Profiling of Oncogenic Driver Events in Lung Adenocarcinoma Revealed MET Mutation as Independent Prognostic Factor.

INTRODUCTION: Oncogenic driver mutations activating receptor tyrosine kinase pathways are promising predictive markers for targeted treatment. We investigated the mutation profile of an updated driver events list on receptor tyrosine kinase/RAS/PI3K axis and the clinicopathologic implications in a cohort of never-smoker predominated Chinese lung adenocarcinoma. METHODS: We tested 154 lung adenocarcinomas and adenosquamous carcinomas for EGFR, KRAS, HER2, BRAF, PIK3CA, MET, NRAS, MAP2K1, and RIT1 mutations by polymerase chain reaction-direct sequencing. MET amplification and ALK and ROS1 translocations were assessed by fluorescent in situ hybridizations. MET and thyroid transcription factor-1 protein expressions were investigated by immunohistochemistry. RESULTS: Seventy percent of lung adenocarcinomas carried actionable driver events. Alterations on EGFR (43%), KRAS (11.4%), ALK (6%), and MET (5.4%) were frequently found. ROS1 translocation and mutations involving BRAF, HER2, NRAS, and PIK3CA were also detected. No mutation was observed in RIT1 and MAP2K1. Patients with EGFR mutations had a favorable prognosis, whereas those with MET mutations had poorer overall survival. Multivariate analysis further demonstrated that MET mutation was an independent prognostic factor. Although MET protein expression was detected in 65% of lung adenocarcinoma, only 10% of the MET-immunohistochemistry positive tumors harbor MET DNA alterations that drove protein overexpression. Appropriate predictive biomarker is essential for selecting patients who might benefit from specific targeted therapy. CONCLUSION: Actionable driver events can be detected in two thirds of lung adenocarcinoma. MET DNA alterations define a subset of patients with aggressive diseases that might potentially benefit from anti-MET targeted therapy. High negative predictive values of thyroid transcription factor-1 and MET expression suggest potential roles as surrogate markers for EGFR and/or MET mutations.

Copy number gain of granulin-epithelin precursor (GEP) at chromosome 17q21 associates with overexpression in human liver cancer.

BACKGROUND: Granulin-epithelin precursor (GEP), a secretory growth factor, demonstrated overexpression in various human cancers, however, mechanism remain elusive. Primary liver cancer, hepatocellular carcinoma (HCC), ranks the second in cancer-related death globally. GEP controlled growth, invasion, metastasis and chemo-resistance in liver cancer. Noted that GEP gene locates at 17q21 and the region has been frequently reported to be amplified in subset of HCC. The study aims to investigate if copy number gain would associate with GEP overexpression. METHODS: Quantitative Microsatellite Analysis (QuMA) was used to quantify the GEP DNA copy number, and fluorescent in situ hybridization (FISH) was performed to consolidate the amplification status. GEP gene copy number, mRNA expression level and clinico-pathological features were analyzed. RESULTS: GEP DNA copy number determined by QuMA corroborated well with the FISH data, and the gene copy number correlated with the expression levels (n = 60, r = 0.331, P = 0.010). Gain of GEP copy number was observed in 20% (12/60) HCC and associated with hepatitis B virus infection status (P = 0.015). In HCC with increased GEP copy number, tight association between GEP DNA and mRNA levels were observed (n = 12, r = 0.664, P = 0.019). CONCLUSIONS: Gain of the GEP gene copy number was observed in 20% HCC and the frequency comparable to literatures reported on the chromosome region 17q. Increased gene copy number contributed to GEP overexpression in subset of HCC.

Detection of ALK rearrangement by immunohistochemistry in lung adenocarcinoma and the identification of a novel EML4-ALK variant.

INTRODUCTION: The echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML4-ALK) fusion gene has been identified as a potent oncogenic driver in non-small-cell lung cancer, in particular adenocarcinoma (ADC). It defines a unique subgroup of lung ADC, which may be responsive to ALK inhibitors. Detection of ALK rearrangement by fluorescence in situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR) is considered to be the standard procedure, but each with its own limitation. We evaluated the practical usefulness of immunohistochemistry (IHC) to detect ALK expression as a reliable detection method of ALK rearrangement in lung ADC. METHODS: We tested 373 lung ADCs for ALK rearrangement by IHC and FISH. Multiplex RT-PCR was performed to confirm the fusion variants. RESULTS: Twenty-two of 373 lung ACs (5.9%) were positive for ALK immunoreactivity. ALK-positive tumor cells demonstrated strong and diffused granular staining in the cytoplasm. All the ALK IHC-positive cases were confirmed to harbor ALK rearrangement, either by FISH, or RT-PCR. Two cases with positive ALK protein expression, but negative for breakapart FISH signal were shown to harbor EML4-ALK variant 1 by RT-PCR. None of the ALK IHC-negative cases were FISH-positive. In addition, we identified a novel EML4-ALK fusion variant (E3:ins53A20), and its potent transformation potential has been confirmed by in vivo tumorigenicity assay. CONCLUSION: IHC can effectively detect ALK rearrangement in lung cancer. It might provide a reliable and cost-effective diagnostic approach in routine pathologic laboratories for the identification of suitable candidates for ALK-targeted therapy.

Identification of a novel 12p13.3 amplicon in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer commonly occurring in southern China. To decipher the molecular basis of this cancer, we performed high-resolution array CGH analysis on eight tumour lines and 10 primary tumours to identify the genes involved in NPC tumorigenesis. In this study, multiple regions of gain were consistently found at 1q21-q24, 7q11-12, 7q21-22., 11q13, 12p13, 12q13, 19p13 and 19q13. Importantly, a 2.1 Mb region at 12p13.31 was highly amplified in a NPC xenograft, xeno-2117. By FISH mapping, we have further delineated the amplicon to a 1.24 region flanked by RP11-319E16 and RP11-433J6. Copy number gains of this amplicon were confirmed in 21/41 (51%) primary tumours, while three cases (7.3%) showed high copy number amplification. Among the 13 genes within this amplicon, three candidate genes, lymphotoxin beta receptor (LTbetaR), tumour necrosis factor receptor superfamily memeber 1A (TNFRSF1R) and FLJ10665, were specifically over-expressed in the NPC xenograft with 12p13.3 amplification. However, only LTbetaR was frequently over-expressed in primary tumours. LTbetaR is a member of the TNF family of receptors, which can modulate NF-kappaB signalling pathways. Over-expression of LTbetaR in nasopharyngeal epithelial cells resulted in an increase of NF-kappaB activity and cell proliferation. In vivo study showed that suppression of LTbetaR by siRNA led to growth inhibition in the NPC tumour with 12p13.3 amplification. These findings implied that LTbetaR is a potential NPC-associated oncogene within the 12p13.3 amplicon and that its alteration is important in NPC tumorigenesis.

Identification of a novel homozygous deletion region at 6q23.1 in medulloblastomas using high-resolution array comparative genomic hybridization analysis.

PURPOSE: The aim of this study is to comprehensively characterize genome copy number aberrations in medulloblastomas using high-resolution array comparative genomic hybridization. EXPERIMENTAL DESIGN: High-density genomic arrays containing 1,803 BAC clones were used to define recurrent chromosomal regions of gains or losses throughout the whole genome of medulloblastoma. A series of 3 medulloblastoma cell lines and 16 primary tumors were investigated. RESULTS: The detected consistent chromosomal aberrations included gains of 1q21.3-q23.1 (36.8%), 1q32.1 (47.4%), 2p23.1-p25.3 (52.6%), 7 (57.9%), 9q34.13-q34.3 (47.4%), 17p11.2-q25.3 (89.5%), and 20q13.31-q13.33 (42.1%), as well as losses of 3q26.1 (57.9%), 4q31.23-q32.3 (42.1%), 6q23.1-25.3 (57.9%), 8p22-23.3 (79%), 10q24.32-26.2 (57.9%), and 16q23.2-q24.3 (63.2%). One of the most notable aberrations was a homozygous deletion on chromosome 6q23 in the cell line DAOY, and single copy loss on 30.3% primary tumors. Further analyses defined a 0.887 Mbp minimal region of homozygous deletion at 6q23.1 flanked by markers SHGC-14149 (6q22.33) and SHGC-110551 (6q23.1). Quantitative reverse transcription-PCR analysis showed complete loss of expression of two genes located at 6q23.1, AK091351 (hypothetical protein FLJ34032) and KIAA1913, in the cell line DAOY. mRNA levels of these genes was reduced in cell lines D283 and D384, and in 50% and 70% of primary tumors, respectively. CONCLUSION: Current array comparative genomic hybridization analysis generates a comprehensive pattern of chromosomal aberrations in medulloblastomas. This information will lead to a better understanding of medulloblastoma tumorigenesis. The delineated regions of gains or losses will indicate locations of medulloblastoma-associated genes. A 0.887 Mbp homozygous deletion region was newly identified at 6q23.1. Frequent detection of reduced expression of AK091351 and KIAA1913 genes implicates them as suppressors of medulloblastoma tumorigenesis.

Detection of oncogene amplifications in medulloblastomas by comparative genomic hybridization and array-based comparative genomic hybridization.

OBJECT: Few studies have been conducted to investigate the genomic survey of oncogene amplification in medulloblastoma. Low frequency of N-myc, C-myc, and epidermal grow factor receptor (EGFR) gene amplification (< 10%) has been reported in medulloblastoma. Previous comparative genomic hybridization (CGH) study of primary medulloblastomas has revealed chromosomal amplification on 2p21, 3p, 5p15.3, 7q, 8q24, 11q22.3, and 17q. The aim of this study was to detect common oncogenes involved in medulloblastoma tumorigenesis. METHODS: The authors studied a series of 14 samples by performing CGH and array-based CGH. The CGH analysis detected nonrandom losses on 8p, 17p, 16q, 8q, and 1p, whereas gains were found on 17q, 12q, 7q, and 1p. Array-based CGH was conducted to investigate amplification of 58 oncogenes throughout the genome of these samples. Gene amplifications identified for the first time included PGY1 at 7q21.1, MDM2 at 12q14.3-q15, and ERBB2 at 17q21.2. The highest frequencies of oncogene gain were detected in D17S1670 (61.5%), PIK3CA (46.2%), PGY1 (38.5%), MET (38.5%), ERBB2 (38.5%), and CSE1L (38.5%). The gain in gene copy numbers was confirmed in 34 additional archival medulloblastoma cases by using fluorescence in situ hybridization analysis. CONCLUSIONS: This is the first genome-wide survey of multiple oncogene amplifications involved in the development of medulloblastoma. Gains of several candidate oncogenes such as D17S1670, ERBB2, PIK3CA, PGY1, MET, and CSE1L were frequently detected. These genes may be used as molecular markers and therapeutic targets of medulloblastomas.

Genome-wide allelotype study of primary glioblastoma multiforme.

OBJECTIVE: To investigate the molecular genetic pathogenesis of primary glioblastoma multiforme (GBM) and identify which chromosomes or chromosomal regions of the entire genome may harbor tumor suppressor genes (TSGs) associated with GBM. METHODS: A high-resolution allelotype study of 21 cases of primary GBM was performed by PCR-based loss of heterozygosity (LOH) analysis. Three hundred and eighty-two fluorescent dye-labeled microsatellite markers covering all 22 autosomes were applied. The mean genetic distance between two flanking markers was about 10 cM. RESULTS: LOH was observed on all 39 nonacrocentric autosomal arms examined in this study. The LOH frequencies of 10q, 10p, 9p, 17p and 13q were the highest (> 50%). Furthermore, high LOH frequencies were detected in the regions containing known TSGs including PTEN, DMBT1, p16, p15, p53 and RB; the LOH frequencies on 14q, 3q, 22q, 11p, 9q, 19q were also high (> 40.5%). Our study observed the following commonly deleted regions: 9p22-23, 10p12.2-14, 10q21.3, 13q12.1-14.1, 13q14.3-31, 17p11.2-12, 17p13, 3q25.2-26.2, 11p12-13, 14q13-31, 14q32.1, 14q11.1-13, 22q13.3, 4q35, 4q31.1-31.2, 6q27 and 6q21-23.3. CONCLUSIONS: The molecular pathogenesis of GBM is very complicated and associated with a variety of genetic abnormalities on many chromosomal arms. The most closely related chromosomal arms to the pathogenesis of GBM are 10q, 10p, 9p, 17p and 13q. Besides the well-known TSGs including PTEN, DMBT1, p16, p15, p53 and RB, multiple unknown TSGs associated with GBM may be present on the commonly deleted regions detected in the present study.

[An allelotype study of primary and corresponding recurrent glioblastoma multiforme].

OBJECTIVE: To investigate molecular genetic alterations associated with primary and corresponding recurrent glioblastoma multiforme(GBM) and to identify which chromosomal regions of the whole genome may be involved in the recurrence of primary GBM. METHODS: A high-resolution allelotyping study of one patient's primary GBM and corresponding recurrent GBM was performed by PCR-based loss of heterozygosity(LOH) analysis with the use of 382 fluorescent dye-labeled polymorphic microsatellite markers covering all 22 autosomes. The mean genetic distance between two flanking markers is 10 cM. RESULTS: LOH at locus D9S157 on 9p21 and at loci D10S537, D10S185, D10S192, D10S597, D10S587, D10S217 on 10q21.3-26.3 was observed in the primary GBM. As for corresponding recurrent tumor, LOH was observed not only in expanded regions on 9p21 and 10q21.3-26.3 but also on multiple other chromosomal arms, including 1q, 7p,7q, 21q, 20p, 20q, 10p, 19p, 19q. CONCLUSION: Chromosome 9p and 10q may be involved in the development of this GBM. Although histopathological diagnoses of the primary and corresponding recurrent tumor are identical, the recurrence of GBM is characterized by an increased involvement of molecular genetic abnormalities and may be accompanied by inactivation of more tumor suppressor genes.

Chromosome 14q may harbor multiple tumor suppressor genes in primary glioblastoma multiforme.

OBJECTIVE: To evaluate whether deletion of chromosome 14q is involved in the carcinogenesis of primary glioblastoma multiforme and to identify possibly common deletion regions. METHJODS: Fourteen fluorescent dye-labeled polymorphic markers were used and polymerase chain reaction-based microsatellite analysis was employed to investigate loss of heterozygosity (LOH) on chromosome 14q in 20 primary glioblastoma multiforme (GBM). RESULTS: Ten of twenty (50%) GBM displayed LOH at one or more of the markers on chromosome 14q. Five tumors showed either LOH or non-informative on all markers tested. The most frequent LOH was observed at locus D14S65 (57.1%) on 14q32.1, and in the chromosomal region spanning from D14S63 (47.1%) to D14S74 (46.7%) on 14q23-31. None of the informative loci exhibited microsatellite instability. CONCLUSIONS: Allelic deletion on chromosome 14q plays an important role in the pathogenesis of GBM. Chromosomal regions at locus D14S65 on 14q32.1 and spanning from D14S63 to D14S74 on 14q23-31 may harbor multiple tumor suppressor genes associated with GBM.

[An allelotype study of human glioblastoma].

OBJECTIVE: To reveal the molecular genetic mechanisms for the pathogenesis of glioblastoma (GBM) and determine which chromosomes or chromosomal regions may play a role in the pathogenesis of GBM or may harbor tumor suppressor genes (TSGs) associated GBM. METHODS: An allelotype study of 21 cases of GBM was performed by polymerase chain reaction and loss of heterozygosity (LOH) analysis. Three hundred and eighty-two microsatellite markers covering all 22 autosomes were used. The mean genetic distance between two flanking markers is about 10 cM. Fluorescent dye-labeled primers and Perkin Elmer 377 DNA Sequencer were applied. RESULTS: LOH was observed on all chromosomal arms examined in this study. The LOH frequencies of 10q, 10p, 13q, 17p and 9p were the highest (>50%), on which high LOH frequencies were detected at the regions resided by the known TSGs including PTEN, DMBT1, p16, p15, p53 and Rb. The following commonly deleted regions were detected: 9p22-23, 10p12.2-14, 10q21.3, 13q12.1-14.1, 13q14.3-31, 17p11.2-12, 17p13, 3q24-27, 11p12-13, 14q31-32.3, 14q21-24.1, 22q13.2-13.3, 4q35, 4q31.1-31.2, 6qtel, 6q16.3. CONCLUSION: This study demonstrated that the pathogenesis of GBM is very complicated and associated with various molecular genetic abnormalities on lots of chromosomes. The chromosomal arms most closely relevant to the pathogenesis of GBM are 10q, 10p, 9p, 17p and 13q. Besides the well-known TSGs, such as PTEN, DMBT1, p16, p15, p53 and Rb, multiple unknown TSGs associated with GBM may be present on the commonly deleted regions observed for the first time in this study.