolution methods, array-CGH and mpFISH. We proved that although our 1Mb chr3 BAC/PAC array could identify single copy number changes even in pentaploid cells, mpFISH provided a more accurate analysis in the dissection of complex karyotypes at high ploidy levels. In heterogeneous or normal cell contaminated samples the most precise analysis can be made by mpFISH due to its ability to give information at single cell level (Darai-Ramqvist et al. 2006). Using high resolution methods we analyzed ten carcinoma cell lines and identified two new hot spots of tumor breakpoints at 3p12-p13 and 3q21. These tumor breakpoint regions carried large segmental duplications, retrotransposable elements and satellite repeats, which participated in recent primate evolution and, as we suggest, are associated with structural chromosomal instability (CIN). CIN is an ongoing dynamic process. Therefore in order to prove that the instability at the breakpoint regions characterizes structural CIN phenotype and it is required for tumor development and progression, dynamic analysis of the tumors must be done. This may elucidate the mechanism of tumor development; and may help to develop CIN phenotype markers useful in choice of consequent treatment. Following the second lead of our study, we have analyzed a putative tumor suppressor gene LIMD1, which is located within the deleted central part of CER1. We found that it binds specifically to pRb and suppresses E2F driven transcription. A tumor suppressor effect of this gene was proven in in vitro and in vivo experiments, as well as in tumor biopsies (Sharp et al. 2004). In another part of the study we analyzed in details chr3 rearrangements in human renal cell carcinoma and nasopharyngeal carcinoma derived monochromosomal (chr3) hybrids and showed that aneuploid tumors maintain a mandatory chromosomal segment balance with stringency concerning no gain of 3p14-21 and no loss of 3q26-27. We concluded that the mechanism of tumor suppression by chr3 transfer is based on the alternative quantitative model. According to this model the tumor cell does not tolerate an increased dosage of the relevant gene or segment, and the lost part can be either of normal cell derived exogeneous or tumor derived endogenous origin (Kost-Alimova et al. 2007). First, we identified and characterized a common eliminated region 1 (CER1) homologous sequence in mouse, where it was divided into two syntenic blocks on chromosome 9. In these blocks, the gene order and content was maintained with exception of two mouse gene duplications. Comparative analysis helped us to characterize five previously not identified mouse genes (Kiss et al. 2002). A more extensive comparative study of CER1 showed that its border regions are characterized by evolutionary plasticity: synteny breaks in several species, recent tandem gene duplications, retroposed pseudogene insertions, and horizontal evolution of the genes. Thus we showed that the cancer associated breakpoint regions have features of evolutionary plasticity. These results and other publications from our group suggested structural instability at the borders of eliminated regions identified by the Elimination Test (Darai et al. 2005) (Kost-Alimova et al. 2003; Kost-Alimova et al. 2004). As a next step, in order to analyze rearrangements of entire chr3 in human tumor cells, we developed and compared two high resolution methods, array-CGH and mpFISH. We proved that although our 1Mb chr3 BAC/PAC array could identify single copy number changes even in pentaploid cells, mpFISH provided a more accurate analysis in the dissection of complex karyotypes at high ploidy levels. In heterogeneous or normal cell contaminated samples the most precise analysis can be made by mpFISH due to its ability to give information at single cell level (Darai-Ramqvist et al. 2006). Using high resolution methods we analyzed ten carcinoma cell lines and identified two new hot spots of tumor breakpoints at 3p12-p13 and 3q21. These tumor breakpoint regions carried large segmental duplications, retrotransposable elements and satellite repeats, which participated in recent primate evolution and, as we suggest, are associated with structural chromosomal instability (CIN). CIN is an ongoing dynamic process. Therefore in order to prove that the instability at the breakpoint regions characterizes structural CIN phenotype and it is required for tumor development and progression, dynamic analysis of the tumors must be done. This may elucidate the mechanism of tumor development; and may help to develop CIN phenotype markers useful in choice of consequent treatment. Following the second lead of our study, we have analyzed a putative tumor suppressor gene LIMD1, which is located within the deleted central part of CER1. We found that it binds specifically to pRb and suppresses E2F driven transcription. A tumor suppressor effect of this gene was proven in in vitro and in vivo experiments, as well as in tumor biopsies (Sharp et al. 2004). In another part of the study we analyzed in details chr3 rearrangements in human renal cell carcinoma and nasopharyngeal carcinoma derived monochromosomal (chr3) hybrids and showed that aneuploid tumors maintain a mandatory chromosomal segment balance with stringency concerning no gain of 3p14-21 and no loss of 3q26-27. We concluded that the mechanism of tumor suppression by chr3 transfer is based on the alternative quantitative model. According to this model the tumor cell does not tolerate an increased dosage of the relevant gene or segment, and the lost part can be either of normal cell derived exogeneous or tumor derived endogenous origin (Kost-Alimova et al. 2007)

Interventional and EBUS cytology in Sweden

Interventional cytology was first introduced in Sweden in the late 1940ies by Sixten Franzén at the Karolinska University Hospital in Solna, Stockholm. In the early 1950ies, Nils Söderström started using the technique at the University Hospital in Lund. Cytology was successively established as common practice at the pathology departments in Sweden, and e.g. Solna and Lund today have a high rate of cytological samples. Over the years new techniques, such as endobronchial ultrasound (EBUS)-guided fine-needle aspirations, and analyses have been introduced, contributing to the maintained value of cytology as a diagnostic method. In this article, we present a brief history and the current situation of cytology in Sweden with focus on interventional and EBUS cytology

Microenvironment-Dependent Phenotypic Changes in a SCID Mouse Model for Malignant Mesothelioma

Background and Aims: Malignant mesothelioma is an aggressive, therapy-resistant tumor. Mesothelioma cells may assume an epithelioid or a sarcomatoid phenotype, and presence of sarcomatoid cells predicts poor prognosis. In this study, we investigated differentiation of mesothelioma cells in a xenograft model, where mesothelioma cells of both phenotypes were induced to form tumors in severe combined immunodeficiency mice. Methods: Xenografts were established and thoroughly characterized using a comprehensive immunohistochemical panel, array comparative genomic hybridization (aCGH) of chromosome 3, fluorescent in situ hybridization, and electron microscopy. Results: Epithelioid and sarcomatoid cells gave rise to xenografts of similar epithelioid morphology. While sarcomatoid-derived xenografts had higher growth rates, the morphology and expression of differentiation-related markers was similar between xenografts derived from both phenotypes. aCGH showed a convergent genotype for both xenografts, resembling the original aggressive sarcomatoid cell sub-line. Conclusion: Human mesothelioma xenografts from sarcomatoid and epithelioid phenotypes converged to a similar differentiation state, and genetic analyses suggested that clonal selection in the mouse microenvironment was a major contributing factor. This thoroughly characterized animal model can be used for further studies of molecular events underlying tumor cell differentiation

Proliferation and Immune Response Gene Signatures Associated with Clinical Outcome to Immunotherapy and Targeted Therapy in Metastatic Cutaneous Malignant Melanoma

Targeted therapy (TT), together with immune checkpoint inhibitors (ICI), has significantly improved clinical outcomes for patients with advanced cutaneous malignant melanoma (CMM) during the last decade. However, the magnitude and the duration of response vary considerably. There is still a paucity of predictive biomarkers to identify patients who benefit most from treatment. To address this, we performed targeted transcriptomics of CMM tumors to identify biomarkers associated with clinical outcomes. Pre-treatment tumor samples from 28 patients with advanced CMM receiving TT (n = 13) or ICI (n = 15) were included in the study. Targeted RNA sequencing was performed using Ion AmpliSeq ™, followed by gene set enrichment analysis (GSEA) using MSigDB’s Hallmark Gene Set Collection to identify gene expression signatures correlating with treatment outcome. The GSEA demonstrated that up-regulation of allograft rejection genes, together with down-regulation of E2F and MYC targets as well as G2M checkpoint genes, significantly correlated with longer progression-free survival on ICI while IFNγ and inflammatory response genes were associated with a better clinical outcome on TT. In conclusion, we identify novel genes and their expression signatures as potential predictive biomarkers for TT and ICI in patients with metastatic CMM, paving the way for clinical use following larger validation studies

Segmental duplications and evolutionary plasticity at tumor chromosome break-prone regions

We have previously found that the borders of evolutionarily conserved chromosomal regions often coincide with tumor-associated deletion breakpoints within human 3p12-p22. Moreover, a detailed analysis of a frequently deleted region at 3p21.3 (CER1) showed associations between tumor breaks and gene duplications. We now report on the analysis of 54 chromosome 3 breaks by multipoint FISH (mpFISH) in 10 carcinoma-derived cell lines. The centromeric region was broken in five lines. In lines with highly complex karyotypes, breaks were clustered near known fragile sites, FRA3B, FRA3C, and FRA3D (three lines), and in two other regions: 3p12.3-p13 (∼75 Mb position) and 3q21.3-q22.1 (∼130 Mb position) (six lines). All locations are shown based on NCBI Build 36.1 human genome sequence. The last two regions participated in three of four chromosome 3 inversions during primate evolution. Regions at 75, 127, and 131 Mb positions carry a large (∼250 kb) segmental duplication (tumor break-prone segmental duplication [TBSD]). TBSD homologous sequences were found at 15 sites on different chromosomes. They were located within bands frequently involved in carcinoma-associated breaks. Thirteen of them have been involved in inversions during primate evolution; 10 were reused by breaks during mammalian evolution; 14 showed copy number polymorphism in man. TBSD sites showed an increase in satellite repeats, retrotransposed sequences, and other segmental duplications. We propose that the instability of these sites stems from specific organization of the chromosomal region, associated with location at a boundary between different CG-content isochores and with the presence of TBSDs and “instability elements,” including satellite repeats and retroviral sequences

Real-world diagnostic accuracy and use of immunohistochemical markers in lung cancer diagnostics

Objectives: Accurate and reliable diagnostics are crucial as histopathological type influ-ences selection of treatment in lung cancer. The aim of this study was to evaluate real-world accuracy and use of immunohistochemical (IHC) staining in lung cancer diagnostics. Materials and Methods: The diagnosis and used IHC stains for small specimens with lung cancer on follow-up resection were retrospectively investigated for a 15-month period at two major sites in Sweden. Additionally, 10 pathologists individually suggested diagnostic IHC staining for 15 scanned bronchial and lung biopsies and cytological specimens. Results: In 16 (4.7%) of 338 lung cancer cases, a discordant diagnosis of potential clinical relevance was seen between a small specimen and the fol-low-up resection. In half of the cases, there was a different small specimen from the same investi-gational work-up with a concordant diagnosis. Diagnostic inaccuracy was often related to a squa-mous marker not included in the IHC panel (also seen for the scanned cases), the case being a neu-roendocrine tumor, thyroid transcription factor-1 (TTF-1) expression in squamous cell carcinomas (with clone SPT24), or poor differentiation. IHC was used in about 95% of cases, with a higher number of stains in biopsies and in squamous cell carcinomas and especially neuroendocrine tumors. Pre-surgical transthoracic samples were more often diagnostic than bronchoscopic ones (72–85% vs. 9–53% for prevalent types). Conclusions: Although a high overall diagnostic accuracy of small specimens was seen, small changes in routine practice (such as consequent inclusion of p40 and TTF-1 clone 8G7G3/1 in the IHC panel for non-small cell cancer with unclear morphology) may lead to improvement, while reducing the number of IHC stains would be preferable from a time and cost perspective

Mandatory chromosomal segment balance in aneuploid tumor cells

BackgroundEuploid chromosome balance is vitally important for normal development, but is profoundly changed in many tumors. Is each tumor dependent on its own structurally and numerically changed chromosome complement that has evolved during its development and progression? We have previously shown that normal chromosome 3 transfer into the KH39 renal cell carcinoma line and into the Hone1 nasopharyngeal carcinoma line inhibited their tumorigenicity. The aim of the present study was to distinguish between a qualitative and a quantitative model of this suppression. According to the former, a damaged or deleted tumor suppressor gene would be restored by the transfer of a normal chromosome. If so, suppression would be released only when the corresponding sequences of the exogenous normal chromosome are lost or inactivated. According to the alternative quantitative model, the tumor cell would not tolerate an increased dosage of the relevant gene or segment. If so, either a normal cell derived, or, a tumor derived endogenous segment could be lost.MethodsFluorescence in Situ Hybridization based methods, as well as analysis of polymorphic microsatellite markers were used to follow chromosome 3 constitution changes in monochromosomal hybrids.ResultsIn both tumor lines with introduced supernumerary chromosomes 3, the copy number of 3p21 or the entire 3p tended to fall back to the original level during both in vitro and in vivo growth. An exogenous, normal cell derived, or an endogenous, tumor derived, chromosome segment was lost with similar probability. Identification of the lost versus retained segments showed that the intolerance for increased copy number was particularly strong for 3p14-p21, and weaker for other 3p regions. Gains in copy number were, on the other hand, well tolerated in the long arm and particularly the 3q26-q27 region.ConclusionThe inability of the cell to tolerate an experimentally imposed gain in 3p14-p21 in contrast to the well tolerated gain in 3q26-q27 is consistent with the fact that the former is often deleted in human tumors, whereas the latter is frequently amplified. The findings emphasize the importance of even minor changes in copy number in seemingly unbalanced aneuploid tumors

The long-term prognostic and predictive capacity of cyclin D1 gene amplification in 2305 breast tumours

Abstract Background Use of cyclin D1 (CCND1) gene amplification as a breast cancer biomarker has been hampered by conflicting assessments of the relationship between cyclin D1 protein levels and patient survival. Here, we aimed to clarify its prognostic and treatment predictive potential through comprehensive long-term survival analyses. Methods CCND1 amplification was assessed using SNP arrays from two cohorts of 1965 and 340 patients with matching gene expression array and clinical follow-up data of over 15 years. Kaplan-Meier and multivariable Cox regression analyses were used to determine survival differences between CCND1 amplified vs. non-amplified tumours in clinically relevant patient sets, within PAM50 subtypes and within treatment-specific subgroups. Boxplots and differential gene expression analyses were performed to assess differences between amplified vs. non-amplified tumours within PAM50 subtypes. Results When combining both cohorts, worse survival was found for patients with CCND1-amplified tumours in luminal A (HR = 1.68; 95% CI, 1.15–2.46), luminal B (1.37; 1.01–1.86) and ER+/LN−/HER2− (1.66; 1.14–2.41) subgroups. In gene expression analysis, CCND1-amplified luminal A tumours showed increased proliferation (P < 0.001) and decreased progesterone (P = 0.002) levels along with a large overlap in differentially expressed genes when comparing luminal A and B-amplified vs. non-amplified tumours. Conclusions Our results indicate that CCND1 amplification is associated with worse 15-year survival in ER+/LN−/HER2−, luminal A and luminal B patients. Moreover, luminal A CCND1-amplified tumours display gene expression changes consistent with a more aggressive phenotype. These novel findings highlight the potential of CCND1 to identify patients that could benefit from long-term treatment strategies