The pharmacogenomics of osteosarcoma.

Osteosarcoma (OS), the most common malignant tumor of bone, is presently treated with multidrug neoadjuvant chemotherapy protocols, which allow to cure 60-65% of patients but also induce toxicity events that cannot be predicted or efficiently prevented. The identification and validation of pharmacogenomic biomarkers is, therefore, absolutely warranted to provide the bases for planning personalized treatments with the aim to increase the therapeutic benefits and to avoid or limit unnecessary toxicities. As several targeted therapies against molecular and immunological markers in OS are presently under clinical investigation, it may be speculated that some new agents for innovative treatments may emerge in the next years. However, the real improvement of therapeutic perspectives for OS is strictly connected to the identification of pharmacogenomic biomarkers that may stratify patients in responders or non-responders and identify those individuals with higher susceptibility to treatment-associated toxicity. This review provides an overview of the pharmacogenomic biomarkers identified so far in OS, which appear to be promising candidates for a translation to clinical practice, after further investigation and/or prospective validation.

Preclinical validation of Aurora kinases-targeting drugs in osteosarcoma.

BACKGROUND: Aurora kinases are key regulators of cell cycle and represent new promising therapeutic targets in several human tumours. METHODS: Biological relevance of Aurora kinase-A and -B was assessed on osteosarcoma clinical samples and by silencing these genes with specific siRNA in three human osteosarcoma cell lines. In vitro efficacy of two Aurora kinases-targeting drugs (VX-680 and ZM447439) was evaluated on a panel of four drug-sensitive and six drug-resistant human osteosarcoma cell lines. RESULTS: Human osteosarcoma cell lines proved to be highly sensitive to both drugs. A decreased drug sensitivity was observed in doxorubicin-resistant cell lines, most probably related to ABCB1/MDR1 overexpression. Both drugs variably induced hyperploidy and apoptosis in the majority of cell lines. VX-680 also reduced in vitro cell motility and soft-agar cloning efficiency. Drug association experiments showed that VX-680 positively interacts with all conventional drugs used in osteosarcoma chemotherapy, overcoming the cross-resistance observed in the single-drug treatments. CONCLUSION: Aurora kinase-A and -B represent new candidate therapeutic targets for osteosarcoma. In vitro analysis of the Aurora kinases inhibitors VX-680 and ZM447439 indicated in VX-680 a new promising drug of potential clinical usefulness in association with conventional osteosarcoma chemotherapeutic agents.

Nucleotide excision repair gene variants and association with survival in osteosarcoma patients treated with neoadjuvant chemotherapy.

The aim of this study was to investigate the role of common polymorphisms in the nucleotide excision repair pathway genes in the tumorigenesis of osteosarcoma and in the response to DNA damaging therapies, such as cisplatin-based neoadjuvant therapy. Excision repair cross-complementing (ERCC) group 2 (XPD; rs13181 and rs1799793), group 5 (XPG; rs17655) and group 1 (XPA; rs3212986 and rs11615) polymorphisms were analyzed in a group of 130 homogenously treated patients with high-grade osteosarcoma, for association with event-free survival (EFS), using the Kaplan-Meier plots and log-rank test. A positive association was observed between both XPD single-nucleotide polymorphisms and an increased EFS (hazards ratio (HR) = 0.34, 95% confidence interval (CI) 0.12-0.98 and HR = 0.19, 95% CI 0.05-0.77, respectively). We had also performed a case-control study for relative risk to develop osteosarcoma. Patients carrying at least one variant allele of XPD rs1799793 had a reduced risk of developing osteosarcoma, compared with wild-type patients (odds ratio = 0.55, 95% CI 0.36-0.84). This study suggests that XPD rs1799793 could be a marker of osteosarcoma associated with features conferring either a better prognosis or a better outcome after platinum therapy, or both.

Clinical impact of the methotrexate resistance-associated genes C-MYC and dihydrofolate reductase (DHFR) in high-grade osteosarcoma.

BACKGROUND: Aims of this study were the validation of C-MYC involvement in methotrexate (MTX) resistance and the assessment of clinical impact of C-MYC and dihydrofolate reductase (DHFR) in osteosarcoma (OS). MATERIALS AND METHODS: The involvement of C-MYC in MTX resistance was validated with an antisense approach. C-MYC and DHFR protein levels at diagnosis were assessed by immunohistochemistry on series of patients treated with either a MTX-based protocol (IOR/OS-1; 72 patients) or with a standard four-drug regimen (ISG/SSG 1; 61 patients). RESULTS: Down-regulation of C-MYC significantly decreased the MTX resistance level of OS cells, demonstrating its causal involvement in this phenomenon. In clinical samples, a worse outcome was associated with increased levels of DHFR and C-MYC at diagnosis in the IOR/OS-1 patients and of C-MYC in the ISG/SSG 1 patients. CONCLUSIONS: Meanwhile the adverse clinical impact of DHFR overexpression appeared to be closely related to the relevance of MTX in the chemotherapeutic protocol, that of C-MYC overexpression was more general and not strictly MTX related. The assessment of C-MYC and DHFR at diagnosis, together with that of other known prognostic markers, can be considered for an early identification of subgroups of OS patients with higher risk of adverse outcome.

Molecular alterations of monophasic synovial sarcoma: loss of chromosome 3p does not alter RASSF1 and MLH1 transcriptional activity.

Differential diagnosis of monophasic synovial sarcoma requires the detection of specific biological markers. In this study we evaluated the presence of molecular alterations in 15 monophasic synovial sarcomas. Multiple changes affecting chromosome arms were detected by CGH-array in all microdissected cases available, and an association between gain or loss of specific regions harbouring cancer progression-associated genes and aneuploid status was found. The most frequent alteration was loss of 3p including 3p21.3-p23 region that, however, did not involve the promoter regions of the corresponding genes, RASSF1 and MLH1. Using Real-Time PCR, mRNA levels of both resulted moderately high compared to normal tissue; however, the weak to absent protein expression suggests RASSF1 and MLH1 post-transcription deregulation. Moreover, immunohistochemical analysis revealed that both mesenchymal and epithelial antigens were present in diploid tumours. These findings confirm the genetic complexity of monophasic synovial sarcoma and underline the need to integrate different analyses for a better knowledge of this tumour, essential to investigate new diagnostic and prognostic markers.

The molecular mechanisms responsible for resistance to ET-743 (Trabectidin; Yondelis) in the Ewing's sarcoma cell line, TC-71.

Identification of new active agents against sarcoma is considered an important challenge in medical oncology. ET-743 (Trabectidin; Yondelis) has recently emerged as the first active drug developed against sarcoma in the last two decades, with promising results especially against soft-tissue sarcoma and Ewing's sarcoma (ES). In this study, we analyzed the molecular mechanisms responsible for resistance to ET-743 in ES cells. Three resistant cell variants (TC/ET 3 nM, TC/ET 6 nM and TC/ET 12 nM) were obtained, showing 28-, 47- and 102-fold increase in ET-743 resistance. Cross-resistance to other drugs was analyzed. Comparative genomic hybridization and cDNA microarray technology were employed to characterize and compare the gene expression profile of two TC/ET variants with the parental cell line. TC/ET cells show a conventional multidrug resistance phenotype and P-glycoprotein overexpression was found to significantly contribute to ET-743 resistance. However, functional studies with the cyclosporine analogue, PSC-833, indicate that other mechanisms are involved in resistance to ET-743. The gene expression profile of TC/ET cells indicated, among up-regulated genes, an increase in expression of insulin-like growth factor receptor-I (IGF-IR) and one of its major intracellular mediators, insulin receptor substrate-1. Functional studies using a neutralizing antibody anti-IGF-IR confirmed involvement of this signaling pathway in resistance to ET-743. Simultaneous blockage of both P-glycoprotein and IGF-IR completely restored sensitivity to ET-743 in ES cells. Overall, these findings provide impetus for future studies testing the therapeutic value of new specific inhibitors of P-glycoprotein and IGF-IR, which could represent a concrete therapeutic option for ES patients refractory to conventional agents.

Analysis of dihydrofolate reductase and reduced folate carrier gene status in relation to methotrexate resistance in osteosarcoma cells.

BACKGROUND: To evaluate the impact of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes on methotrexate (MTX) resistance in osteosarcoma cells in relation to retinoblastoma (RB1) gene status. MATERIALS AND METHODS: A series of human osteosarcoma cell lines-either sensitive or resistant to MTX-and 16 osteosarcoma tumour samples were used in this study. RESULTS: In U-2OS MTX-resistant variants, and in other RB1-positive cell lines, MTX resistance was associated with increased levels of DHFR and with a slight decrease of RFC gene expression. In Saos-2 MTX-resistant variants, and in another RB1-negative cell line, development of MTX resistance was associated with a decrease in expression of RFC, without any significant involvement of DHFR. In osteosarcoma clinical samples, amplification of the DHFR gene at clinical onset appeared to be more frequent in RB1-positive compared with RB1-negative tumours. CONCLUSIONS: Amplification of the DHFR gene may occur more frequently in the presence of RB1-mediated negative regulation of its activity and can be present at clinical onset in osteosarcoma patients. Simultaneous evaluation of RFC, DHFR and RB1 gene status at the time of diagnosis may become the basis for the identification of potentially MTX-unresponsive osteosarcoma patients, who could benefit from treatment protocols with alternative antifolate drugs.

Quality assessment of genetic markers used for therapy stratification.

PURPOSE: Therapy stratification based on genetic markers is becoming increasingly important, which makes commitment to the highest possible reliability of the involved markers mandatory. In neuroblastic tumors, amplification of the MYCN gene is an unequivocal marker that indicates aggressive tumor behavior and is consequently used for therapy stratification. To guarantee reliable and standardized quality of genetic features, a quality-assessment study was initiated by the European Neuroblastoma Quality Assessment (ENQUA; connected to International Society of Pediatric Oncology) Group. MATERIALS AND METHODS: One hundred thirty-seven coded specimens from 17 tumors were analyzed in 11 European national/regional reference laboratories using molecular techniques, in situ hybridization, and flow and image cytometry. Tumor samples with divergent results were re-evaluated. RESULTS: Three hundred fifty-two investigations were performed, which resulted in 23 divergent findings, 17 of which were judged as errors after re-evaluation. MYCN analyses determined by Southern blot and in situ hybridization led to 3.7% and 4% of errors, respectively. Tumor cell content was not indicated in 32% of the samples, and 11% of seemingly correct MYCN results were based on the investigation of normal cells (eg, Schwann cells). Thirty-eight investigations were considered nonassessable. CONCLUSION: This study demonstrated the importance of revealing the difficulties and limitations for each technique and problems in interpreting results, which are crucial for therapeutic decisions. Moreover, it led to the formulation of guidelines that are applicable to all kinds of tumors and that contain the standardization of techniques, including the exact determination of the tumor cell content. Finally, the group has developed a common terminology for molecular-genetic results.

Prognostic impact of chromosomal aberrations in Ewing tumours.

Although greater than 50% of Ewing tumours contain non-random cytogenetic aberrations in addition to the pathognomonic 22q12 rearrangements, little is known about their prognostic significance. To address this question, tumour samples from 134 Ewing tumour patients were analysed using a combination of classical cytogenetics, comparative genomic and fluorescence in situ hybridisation. The evaluation of the compiled data revealed that gain of chromosome 8 occurred in 52% of Ewing tumours but was not a predictive factor for outcome. Gain of 1q was associated with adverse overall survival and event-free survival in all patients, irrespective of whether the tumour was localised or disseminated (overall survival: P=0.002 and P=0.029; event-free survival: P=0.018 and P=0.010). Loss of 16q was a significant predictive factor for adverse overall survival in all patients (P=0.008) and was associated with disseminated disease at diagnosis (P=0.039). Gain of chromosome 12 was associated with adverse event-free survival (P=0.009) in patients with localised disease. These results indicate that in addition to a 22q12 rearrangement confirmation in Ewing tumours it is important to assess the copy number of 1q and 16q to identify patients with a higher probability of adverse outcome.

The EWS protein is dispensable for Ewing tumor growth.

EWS encodes a ubiquitously expressed RNA binding protein with largely unknown function. In Ewing sarcoma family tumors (EFT), one allele is rearranged with an ETS gene. This is the first description of an EFT with a complete EWS deficiency in the presence of two copies of a rearranged chromosome 22 carrying an interstitial EWS-FLI1 translocation. Absence of EWS protein suggested that it is dispensable for EFT growth. By sequencing of EWS cDNA from unrelated EFTs, we excluded inactivation of EWS as a general mechanism in EFT pathogenesis. Rather, EWS was found to be uniformly expressed in two splicing variants of similar abundancy, EWSalpha and EWSbeta, which differ in a single amino acid. Three EWS negative cell lines were established, which will serve as valuable models to study normal and aberrant EWS function upon reintroduction into the tumor cells.

Fine-mapping of cytogenetically undetectable EWS/ERG fusions on DNA fibers of Ewing tumors.

In contrast to the EWS/FLI1 fusion which is represented by a t(11;22)(q24;q12), EWS/ERG fusions are frequently cytogenetically not detectable. Three Ewing tumors (ET), two with apparently normal chromosomes 21 and 22, and one ET with a t(2;22)(p25;q12), were studied by FISH on interphase nuclei, metaphase chromosomes and on DNA fibers. EWS/ERG transcripts were detected by RT-PCR in all cases. FISH, using cosmids located proximally (F10, G9) and distally (F7) to the EWS breakpoint region, revealed no detectable separation of these probes in two cases. In contrast, co-hybridization of probe PT1526 containing the ERG breakpoint region with G9 revealed the juxtaposition of two signals per interphase nucleus in all three cases indicating the EWS/ERG fusions. Chromosome preparations displayed the juxtaposed signals on the der(22), and hybridization signals of the probes PT1526 and G9 on the non-rearranged chromosomes 21 and 22 in all cases, respectively. The PT1526 signal on the der(21) was seen only in cases 1 and 2. These results were confirmed by triple-target FISH on tumor DNA fibers. In all three cases, the hybridization pattern F10 - G9 - PT1526 indicates a centromere to telomere orientation. This finding suggests that EWS/ERG fusions in ETs may be generated by an inversion of the ERG gene or a part thereof followed by an insertion into the EWS gene on the der(22). Double-target FISH on interphase nuclei using probes flanking the EWS breakpoint region and probe PT1526 enables the detection of virtually all 22q12 rearrangements in ETs, thus providing a reliable diagnostic assay.

Automatic detection and genetic profiling of disseminated neuroblastoma cells.

BACKGROUND: Rare tumor cells circulating in the hematopoietic system can escape identification. On the other hand, the nature of these cells, positive for an immunologiCal tumor marker, cannot be determined without any genetic information. PROCEDURE: To overcome these problems a novel computer assisted scanning system for automatic cell search, analysis, and sequential repositioning was developed. This system allows an exact quantitative analysis of rare tumor cells in the bone marrow and peripheral blood by sequential immunological and molecular cytogenetic characterization. RESULTS AND CONCLUSIONS: In that virtually all tumor cells in a mixing experiment could be recovered unambiguously, we can conclude that the sensitivity of this approach is set by the number of cells available for analysis. Sequential FISH analyses of immunologically positive cells improve both the specificity and the sensitivity of the microscopic minimal residual disease detection.

Peripheral primitive neuroectodermal tumour of the cervix.

Peripheral primitive neuro-ectodermal tumours (PNET) of the cervix are very rare. Here, we report the clinical, pathological, immunohistochemical and genetic features of a case of a PNET located in the cervix. Hysterectomy revealed a cervical tumour. On microscopic examination, a vaguely lobular arrangement of uniformly appearing neoplastic cells, with round to oval nuclei, distinct nuclear membranes and a clear, moderately glycogen-rich cytoplasm was seen. Cells stained positive for LEU 7, S 100, monoclonal NSE and particularly for MIC2. Neurogenic differentiation was also seen by electron microscopic examination. The genetic hallmark of PNET, a 22q12 rearrangement was demonstrated by fluorescence in situ hybridisation experiments, supporting the diagnosis. Awareness of the existence of primary PNET of the cervix is important to avoid confusion with other tumours of the cervix.

Molecular Cytogenetics in Ewing Tumors: Diagnostic and Prognostic Information.

Ewing tumors (ETs) are characterized by rearrangements of the EWS gene located in 22q12 and a high MIC2 expression. In 85% of ETs, the t(11;22)(q24;q12) generates chimeric fusion transcripts between the EWS and the FLI1 gene, whereas in the remaining cases the EWS gene is rearranged with different partners of the ETS oncogene family. Besides classical cytogenetic analysis, fluorescence in situ hybridization (FISH) and RT-PCR can be used to demonstrate these 22q12 rearrangements which are pathognomonic for ETs. To visualize 22q12 rearrangements in individual cells, DNA probes flanking the EWS-R1 breakpoint region on chromosome 22 can be hybridized in double-target FISH experiments on tumor cell preparations. Intact chromosomes 22 are indicated by juxtaposition of the DNA probes, whereas rearrangements of the EWS gene separate the hybridization signals. In addition to 22q12 rearrangements, numerical aberrations of chromosomes 8 and 12 can be observed in about 50% of ETs, deletions at the short arm of chromosome 1 and der (16)t(1;16)(q12;q11.2) chromosomes in about 20% of the cases. Numerical aberrations, deletions at 1p36.3, and the t(1;16) were detected by using double-target FISH on touch, cytospin, and chromosome preparations, on frozen and paraffin sections and isolated deparaffinized nuclei. So far, numerical aberrations of chromosomes 8 and 12 did not show prognostic impact. However, deletions at 1p36.3 and imbalances between the long and short arms of chromosome 1 were associated with adverse clinical outcome in a group of patients with localized disease. Copyright 2000 S. Karger GmbH, Freiburg

Prognostic impact of deletions at 1p36 and numerical aberrations in Ewing tumors.

Ewing's sarcoma, peripheral primitive neuroectodermal tumors, and Askin tumors are referred to as Ewing tumors (ETs), and are characterized by high MIC2 expression and a t(11;22)(q24;q12) or other rearrangements involving 22q12. In addition to these constant aberrations, facultative numerical and structural aberrations have been reported: gains of chromosomes 8 and 12, the unbalanced translocation t(1;16), and deletions at the short arm of chromosome 1. To evaluate the frequency and to study the biological impact of these facultative aberrations, we analyzed tumor specimens from 58 ET patients by classical cytogenetics and/or in situ hybridization techniques and compared these data with clinical parameters. Gains of chromosomes 8 and 12 were detected in 55% (32/58) and 24% (14/58) of the cases, respectively. Loss of chromosome 16 or der (16)t(1;16) chromosomes were found in 20% (10/51); deletions at 1p36 were observed in 18% (9/51) of the cases evaluated. The presence of these aberrations did not correlate with age and sex of the patients, with the location of the primary tumor or with the extent of disease at diagnosis by chi-square analysis and Fisher's exact test. Patients with tumors harboring gains of chromosome 8 showed a slightly better clinical outcome (n = 14/30, P = 0.17), whereas gains of chromosome 12 did not influence the clinical outcome (n = 7/30, P = 0.63). However, Kaplan and Meier analysis revealed that deletions at the short arm of chromosome 1 were associated with an unfavorable outcome in patients with localized disease (n = 6/22; P = 0.004).

Synthesis, processing, and transport of RNA within the three-dimensional context of the cell nucleus.

Fluorescence in situ hybridization and immunocytochemical techniques have contributed significantly to our current understanding of how transcription, RNA processing, and RNA transport are spatially and temporally organized in the cell nucleus. New technologies enabling the visualization of nuclear components in living cells specifically advanced our knowledge of the dynamic aspects of these nuclear processes. The picture that emerges from the work reviewed here shows that the positioning of genes within the three-dimensional nuclear space is of crucial importance, not only for its expression, but also for the efficient processing of its transcripts. Splicing factors are recruited from speckles to sites of active transcription, which can be present within, at the periphery, or at a relatively large distance from speckles. Furthermore, results are discussed showing that transcripts are exported by means of random diffusion.

Sensitive mRNA detection by fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification.

With the ongoing progress in human genome projects, many genes are discovered whose function and/or expression pattern are not known. Most of these genes are expressed in relatively low abundance compared to housekeeping genes such as elongation factor-1alpha and beta-actin. Gene expression is studied by Northern blot assays or by semiquantitative PCR methods. Another method is the visualization of transcripts in tissue or cell cultures by fluorescence in situ hybridization (FISH). However, for low-abundance RNA detection, this method is hampered by its limited detection sensitivity and by the interference of background signals with specific hybridization signals. Background signals are introduced by nonspecific hybridization of probe sequences or nonspecific binding of antibodies used for visualization. To eliminate background signals derived from both sources and to benefit from the peroxidase-driven tyramide signal amplification (TSA), we directly conjugated horseradish peroxidase (HRP) to oligodeoxynucleotides (ODNs) and used these probes to study in the bladder cancer cell line 5637 the expression of various cytokine genes which, according to Northern hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) assays, are expressed at levels up to 10,000-fold less than abundantly expressed housekeeping genes. The results show that reduction of probe complexity and the limited use of immunocytochemical detection layers strongly reduces noise signals derived from nonspecific binding of nucleic acid probe and antibodies. The use of the HRP-ODNs in combination with TSA allowed detection of low-abundance cytokine mRNAs by FISH.

Neuroblastoma cells can actively eliminate supernumerary MYCN gene copies by micronucleus formation--sign of tumour cell revertance?

Human neuroblastoma cell lines frequently exhibit MYCN amplification and many are characterised by the presence of morphologically distinct cell types. The neuronal cells (N-cells) and the so-called flat cells (F-cells) are thought to represent manifestations of different neural crest cell lineages and are considered to be the consequence of neuroblastoma cell pluripotency. In this study, various neuroblastoma cell lines were examined for micronuclei. In F-cells of neuroblastoma cell lines with extrachromosomally amplified MYCN, we observed the frequent occurrence of micronuclei. Using fluorescence in situ hybridisation (FISH) with a MYCN specific probe, we demonstrated that these micronuclei were packed with MYCN hybridisation signals. In addition, in a minor percentage of cells, MYCN signals occurred in clusters, adhered to the nuclear membrane and aggregated in nuclear protrusions. In F-cells, a substantial reduction or lack of amplified MYCN copies was observed. These observations let us conclude that extrachromosomally amplified genes can be actively eliminated from the nucleus resulting in a dramatic loss of amplified sequences in the F-cells. Moreover, reduction or loss of amplified sequences in F-cells was shown to be accompanied by downregulation of MYCN expression, by a decrease in proliferative activity and by upregulation of molecules of the major histocompatibility complex class I (MHC I). Interestingly, F-cells are not restricted to neuroblastoma cell cultures, but also occur in cell lines of other tissue origin. All F-cells share important biological features, interpreted as cell revertance, i.e. loss of the malignant phenotype and properties. This fact, together with the demonstration that neuroblastoma cells do not differentiate into Schwann cells in vivo [1] Ambros et al. NEJM 1996, 334, 1505-1511, do not support the hypothesis that F-cells represent Schwannian/glial differentiation in vitro. We therefore postulate that the elimination of amplified MYCN gene copies in cultivated neuroblastoma cells is in line with the phenomenon of tumour cell revertance.

Demonstration of the translocation der(16)t(1;16)(q12;q11.2) in interphase nuclei of Ewing tumors.

The der(16)t(1;16) has been detected cytogenetically in a number of malignancies including Ewing tumors (ETs). To enable fast and reliable analysis of der(16) chromosomes, we established an interphase cytogenetic approach. By using two DNA probes hybridizing to the heterochromatic portions on the long arms of chromosomes 1 and 16, this technique allows the detection of this chromosomal aberration in nonproliferating cells. Formation of the der(16) leads to partial excess of 1q material and partial loss of the long arm of chromosome 16. Double-target fluorescence in situ hybridization (FISH) experiments were performed on cytospin slides of 13 ETs, near-triploid tumor cells and normal cells to assess whether the FISH technique used permits the discrimination of nuclei harboring this aberration from nuclei without a der(16) chromosome. In five ETs, we found evidence for the presence of one or two der(16)t(1;16) chromosomes both by FISH and by conventional cytogenetics. Tumor cells displayed two signals for intact chromosomes 1, one or two additional fused signals for the der(16) chromosomes, and one signal for the intact chromosome 16. In one case without fused signals, the presence of a der(16) was demonstrated by hybridizing a painting probe for chromosome 16 simultaneously with the paracentromeric probe for chromosome 1. Our results suggest that double-target FISH on interphase nuclei offers an ideal tool for analyzing tumors prospectively and retrospectively to assess the biological role and the possible prognostic impact of the der(16) in ETs and in other solid tumors.