Olga Ludkovski

Absence of TMPRSS2:ERG fusions and PTEN losses in prostate cancer is associated with a favorable outcome

TMPRSS2:ERG gene fusions and PTEN deletions are the most common genomic aberrations in prostate cancer. Recent work has suggested that the TMPRSS2:ERG fusion is associated with a more aggressive phenotype. Similarly, PTEN deletion has been associated with biochemical recurrence and lymph node metastasis. To date, there has been no systematic analysis of the combined influence of genomic PTEN deletion with TMPRSS2:ERG gene fusions on clinical parameters of prostate cancer progression. We carried out a retrospective analysis of 125 prostate cancers with known clinical outcome using interphase fluorescence in situ hybridization to detect the relative prevalence of TMPRSS2:ERG rearrangements and/or PTEN genomic deletions. TMPRSS2:ERG rearrangement was found in 60 of 125 (48%) prostate cancers. Duplication of TMPRSS2:ERG fusion was observed in seven (6%) tumors. Gleason grade (P=0.0002)/score (P=0.001), median tumor volume (P=0.0024), preoperative PSA (P=0.001) and perineural invasion (P=0.0304) were significantly associated with biochemical recurrence by univariate analysis with TMPRSS2:ERG approaching significance (P=0.0523). By multivariate analysis, relevant factors associated with recurrence were Gleason scores 7 (P=0.001) and 8–10 (P=0.015), PTEN homozygous deletion (P=0.013) and concurrent TMPRSS2:ERG fusion and PTEN deletion (P=0.036). Kaplan–Meier analysis indicated that the presence of TMPRSS2:ERG fusion was marginally less favorable in comparison to no fusion. Duplication of fusion gene showed worse prognosis. It was possible to determine the relative frequencies of PTEN deletion and/or TMPRSS2:ERG fusions in 82 of 125 prostate cancers. With biochemical recurrence as an endpoint, the genomic biomarkers identified three patient groups: (1) ‘poor genomic grade’ characterized by both PTEN deletion and TMPRSS2:ERG fusions (23/82, 28%); (2) ‘intermediate genomic grade’ with either PTEN deletion or TMPRSS2:ERG fusion (35/82, 43%) and (3) ‘favorable genomic grade’ in which neither rearrangement was present (24/82, 29%). Kaplan–Meier and multivariate analysis indicate that TMPRSS2:ERG fusion and PTEN loss together are a predictor of earlier biochemical recurrence of disease.

The association between EGFR variant III, HPV, p16, c-MET, EGFR gene copy number and response to EGFR inhibitors in patients with recurrent or metastatic squamous cell carcinoma of the head and neck

BackgroundWe examine the potential prognostic and predictive roles of EGFR variant III mutation, EGFR gene copy number (GCN), human papillomavirus (HPV) infection, c-MET and p16INK4Aprotein expression in recurrent or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN).MethodsWe analyzed the archival tumor specimens of 53 patients who were treated in 4 phase II trials for R/M SCCHN. Two trials involved the EGFR inhibitor erlotinib, and 2 trials involved non-EGFR targeted agents. EGFRvIII mutation was determined by quantitative RT-PCR, HPV DNA by Linear Array Genotyping, p16 and c-MET protein expression by immunohistochemistry, and EGFR GCN by FISH.ResultsEGFRvIII mutation, detected in 22 patients (42%), was associated with better disease control, but no difference was seen between erlotinib-treated versus non-erlotinib treated patients. EGFRvIII was not associated with TTP or OS. The presence of HPV DNA (38%), p16 immunostaining (32%), c-MET high expression (58%) and EGFR amplification (27%), were not associated with response, TTP or OS.ConclusionEGFRvIII mutation, present in about 40% of SCCHN, appears to be an unexpected prognostic biomarker associated with better disease control in R/M SCCHN regardless of treatment with erlotinib. Larger prospective studies are required to validate its significance.

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

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

Phase II Study of Preoperative Gefitinib in Clinical Stage I Non–Small-Cell Lung Cancer

PURPOSEnEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have proven efficacy in advanced non-small-cell lung cancer (NSCLC). Their role in early-stage NSCLC has not been established. Our purpose was to explore the use of preoperative gefitinib in clinical stage I NSCLC to assess tumor response, toxicity, and clinical and molecular predictors of response.nnnPATIENTS AND METHODSnPatients received gefitinib 250 mg/d for up to 28 days, followed by mediastinoscopy and surgical resection in an open-label, single-arm study. Tumor response was evaluated by Response Evaluation Criteria in Solid Tumors. Blood samples and tumor biopsies were collected and analyzed for transforming growth factor alpha level, EGFR protein expression, EGFR gene copy number, and EGFR (exon 19 to 21) and KRAS mutations.nnnRESULTSnThirty-six patients completed preoperative treatment (median duration, 28 days; range, 27 to 30 days). Median follow-up time is 2.1 years (range, 0.86 to 3.46 years). Three patients experienced grade 3 toxicities (rash, diarrhea, and elevated ALT). Tumors demonstrated EGFR-positive protein expression in 83%, high gene copy number in 59%, EGFR mutations in 17%, and KRAS mutations in 17%. Tumor shrinkage was more frequent among women and nonsmokers. Partial response was seen in four patients (11%), and disease progression was seen in three patients (9%). The strongest predictor of response was EGFR mutation.nnnCONCLUSIONnPreoperative window therapy with gefitinib is a safe and feasible regimen in early NSCLC and provides a trial design that may better inform predictors of treatment response or sensitivity.

PTEN losses exhibit heterogeneity in multifocal prostatic adenocarcinoma and are associated with higher Gleason grade.

Prostatic adenocarcinoma is an epithelial malignancy characterized by marked histological heterogeneity. It most often has a multifocal distribution within the gland, and different Gleason grades may be present within different foci. Data from our group and others have shown that the genomic deletion of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor gene and the disruption of the ETS gene family have a central role in prostate cancer and are likely to be associated with Gleason grade. In this study, prostate cancer samples were systematically analyzed to determine whether there was concordance between PTEN losses and TMPRSS2–ERG fusion rearrangements, within or between foci in multifocal disease, using well-annotated tissue microarrays (TMAs) consisting of 724 cores derived from 142 radical prostatectomy specimens. Three-color fluorescence in situ hybridization analysis of both the PTEN deletion and the TMPRSS2–ERG fusion was used to precisely map genetic heterogeneity, both within and between tumor foci represented on the TMA. PTEN deletion was observed in 56 of 134 (42%) patients (hemizygous=42 and homozygous=14). TMPRSS2–ERG fusion was observed in 63 of 139 (45%) patients. When analyzed by Gleason pattern for a given TMA core, PTEN deletions were significantly associated with Gleason grades 4 or 5 over grade 3 (P<0.001). Although TMPRSS2–ERG fusions showed a strong relationship with PTEN deletions (P=0.007), TMPRSS2–ERG fusions did not show correlation with Gleason grade. The pattern of genetic heterogeneity of PTEN deletion was more diverse than that observed for TMPRSS2–ERG fusions in multifocal disease. However, the marked interfocal discordance for both TMPRSS2–ERG fusions and PTEN deletions was consistent with the concept that multiple foci of prostate cancer arise independently within the same prostate, and that individual tumor foci can have distinct patterns of genetic rearrangements.

Prognostic and Predictive Value of Epidermal Growth Factor Receptor Tyrosine Kinase Domain Mutation Status and Gene Copy Number for Adjuvant Chemotherapy in Non-small Cell Lung Cancer

Purpose: Patients with non-small cell lung carcinoma with epidermal growth factor receptor (EGFR) mutations may have a more favorable prognosis and greater response to chemotherapy. The effect of EGFR mutation and gene copy on patients with early-stage non-small cell lung carcinoma receiving adjuvant chemotherapy has not been reported. Patients and Methods: Tumor samples from NCIC Clinical Trials Group JBR.10, an adjuvant trial of vinorelbine/cisplatin adjuvant chemotherapy [ACT] versus observation (OBS), were analyzed for EGFR mutation by multiple sensitive methods and copy number by fluorescent in situ hybridization. Their prognostic and predictive roles were explored in correlation with survival. Results: Mutation results were available in 221 OBS and 215 ACT and fluorescent in situ hybridization results in 159 OBS and 163 ACT patients. Mutations were identified in 43 (27 OBS and 16 ACT) patients (36 sensitizing exon 19 deletions or L858R mutations). Compared with wild-type, sensitizing mutations were not significantly prognostic in OBS patients (hazard ratio [HR]: 0.79, 95% confidence interval [CI]: 0.38–1.63, p = 0.53). Although the presence of sensitizing mutations resulted in relatively greater benefit in ACT patients (HR: 0.44, 95% CI: 0.11–1.70, p = 0.22) compared with wild-type patients (HR: 0.78, 95% CI: 0.58–1.06, p = 0.12), this quantitative difference was not significant (interaction p = 0.50). Similarly, high EGFR copy was neither significantly prognostic nor predictive, although quantitatively it was associated with greater benefit from ACT. Conclusions: Trends toward longer survival and a greater benefit from chemotherapy were observed in patients with exon 19/21 mutations and high EGFR copy, although the differences were not statistically significant. The interpretation of the results was limited by the low EGFR mutation rate in this study of mainly white patients.

Chronic hypoxia compromises repair of DNA double-strand breaks to drive genetic instability

Hypoxic cells have been linked to genetic instability and tumor progression. However, little is known about the exact relationship between DNA repair and genetic instability in hypoxic cells. We therefore tested whether the sensing and repair of DNA double-strand breaks (DNA-dsbs) is altered in irradiated cells kept under continual oxic, hypoxic or anoxic conditions. Synchronized G0–G1 human fibroblasts were irradiated (0–10 Gy) after initial gassing with 0% O2 (anoxia), 0.2% O2 (hypoxia) or 21% O2 (oxia) for 16 hours. The response of phosphorylated histone H2AX (γ-H2AX), phosphorylated ataxia telangiectasia mutated [ATM(Ser1981)], and the p53 binding protein 1 (53BP1) was quantified by intranuclear DNA repair foci and western blotting. At 24 hours following DNA damage, residual γ-H2AX, ATM(Ser1981) and 53BP1 foci were observed in hypoxic cells. This increase in residual DNA-dsbs under hypoxic conditions was confirmed using neutral comet assays. Clonogenic survival was also reduced in chronically hypoxic cells, which is consistent with the observation of elevated G1-associated residual DNA-dsbs. We also observed an increase in the frequency of chromosomal aberrations in chronically hypoxic cells. We conclude that DNA repair under continued hypoxia leads to decreased repair of G1-associated DNA-dsbs, resulting in increased chromosomal instability. Our findings suggest that aberrant DNA-dsb repair under hypoxia is a potential factor in hypoxia-mediated genetic instability.

FISH assay development for the detection of p16/CDKN2A deletion in malignant pleural mesothelioma

Aims To develop a fluorescence in-situ hybridisation (FISH) assay for detecting p16/CDKN2A deletion on paraffin tissue sections for use as an ancillary test to distinguish reactive from malignant mesothelial proliferations. Method Dual-colour FISH for p16/CDKN2A and chromosome 9 (CEP-9) was performed on 11 benign mesothelial proliferations and 54 malignant pleural mesothelioma (MPM) cases to establish cut-off values for p16/CDKN2A deletion. A third MYC probe was used to verify cases showing homozygous deletion. Eight equivocal biopsies were used for assay testing. Results Cut-off values for p16/CDKN2A deletion were calculated based on FISH signalling patterns obtained from the benign controls (mean percent nuclei plus three standard deviations). Hemizygous deletion was defined as >44% of nuclei showing the hemizygous (one p16/CDKN2A, two CEP-9 signals) or >15% of nuclei showing the monosomy (one p16/CDKN2A, one CEP-9 signal) deletion patterns. None of the benign cases showed a homozygous deletion pattern (no p16/CDKN2A, at least one CEP-9 signal). In the malignant cases, the percentage of nuclei showing homozygous deletion ranged from 1% to 87%. Therefore, the cut-off value for homozygous deletion was defined as >10%. P16/CDKN2A deletion was detected in 61% (33/54) of MPM cases. Among the equivocal biopsies, four showed homozygous and one showed hemizygous p16/CDKN2A deletion. Age over 60u2005years, asbestos exposure and p16/CDKN2A deletion were associated with a worse prognosis. Conclusion Distinction between benign and malignant mesothelial proliferations can be diagnostically challenging. FISH for p16/CDKN2A deletion is a useful test for confirming the diagnosis of MPM.

Microdeletion and concurrent translocation associated with a complex TMPRSS2:ERG prostate cancer gene fusion.

In prostate cancer, >50% of tumors are characterized by a genomic rearrangement of TMPRSS2 and ERG (Tomlins et al., 2005, 2006; Ahlers and Figg, 2006; Perner et al., 2006; Wang et al., 2006). The elevated expression of TMPRSS2:ERG fusion transcripts in prostate cancers has been shown to arise by intrachromosomal rearrangement with 50 TMPRSS2 fusing in-frame with 30 ERG (Tomlins et al., 2005). Since both genes have the same transcriptional orientation and are located *2.9 Mb apart on chromosome 21, it follows that an interstitial deletion or a more complex alteration of the intervening DNA is required. Using a break-apart three-color fluorescence in situ hybridization (FISH) strategy, modifying the probe configuration previously described by Tomlins et al. (2005), we and others (Perner et al., 2006) deduced that the TMPRSS2:ERG fusion is often accompanied by a small hemizygous interstitial deletion within band 21q22, between the ERG and TMPRSS2 genes (Yoshimoto et al., 2006). Recent findings have provided additional evidence of intrachromosomal rearrangement and concurrent microdeletion in the majority of TMPRSS2:ERG fusion positive prostate cancers, using FISH, arraybased comparative genomic hybridization (aCGH) and high-resolution single nucleotide polymorphism (SNP) array analyses (Hermans et al., 2006; Iljin et al., 2006; Liu et al., 2006; Perner et al., 2006; Yoshimoto et al., 2006; Mehra et al., 2007). One limitation of the array mapping approaches is that a precise definition of the deletion interval cannot be deduced, because of the heterogeneity of the gene fusion isoforms combined with the relatively low density of SNPs within the TMPRSS2 region. In this study, we show that the systematic application of multicolor FISH methods can provide comprehensive information on the complexity of genomic alterations associated with TMPRSS2:ERG fusions in prostate cancer. Furthermore, we present a detailed FISH analysis using bacterial artificial chromosome (BAC) probes covering the entire deleted region between the TMPRSS2 and ERG loci (Fig. 1). The value of this approach is illustrated by the detection of a novel complex TMPRSS2:ERG rearrangement involving concurrent deletion and translocation. All prostatic adenocarcinoma specimens used in this study were obtained from radical prostatectomies, collected according to the Research Ethics Guidelines at the Princess Margaret Hospital (Toronto, Canada). Analysis of RNA from the 15 tumors used in this study confirmed that they all expressed the common TMPRSS2:ERG fusion transcript by RT-PCR (Tomlins et al., 2005). As expected, the 50 BAC probe for the TMPRSS2 signal colocalized with the signal of the 30 ERG BAC, confirming the presence of the typical 50 TMPRSS2:30 ERG rearrangements (not shown). In all cases except one, microdeletion defined by the 30 TMPRSS2 signal loss concurrent with 50 ERG loss was also detected. The atypical tumor presented in Figure 2 lost the 50 ERG (green) signal consistent with a microdeletion, but failed to exhibit the expected colocalization of the 50 end of TMPRSS2 (blue) with the 30 ERG fusion signal (red). Instead, an extreme nuclear separation of these two probes was apparent. Sequential FISH analysis using the BAC probes shown in Figure 1 identified a split of the typical colocalized

Analysis of segmental duplications, mouse genome synteny and recurrent cancer-associated amplicons in human chromosome 6p21-p12.

It has been proposed that regions of microhomology in the human genome could facilitate genomic rearrangements, copy number transitions, and rapid genomic change during tumor progression. To investigate this idea, this study examines the role of repetitive sequence elements, and corresponding syntenic mouse genomic features, in targeting cancer-associated genomic instability of specific regions of the human genome. Automated database-mining algorithms designed to search for frequent copy number transitions and genomic breakpoints were applied to 2 publicly-available online databases and revealed that 6p21-p12 is one of the regions of the human genome most frequently involved in tumor-specific alterations. In these analyses, 6p21-p12 exhibited the highest frequency of genomic amplification in osteosarcomas. Analysis of repetitive elements in regions of homology between human chromosome 6p and the syntenic regions of the mouse genome revealed a strong association between the location of segmental duplications greater than 5 kilobase-pairs and the position of discontinuities at the end of the syntenic region. The presence of clusters of segmental duplications flanking these syntenic regions also correlated with a high frequency of amplification and genomic alteration. Collectively, the experimental findings, in silico analyses, and comparative genomic studies presented here suggest that segmental duplications may facilitate cancer-associated copy number transitions and rearrangements at chromosome 6p21-p12. This process may involve homology-dependent DNA recombination and/or repair, which may also contribute towards the overall plasticity of the human genome.