A renewed model of pancreatic cancer evolution based on genomic rearrangement patterns.

Pancreatic cancer, a highly aggressive tumour type with uniformly poor prognosis, exemplifies the classically held view of stepwise cancer development. The current model of tumorigenesis, based on analyses of precursor lesions, termed pancreatic intraepithelial neoplasm (PanINs) lesions, makes two predictions: first, that pancreatic cancer develops through a particular sequence of genetic alterations (KRAS, followed by CDKN2A, then TP53 and SMAD4); and second, that the evolutionary trajectory of pancreatic cancer progression is gradual because each alteration is acquired independently. A shortcoming of this model is that clonally expanded precursor lesions do not always belong to the tumour lineage, indicating that the evolutionary trajectory of the tumour lineage and precursor lesions can be divergent. This prevailing model of tumorigenesis has contributed to the clinical notion that pancreatic cancer evolves slowly and presents at a late stage. However, the propensity for this disease to rapidly metastasize and the inability to improve patient outcomes, despite efforts aimed at early detection, suggest that pancreatic cancer progression is not gradual. Here, using newly developed informatics tools, we tracked changes in DNA copy number and their associated rearrangements in tumour-enriched genomes and found that pancreatic cancer tumorigenesis is neither gradual nor follows the accepted mutation order. Two-thirds of tumours harbour complex rearrangement patterns associated with mitotic errors, consistent with punctuated equilibrium as the principal evolutionary trajectory. In a subset of cases, the consequence of such errors is the simultaneous, rather than sequential, knockout of canonical preneoplastic genetic drivers that are likely to set-off invasive cancer growth. These findings challenge the current progression model of pancreatic cancer and provide insights into the mutational processes that give rise to these aggressive tumours.

Correction to: Use of multicolor fluorescence in situ hybridization to detect deletions in clinical tissue sections.

Figure 2 is incorrect in the original version of this article. The correct figure 2 is provided below.

Use of multicolor fluorescence in situ hybridization to detect deletions in clinical tissue sections.

A variety of laboratory methods are available for the detection of deletions of tumor suppressor genes and losses of their proteins. The clinical utility of fluorescence in situ hybridization (FISH) for the identification of deletions of tumor suppressor genes has previously been limited by difficulties in the interpretation of FISH signal patterns. The first deletion FISH assays using formalin-fixed paraffin-embedded tissue sections had to deal with a significant background level of signal losses affecting nuclei that are truncated by the cutting process of slide preparation. Recently, more efficient probe designs, incorporating probes adjacent to the tumor suppressor gene of interest, have increased the accuracy of FISH deletion assays so that true chromosomal deletions can be readily distinguished from the false signal losses caused by sectioning artifacts. This mini-review discusses the importance of recurrent tumor suppressor gene deletions in human cancer and reviews the common FISH methods being used to detect the genomic losses encountered in clinical specimens. The use of new probe designs to recognize truncation artifacts is illustrated with a four-color PTEN FISH set optimized for prostate cancer tissue sections. Data are presented to show that when section thickness is reduced, the frequency of signal truncation losses is increased. We also provide some general guidelines that will help pathologists and cytogeneticists run routine deletion FISH assays and recognize sectioning artifacts. Finally, we summarize how recently developed sequence-based approaches are being used to identify recurrent deletions using small DNA samples from tumors.

Canadian Cancer Trials Group IND197: a phase II study of foretinib in patients with estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2-negative recurrent or metastatic breast cancer.

In murine models, overexpression of the MET receptor transgene induces tumors with human basal gene expression characteristics supporting MET inhibition as a treatment strategy for triple-negative breast cancer (TNBC). Foretinib is an oral multi-kinase inhibitor of MET, RON, AXL, TIE-2, and VEGF receptors with anti-tumor activity in advanced HCC and papillary renal cell cancer. Patients with centrally reviewed primary TNBC and 0-1 prior regimens for metastatic disease received daily foretinib 60 mg po in a 2-stage single-arm trial. Primary endpoints were objective response and early progression rates per RECIST 1.1. In stage 2, correlative studies of MET, PTEN, EGFR, and p53 on archival and fresh tumor specimens were performed along with enumeration of CTCs. 45 patients were enrolled with 37 patients having response evaluable and centrally confirmed primary TNBC (cTNBC). There were 2 partial responses (ITT 4.7 % response evaluable cTNBC 5.4 %) with a median duration of 4.4 months (range 3.7-5 m) and 15 patients had stable disease (ITT 33 %, response evaluable cTNBC 40.5 %) with a median duration of 5.4 months (range 2.3-9.7 m). The most common toxicities (all grades/grade 3) were nausea (64/4 %), fatigue (60/4 %), hypertension (58/49 %), and diarrhea (40/7 %). Six serious adverse events were considered possibly related to foretinib and 4 patients went off study due to adverse events. There was no correlation between MET positivity and response nor between response and PTEN, EGFR, p53, or MET expression in CTCs. Although CCTG IND 197 did not meet its primary endpoint, the observation of a clinical benefit rate of 46 % in this cTNBC population suggests that foretinib may have clinical activity as a single, non-cytotoxic agent in TNBC (ClinicalTrials.gov number, NCT01147484).

Analytic validation of a clinical-grade PTEN immunohistochemistry assay in prostate cancer by comparison with PTEN FISH.

PTEN loss is a promising prognostic and predictive biomarker in prostate cancer. Because it occurs most commonly via PTEN gene deletion, we developed a clinical-grade, automated, and inexpensive immunohistochemical assay to detect PTEN loss. We studied the sensitivity and specificity of PTEN immunohistochemistry relative to four-color fluorescence in situ hybridization (FISH) for detection of PTEN gene deletion in a multi-institutional cohort of 731 primary prostate tumors. Intact PTEN immunostaining was 91% specific for the absence of PTEN gene deletion (549/602 tumors with two copies of the PTEN gene by FISH showed intact expression of PTEN by immunohistochemistry) and 97% sensitive for the presence of homozygous PTEN gene deletion (absent PTEN protein expression by immunohistochemistry in 65/67 tumors with homozygous deletion). PTEN immunohistochemistry was 65% sensitive for the presence of hemizygous PTEN gene deletion, with protein loss in 40/62 hemizygous tumors. We reviewed the 53 cases where immunohistochemistry showed PTEN protein loss and FISH showed two intact copies of the PTEN gene. On re-review, there was ambiguous immunohistochemistry loss in 6% (3/53) and failure to analyze the same tumor area by both methods in 34% (18/53). Of the remaining discordant cases, 41% (13/32) revealed hemizygous (n=8) or homozygous (n=5) PTEN gene deletion that was focal in most cases (11/13). The remaining 19 cases had two copies of the PTEN gene detected by FISH, representing truly discordant cases. Our automated PTEN immunohistochemistry assay is a sensitive method for detection of homozygous PTEN gene deletions. Immunohistochemistry screening is particularly useful to identify cases with heterogeneous PTEN gene deletion in a subset of tumor glands. Mutations, small insertions, or deletions and/or epigenetic or microRNA-mediated mechanisms may lead to PTEN protein loss in tumors with normal or hemizygous PTEN gene copy number.

Depletion of bone marrow CCSP-expressing cells delays airway regeneration.

The contribution of bone marrow cells (BMC) in lung repair is controversial. We previously reported a subpopulation of BMC that express Clara cell secretory protein (CCSP). To determine the contribution of endogenous CCSP(+) BMC to airway regeneration, we performed bone marrow transplantation studies using the CCtk mouse, which expresses a thymidine kinase suicide gene under regulation of the CCSP promoter. Mice were transplanted with wild-type or CCtk BMC and treated with ganciclovir to eliminate CCSP(+) cells. After airway injury using naphthalene, mice depleted of CCSP(+) BMC had more inflammatory cells in lung and decreased levels of oxygen in arterial blood. They also had reduced expression of airway epithelial genes and less Clara cells compared to control mice that had intact CCSP(+) BMC and bone marrow derived CCSP(+) cells in the airways. After naphthalene injury, administration of CCSP reproduced the beneficial effect of CCSP(+) BMC by improving recovery of airway epithelium, reducing lung inflammation and increasing oxygen in arterial blood from mice depleted of CCSP(+) BMC. Our data demonstrate that ablation of CCSP(+) BMC delays airway recovery and suggests the beneficial effect of CCSP(+) BMC in lung recovery is in part due to production of CCSP itself.

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% O(2) (anoxia), 0.2% O(2) (hypoxia) or 21% O(2) (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.

Bone marrow cells expressing clara cell secretory protein increase epithelial repair after ablation of pulmonary clara cells.

We have previously reported a subpopulation of bone marrow cells (BMC) that express Clara cell secretory protein (CCSP), generally felt to be specific to lung Clara cells. Ablation of lung Clara cells has been reported using a transgenic mouse that expresses thymidine kinase under control of the CCSP promoter. Treatment with ganciclovir results in permanent elimination of CCSP(+) cells, failure of airway regeneration, and death. To determine if transtracheal delivery of wild-type bone marrow CCSP(+) cells is beneficial after ablation of lung CCSP(+) cells, transgenic mice were treated with ganciclovir followed by transtracheal administration of CCSP(+) or CCSP(-) BMC. Compared with mice administered CCSP(-) cells, mice treated with CCSP(+) cells had more donor cells lining the airway epithelium, where they expressed epithelial markers including CCSP. Although donor CCSP(+) cells did not substantially repopulate the airway, their administration resulted in increased host ciliated cells, better preservation of airway epithelium, reduction of inflammatory cells, and an increase in animal survival time. Administration of CCSP(+) BMC is beneficial after permanent ablation of lung Clara cells by increasing bronchial epithelial repair. Therefore, CCSP(+) BMC could be important for treatment of lung diseases where airways re-epithelialization is compromised.

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.

PTEN genomic deletions that characterize aggressive prostate cancer originate close to segmental duplications.

Deletion of PTEN at 10q23.3 occurs in ∼40% of human prostate cancers and is associated with aggressive metastatic potential, poor prognosis, and androgen-independence. This high frequency of recurrent PTEN deletions in prostate cancer suggests there may be unusual genomic features close to this locus that facilitate DNA alteration at 10q23.3. To explore possible mechanisms for deletions in the PTEN region, a meta-analysis of 311 published human genome array datasets was conducted and determined that the minimal prostate cancer-associated deletion at 10q23.3 corresponds to ∼2.06 MB region flanked by BMPR1A and FAS. On a separate cohort comprising an additional 330 tumors, four-color fluorescence in situ hybridization analysis using probes for BMPR1A, FAS, cen(10), and PTEN showed that 132 of 330 (40%) tumors had PTEN loss, 50 (15%) of which were homozygous losses (comprising in total 100 deletion events). Breakpoints between PTEN and BMPR1A or FAS were subsequently mapped in 100 homozygous and 82 hemizygous PTEN losses, revealing that 125/182 PTEN microdeletions occurred within the 940 kB interval between BMPR1A and PTEN. Furthermore, this breakpoint interval coincides with a repeat-rich region of 414 kB containing the SD17 and SD18 segmental duplications, which contain at least 13 homologous inverted repeat sequences. Together, these data suggest that a strong selective growth advantage for loss of PTEN and upregulation of PI3K/AKT, combined with the close proximity of PTEN to a large unstable segment of repeated DNA comprising SD17 and SD18, can lead to recurrent microdeletions of the PTEN gene in prostate cancer. © 2011 Wiley Periodicals, Inc.

Lymphomas with concurrent BCL2 and MYC translocations: the critical factors associated with survival.

BCL2 and MYC are oncogenes commonly deregulated in lymphomas. Concurrent BCL2 and MYC translocations (BCL2(+)/MYC(+)) were identified in 54 samples by karyotype and/or fluorescence in situ hybridization with the aim of correlating clinical and cytogenetic characteristics to overall survival. BCL2(+)/MYC(+) lymphomas were diagnosed as B-cell lymphoma unclassifiable (BCLU; n = 36) with features intermediate between Burkitt lymphoma and diffuse large B-cell lymphoma (DLBCL); DLBCL (n = 17), or follicular lymphoma (n = 1). Despite the presence of a t(14;18), 5 cases were BCL2 protein-negative. Nonimmunoglobulin gene/MYC (non-IG/MYC) translocations occurred in 24 of 54 cases (44%) and were highly associated with DLBCL morphology (P < .001). Over a median follow-up of 5.3 years, 6 patients remained in remission and 32 died within 6 months of the MYC(+) rearrangement, irrespective of whether MYC(+) occurred at diagnosis (31 of 54) or transformation (23 of 54; P = .53). A non-IG/MYC translocation partner, absent BCL2 protein expression and treatment with rituximab-based chemotherapy, were associated with a more favorable outcome, but a low International Prognostic Index score and DLBCL morphology were independent predictors of overall survival. A comprehensive cytogenetic analysis of BCL2 and MYC status on all aggressive lymphomas may identify a group of high-risk patients who may benefit from chemotherapeutic regimens that include rituximab and/or BCL2-targeted therapy.

BRAF V600E mutation and MET amplification as resistance pathways of the second-generation anaplastic lymphoma kinase (ALK) inhibitor alectinib in lung cancer.

BACKGROUND: Anaplastic lymphoma kinase (ALK) targeted therapies have demonstrated remarkable efficacy in ALK-positive lung adenocarcinomas. However, patients inevitably develop resistance to such therapies. To investigate novel mechanisms of resistance to second generation ALK inhibitors, we characterized and modeled ALK inhibitor resistance of ALK-positive patient-derived xenograft (PDX) models established from advanced-stage lung adenocarcinoma patients who have progressed on one or more ALK inhibitors. METHODS: Whole exome sequencing was performed to identify resistance mechanisms to ALK inhibitors in PDXs generated from biopsies at the time of relapse. ALK fusion status was confirmed using fluorescent in situ hybridization, immunohistochemistry, RNA-sequencing, RT-qPCR and western blot. Targeted therapies to overcome acquired resistance were then tested on the PDX models. RESULTS: Three PDX models were successfully established from biopsies of two patients who had progressed on crizotinib and/or alectinib. The PDX models recapitulated the histology and ALK status of their patient tumors, as well as their matched patients' clinical treatment outcome to ALK inhibitors. Whole exome sequencing identified MET amplification and previously unreported BRAF V600E mutation as independent mechanisms of resistance to alectinib. Importantly, PDX treatment of inhibitors specific for these targets combined with ALK inhibitor overcame resistance. CONCLUSIONS: Bypass signaling pathway through c-MET and BRAF are independent mechanisms of resistance to alectinib. Individualized intervention against these resistance pathways could be viable therapeutic options in alectinib-refractory lung adenocarcinoma.

Phase II Trial of Cabozantinib in Recurrent/Metastatic Endometrial Cancer: A Study of the Princess Margaret, Chicago, and California Consortia (NCI9322/PHL86).

PURPOSE: The relevance of the MET/hepatocyte growth factor pathway in endometrial cancer tumor biology supports the clinical evaluation of cabozantinib in this disease. PATIENTS AND METHODS: PHL86/NCI#9322 (NCT01935934) is a single arm study that evaluated cabozantinib (60 mg once daily) in women with endometrial cancer with progression after chemotherapy. Coprimary endpoints were response rate and 12-week progression-free-survival (PFS). Patients with uncommon histology endometrial cancer (eg, carcinosarcoma and clear cell) were enrolled in a parallel exploratory cohort. RESULTS: A total of 102 patients were accrued. Among 36 endometrioid histology patients, response rate was 14%, 12-week PFS rate was 67%, and median PFS was 4.8 months. In serous cohort of 34 patients, response rate was 12%, 12-week PFS was 56%, and median PFS was 4.0 months. In a separate cohort of 32 patients with uncommon histology endometrial cancer (including carcinosarcoma), response rate was 6% and 12-week PFS was 47%. Six patients were on treatment for >12 months, including two for >30 months. Common cabozantinib-related toxicities (>30% patients) included hypertension, fatigue, diarrhea, nausea, and hand-foot syndrome. Gastrointestinal fistula/perforation occurred in four of 70 (6%) patients with serous/endometrioid cancer and five of 32 (16%) patients in exploratory cohort. We observed increased frequency of responses with somatic CTNNB1 mutation [four partial responses (PRs) in 10 patients, median PFS 7.6 months] and concurrent KRAS and PTEN/PIK3CA mutations (three PRs in 12 patients, median PFS 5.9 months). CONCLUSIONS: Cabozantinib has activity in serous and endometrioid histology endometrial cancer. These results support further evaluation in genomically characterized patient cohorts.

Cytogenetic abnormalities and clinical correlations in peripheral T-cell lymphoma.

Cytogenetic correlations among most types of peripheral T-cell lymphoma (PTCL) have not been very informative to date. This study aimed to identify recurrent chromosomal abnormalities in angioimmunoblastic T-cell lymphoma (AITL), ALK-negative anaplastic large cell lymphoma (ALK-ALCL) and peripheral T-cell lymphoma, unspecified (PTCL-US), and to evaluate their prognostic value. We reviewed the cytogenetic findings of 90 previously-diagnosed cases of PTCL and correlated the cytogenetic findings with the specific histological subtype. The most common abnormalities for AITL were 5q (55%), 21 (41%) and 3q (36%) gains, concurrent trisomies of 5 and 21 (41%), and loss of 6q (23%). In ALK(-) ALCL, gains of 1q (50%) and 3p (30%), and losses of 16pter (50%), 6q13q21 (30%), 15 (30%), 16qter (30%) and 17p13 (30%) were frequent findings. In PTCL-US, frequent gains involved 7q22q31 (33%), 1q (24%), 3p (20%), 5p (20%), and 8q24qter (22%), and losses of 6q22q24 (26%) and 10p13pter (26%). We did not observe any association between specific chromosomal abnormalities and overall survival (OS). However, cases with complex karyotypes, most frequently observed in ALK(-) ALCL and PTCL-US, had a significantly shorter OS. Although, genetic differences were noted in these subtypes, further studies are needed to determine the key pathogenetic events in PTCL.

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.

An Anaplastic Lymphoma Kinase Immunohistochemistry-Negative but Fluorescence In Situ Hybridization-Positive Lung Adenocarcinoma Is Resistant to Crizotinib.

INTRODUCTION: Oncogenic fusion of anaplastic lymphoma kinase (ALK) with echinoderm microtubule associated protein like 4 protein or other partner genes occurs in 3% to 6% of lung adenocarcinomas. Although fluorescence in situ hybridization (FISH) is the accepted standard for detecting anaplastic lymphoma receptor tyrosine kinase gene (ALK) gene rearrangement that gives rise to new fusion genes, not all ALK FISH-positive patients respond to ALK inhibitor therapies. We report here an ALK FISH-positive patient-derived xenograft (PDX) that was nonresponsive to crizotinib therapy. METHODS: The PDX patient human lung cancer (PHLC402) was established in NOD/SCID mice from a patient with resected pT4N1M0 lung adenocarcinoma. ALK gene status was investigated using the standard FISH break-apart assay, reverse-transcriptase quantitative polymerase chain reaction, RNA sequencing and immunohistochemical assay using the 5A4 antibody. PHLC402 was treated with crizotinib (50 mg/kg) by daily oral gavage. RESULTS: ALK FISH assay was positive in both the primary patient tumor and PDX, which were negative for ALK protein expression by immunohistochemical analysis. ALK fusion product was not detected by RNA sequencing and reverse-transcriptase quantitative polymerase chain reaction comparing the 5' and 3' ALK transcript levels. Crizotinib treatment of PHLC402 grown in mice resulted in no tumor response. CONCLUSION: ALK protein expression may be necessary for ALK FISH-positive lung cancer to be responsive to ALK inhibitor therapy.

Inhibiting MDM2-p53 Interaction Suppresses Tumor Growth in Patient-Derived Non-Small Cell Lung Cancer Xenograft Models.

BACKGROUND: The tumor suppressor p53 is frequently inactivated in non-small cell lung cancer (NSCLC). Activation of the p53 pathway by inhibition of its negative regulator MDM2 may offer an attractive approach for NSCLC therapy. We evaluated the antitumor activity of the small-molecule MDM2 inhibitor RG7388 in patient-derived xenograft (PDX) models of NSCLC. METHODS: We investigated the effect of RG7388 treatment on cell proliferation, cell cycle arrest, and apoptosis using a panel of human NSCLC cell lines (A549, H157, H1650, H1395, and H358) and PDX cell lines (human lung cell lines 12, 137, 277, and 196). PDX-bearing mice were used to test the therapeutic efficacy and pharmacodynamic effects of RG7388 treatment. RESULTS: We demonstrated that RG7388 promotes low nanomolar antiproliferative activity selectively in cell lines with wild-type p53 and p53 pathway activation, resulting in cell cycle arrest and apoptosis. In PDX models, oral administration of RG7388 led to potent dose-dependent and time-dependent activation of p53 and had a significant impact on p53 downstream targets. Daily treatment of RG7388 in mice at 50 and 80 mg/kg/day inhibited tumor growth in three wild-type p53 PDX models. Activation of the p53 pathway inhibited cell proliferation as observed by reduced Ki-67-positive cells in xenograft tumors. However, induction of apoptotic caspase activity was not observed in these tumors. Notably, RG7388 treatment remains effective in tumors lacking MDM2 amplification but expressing wild-type p53. CONCLUSIONS: MDM2 small-molecule inhibitor is effective in treating NSCLC tumors with wild-type p53, supporting further clinical investigation as a potential NSCLC therapy.

Coamplification of 12p11 and 12q13 approximately q22 in multiple ring chromosomes in a spindle cell sarcoma resolved by novel multicolor fluorescence in situ hybridization analysis.

An 80-year-old male presented with a lobulated mass in the lower abdominal wall. A diagnosis of an intermediate grade myofibroblastic spindle cell sarcoma was made. Cytogenetic analysis demonstrated a complex karyotype with a der(6), a small marker and five, different in size, ring chromosomes. Fluorescence in situ hybridization (FISH), multiplex FISH, and multicolor banding analysis was used to further delineate this complex karyotype. The der(6) was shown to be a der(18)t(6;18;9;12;18), the marker chromosome was identified as del(17), and the ring chromosomes as r(9) and r(12;18)x4. Amplification of 18 and coamplification of 12p and 12q was detected in the ring and marker chromosomes. No intercellular heterogeneity was observed although a few micronuclei containing chromosome 18 and anaphase bridges, containing chromosome 12 material, the result of bridge-fusion-bridge (BFB) cycles, were observed. Our findings combined with results from others indicate that amplification of chromosomes 12 and 18 as well as BFB phenomena characterize this type of sarcoma.

Prognostic value of fibroblast growth factor receptor 1 gene locus amplification in resected lung squamous cell carcinoma.

INTRODUCTION: Fibroblast growth factor receptor 1 (FGFR1) gene amplification was recently reported as a recurrent abnormality in 10% to 20% of primary lung squamous cell carcinomas (SqCCs), and has attracted significant interest as a potential therapeutic target. Limited data are available for its prognostic impact in early-stage SqCC. METHODS: Tissue microarrays containing 135 primary lung SqCCs and 58 matching lymph node metastases were tested by interphase fluorescence in situ hybridization for DNA copy number (CN) abnormalities at the 8p12 region including FGFR1. RESULTS: FGFR1amplification was found in 18.2% (22 of 121 evaluable) of primary SqCC, using a definition of average copies of FGFR1 per cell of 5.0 or more. Concordance rate between primaries and matching lymph node metastases was 97.7% (43 of 44; 7 amplified and 37 nonamplified), with the only discordant case showing CN at approximately the dichotomous cutoff. Similarly, concordance between two separate lymph node metastases in each of 10 patients was 100% (1 amplified and 9 nonamplified). Using various CN cutoffs, we found no statistically significant association between FGFR1 CN abnormalities and patient age, sex, tumor grade, stage, smoking status, disease-free survival, cause-specific survival, or overall survival. CONCLUSION: FGFR1 amplification is not prognostic in resected lung squamous cell carcinoma patients.