Case Report: Neoadjuvant and Adjuvant Crizotinib Targeted Therapy in Stage IIIA-N2 ALK-Positive Non-Small-Cell Lung Cancer.

The treatment of anaplastic lymphoma kinase (ALK)-positive locally advanced non-small-cell lung cancer (NSCLC) is challenging because there is no randomized controlled trial has been reported. The value of neoadjuvant and adjuvant targeted therapy remains unclear. Herein, we show that systemic treatment with ALK inhibitor crizotinib before surgery can provide the potential to cure the initially inoperable tumor. A 27-year-old man was diagnosed with a stage IIIAcT3N2M0 (7thUICC/AJCC) upper left lung adenocarcinoma harboring EML4-ALK fusion gene. Clinically, the patient had a large primary lesion adjacent to the pericardium and regional lymph node metastasis at the ipsilateral mediastinum. Poor tumor response was observed after 3 cycles of chemotherapy (gemcitabine plus cisplatin), and upon multidisciplinary discussion, the patient was started with 250 mg crizotinib twice daily. Successive clinical examinations showed a progressive reduction of the lesions. After 2 months of therapy, the patient was downstaged to cT2aN2M0, then video-assisted thoracic surgery was performed and the final histopathological stage was ypT2aN2M0. The treatment with crizotinib (250 mg, qd) was continued more than 30 months post surgery and stopped until intracranial oligometastasis. The patient's overall survival (OS) time is 68 months at last follow-up. This case presented here supports the use of neoadjuvant and adjuvant treatment with ALK inhibitors in ALK positive locally advanced NSCLC.

Validation of human small airway measurements using endobronchial optical coherence tomography.

BACKGROUND: Small airway remodeling is the cardinal feature underlying chronic airway diseases. There is no modality which identifies small airway pathological changes, which is crucial for early diagnosis, efficacy and prognostic assessment. OBJECTIVE: To evaluate the usefulness of endobronchial optical coherence tomography (EB-OCT) in assessing small airways morphology in vivo. METHODS: Twelve patients with pulmonary nodules scheduled for lung resection underwent spirometry, multi-detector computed tomography (MDCT) and EB-OCT. We measured D(mean) (mean luminal diameter), Ai (inner luminal area), Aw (airway wall area) and Aw% [Aw/(Ai + Aw) × 100%] from the 3rd to 5th generation bronchi of RB9 segment by MDCT. D(mean), Ai, Aw and Aw% from the 3rd to 9th generation bronchi of RB9 segment were measured by EB-OCT and histology. Correlations of these parameters, measured by three different methods, were evaluated. We recruited 4 COPD patients to determine if EB-OCT could identify peripheral airway remodeling. RESULTS: The 4 parameters, measured by CT and EB-OCT, correlated significantly [D(mean) (r = 0.991), Ai (r = 0.997), Aw (r = 0.997), Aw% (r = 0.991), all P < 0.01]. Significant correlation were found for these parameters, measured by histology and EB-OCT, from the 3rd to 5th generation bronchi [D(mean) (r = 0.989), Ai (r = 0.997), Aw (r = 0.999), Aw% (r = 0.988), all P < 0.01], and from the 6th to 9th generation bronchi [D(mean) (r = 0.979), Ai (r = 0.997), Aw (r = 0.994) and Aw% (r = 0.988), all P < 0.01]. Significant small airways morphological abnormalities were observed in COPD patients. CONCLUSIONS: EB-OCT, a minimally invasive imaging modality with high-resolution, is useful and clinically practical for assessing proximal and distal airways of human compared with CT and histology.

CHRNA3 polymorphism modifies lung adenocarcinoma risk in the Chinese Han population.

Recent genome-wide association studies (GWASs) have identified 15q25.1 as a lung cancer susceptibility locus. Here, we sought to explore the direct carcinogenic effects of genetic variants in this region on the risk of developing lung adenocarcinoma (ADC). Five common SNPs (rs8034191, rs16969968, rs1051730, rs938682, and rs8042374) spanning the 15q25.1 locus were assayed in a case-control study examining a cohort of 301 lung ADCs and 318 healthy controls. Stratification analysis by gender, smoking status, and tumor, node, metastasis (TNM) classification, was performed. In addition, sections from ADC tissue and normal tissue adjacent to tumors were stained with an anti-CHRNA3 (cholinergic receptor nicotinic α3) antibody by immunohistochemistry in 81 cases. Our results demonstrate that rs8042374, a variant of the CHRNA3 gene, is associated with an increased risk of ADC with an OR of 1.76 (95% CI: 1.17-2.65, p=0.024). This variant was linked to a greater risk of ADC in female nonsmokers (OR (95% CI): 1.81 (1.05-3.12), p=0.032) and female stage I+II cases (OR (95% CI): 1.92 (1.03-3.57), p=0.039). Although located within the same gene, rs938682 showed protective effects for smokers, stage III+IV cases, and male stage III+IV cases. Additionally, the CHRNA3 protein level in ADC tissue was slightly higher than in the surrounding normal lung tissue, based on immunohistochemical analysis. Our results suggest that the CHRNA3 polymorphism functions as a genetic modifier of the risk of developing lung ADC in the Chinese population, particularly in nonsmoking females.

Malignant perivascular epithelioid cell tumor of mesentery with lymph node involvement: a case report and review of literature.

Perivascular epithelioid cell tumor (PEComa) is a rare but distinct mesenchymal neoplasm composed of histologically and immunohistochemically unique perivascular epithelioid cells. Due to its relative rarity, little is known about the histogenesis and prognostic factors of this tumor. We describe a case of unusual mesenteric PEComa in a 38-year-old female patient with regional lymph node involvement. Histologically, the tumor was composed of sheet of epithelioid cells with abundant clear or eosinophillic cytoplasms. Extensive coagulative necrosis and a few mitotic figures (2/50 high power field) could be found in tumor. The epithelioid tumor cells were diffusely positive for HMB-45, Melan-A, and focally positive for calponin. One of enlarged mesenteric lymph nodes was observed to be involved by tumor. A diagnosis of malignant mesenteric PEComa with lymph node involvement was made. The patient received chemotherapy after total resection of tumor and segmental resection of involved jejunum. There was no sign of recurrence of tumor found in period of 6-month regular follow-up after chemotherapy. To our knowledge, this is the first case of malignant PEComa in mesentery accompanied with regional lymph node involvement. The literature on this rare tumor is reviewed and diagnostic criteria of malignant PEComa are discussed. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1309992178882788.

[Study on genetic polymorphisms of CYP2F1 gene in Guangdong population of China].

OBJECTIVE: To investigate the genetic polymorphism of CYP2F1 gene, a member of CYP450 gene family in the healthy population and the patients with nasopharyngeal carcinoma (NPC) of Guangdong province, and furthermore analyze the relationship between CYP2F1 genetic polymorphism and the risk of developing NPC. METHODS: By direct gene sequencing, all of 10 exons of CYP2F1 gene were detected in 40 peripheral blood specimens of patients with primary NPC. For the genetic polymorphism with high allelic frequency, mismatch PCR-RFLP technique was developed to identify the different frequency between 368 NPC cases and 344 cancer-free controls. RESULTS: There were totally 35 SNPs identified in all of 10 exons and exon-intron junctions of CYP2F1 gene from 40 NPC patients, which included 10 missense mutations and 1 frame shift mutation. The most important mutation was C insertion located in 15-16 bp, which caused the frame shift. The allelic frequency of C insertion was 25%. However, there was no significant difference found between 368 NPC cases and 344 controls in allelic frequency of 15-16 bp C insertion mutation (P>0.05). CONCLUSION: A lot of genetic polymorphism of CYP2F1 gene is found in Guangdong population of China. However, no single genetic polymorphism associated with the individual susceptibility to NPC can be identified. The cooperated operations with multiple genetic polymorphisms of one or more genes may be critical factors contributing to the development and progression of NPC.

Hypoxia can contribute to the induction of the Epstein-Barr virus (EBV) lytic cycle.

BACKGROUND: Like other herpes viruses, latent Epstein-Barr virus (EBV) infection can be reactivated to lytic replication. Reactivation can be achieved by treatment with various reagents, including tetradecanoyl phorbol acetate (TPA) and Ca2+ ionophores. Relatively little is known about the physiological factors related to reactivation of EBV. Previous studies have demonstrated that G0/G1 cell cycle arrest is associated with EBV activation, and that hypoxic conditions can induce cell cycle arrest. In the present study we investigated the effect of hypoxia on reactivation of EBV. OBJECTIVE AND METHODS: Hypoxic culture conditions were established and the expression of Zta protein and the number of EBV DNA copies were measured in B95-8 cells maintained under these conditions. RESULTS: Hypoxia treatment not only increased the expression of the EBV immediate-early protein Zta (which mediates the switch between the latent and lytic form of infection), but also increased the number of EBV DNA copies in B95-8 cells. CONCLUSIONS: EBV in latent infection can be activated to lytic infection by hypoxia treatment.

High frequency somatic mutations in RASSF1A in nasopharyngeal carcinoma.

High frequency loss of 3p21.3 region is a common event in various kinds of tumors including nasopharyngeal carcinoma (NPC). RASSF1A has been identified as a putative tumor suppressor gene residing in this region. Chromosome alterations and epigenetic changes are commonly observed as mechanisms for inactivation of RASSF1A function. In this study, we applied the PCR-cloning-sequencing strategy to examine somatic mutations in RASSF1A in NPC tissues as compared with the sequences detected in the matched peripheral blood lymphocytes. Our results revealed a high incidence of RASSF1A mutation in primary tumor tissues of NPC. There are totally 35 mutations identified in 74% (17/23) of these NPC cases, including 30 transitions, three transversions and two deletions. Most of these mutations result in amino acid changes: three nonsense (stop codon) mutations, two-1 bp deletion (frameshift), 26 missense and the remaining four are synonymous (silent). No obvious 'hot-spot' mutations were observed in this study. A similarly high rate (74%) of promoter methylation of RASSF1A was also detected in the same group of NPC tissues, but no significant correlation between mutation and methylation was detected. Our results suggest various mechanisms involved in inactivation of RASSF1A function and indicate a critical role of RASSF1A in NPC development.

Transcriptional expression of RPMS1 in nasopharyngeal carcinoma and its oncogenic potential.

The Epstein-Barr virus (EBV) BamHI A rightward transcripts (BARTs) were originally identified in C15 xenograft of nasopharyngeal carcinoma (NPC) and easily detected in a wide variety of EBV latent infection and EBV-associated tumors. It had been reported that p31 cosmid containing BARTs immortalized monkey epithelial cells, but which particular gene among BARTs family participates in the transformation procedure remains to be identified. RPMS1 is the only full-length cDNA confirmed so far and one of the most abundant spliced forms in BARTs family. To investigate the involvement of RPMS1 gene in NPC, we examined the expression of RPMS1 transcripts in NPC biopsies from Guangdong and its oncogenic potential. Our results revealed that RPMS1 mRNA preferentially expressed in primary NPC to non-carcinoma tissue of nasopharynx and peripheral blood lymphocytes (PBLs) of NPC patients. Furthermore, by introducing RPMS1 ORF into HEK293 cells, these transfectants enhanced the anchorage-independent growth and produced tumors in nude mice. These data imply that RPMS1 gene might play an important role in the development of NPC.

[Expression of cytochrome P450 enzymes in human nasopharyngeal carcinoma and non-cancerous nasopharynx tissue].

BACKGROUND & OBJECTIVE: The etiology of nasopharyngeal carcinoma (NPC) is associated with environmental and hereditary factors. Xenobiotics from environment must be activated to derive carcinogens. Several cytochrome P450 (CYP450) metabolic enzymes participate to the activation of pre-carcinogens, and the genetic polymorphism of those genes is associated with metabolic polymorphism and susceptibility to cancer. We performed a preliminary investigation on the xenobiotics metabolism of human nasopharynx by determining the expression of CYP450 genes in NPC and non-cancerous nasopharynx tissues. METHODS: The following two methods were used: (1)A cDNA library from the mixed RNA sample of seven non-cancerous nasopharynx tissues was generated, followed by clone sequencing and bioinformatics analysis. (2)RNA of 14 NPC and 8 non-cancerous nasopharynx tissues were reversely transcribed, and the expression of CYP450 genes in those samples was determined by PCR amplification. RESULTS: Eight ESTs of CYP450 genes including CYP1B1, CYP2F1, CYP2J2, CYP4B1, CYP4F12, CYP5A(TBXAS1), CYP20A1, and CYP51A1 were detected in non-cancerous nasopharynx cDNA library, among these CYP4B1 exhibited the highest expression level with 16 copies of ESTs. Positive expression of fourteen CYP450 genes including CYP1A1, CYP1B1, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2F1, CYP3A4, CYP3A5, CYP3A7, and CYP4B1 were detected by RT-PCR, among these, CYP1B1, CYP2B6, and CYP4B1 had also been detected in the cDNA library. A total of 19 CYP450 genes expression were detected in NPC and non-cancerous nasopharynx tissues, and the expression levels of CYP1B1, CYP2C8, CYP2C9, CYP2D6, CYP3A5, and CYP4B1 were higher than those of the other genes. CONCLUSION: The expression of a great number of CYP450 genes was detected in human nasopharynx, some of which might participate to the activation of pre-carcinogen in human nasopharynx. Contribution of these genes to the risk of NPC needs further investigation.

Genetic polymorphisms of CYP2A13 and its relationship to nasopharyngeal carcinoma in the Cantonese population.

Nasopharyngeal carcinoma (NPC) is characterized by a high prevalence in Southern China, especially among Cantonese individuals of the Guangdong Province. Epidemiological studies have suggested that frequent exposure to high levels of nitrosamine from preserved foods such as salted fish could be a risk factor for NPC. Cytochrome P450 encompasses a family of enzymes that metabolize carcinogens and CYP2A13, a member of this family, is expressed predominantly in the respiratory tract with the highest levels in the nasal mucosa. In an effort to test whether a correlation exists between CYP2A13 genetic polymorphism and the risk of developing NPC, we sequenced all nine exons and the exon-intron junctions of the CYP2A13 gene in 45 NPC patients. We identified a total of 21 single nucleotide polymorphism (SNPs), including 7 novel SNPs. The most frequent functional variant allele was 74A-1757G-3375T-7233G with a haplotype frequency of 7.8% in the 45 NPC cases. In addition, a stop codon mutation was detected in one case. We then selected the 3 most frequent SNPs and one stop codon mutation to expand our study to a case-control analysis within the Cantonese population. A novel haplotype consisting 8 SNPs in introns, and four additional novel SNPs were identified; but no correlation between CYP2A13 genetic polymorphism and individual susceptibility to NPC was observed.

[Expression of Epstein-Barr virus A73 gene in nasopharyngeal carcinoma tissues and its subcellular location].

BACKGROUND & OBJECTIVE: Epstein-Barr virus (EBV) A73 gene encodes for a main mRNA of BamHI A rightward transcripts (BARTs) family which is widely transcribed in EBV-carrying cells and tumors. This study was designed to investigate the expression of A73 mRNA in nasopharyngeal carcinoma(NPC) from Guangdong region,from which A73 coding sequence(CDS) was cloned and introduced into epithelial cell line to observe the subcellular fraction of its encoding protein. METHODS: DNA and total RNA were isolated from NPC and noncarcinoma nasopharyngeal tissues,peripheral blood lymphocyte of NPC, SUNE1, B95.8, Raji, and BJAB cells; Nested PCR was designed to identify the EBV infection by amplifying the W sequence of EBV DNA in above- mentioned samples, and RT-PCR was used to analyze the transcription of A73 gene in the same samples. A73 CDS amplifying from a NPC tissue was cloned into pEGFP-C2 on special direction. After being transfected with this recombinant plasmid by lipofectin mediation, human embryo kidney(HEK293) cells were analyzed by RT-PCR and laser confocal microscope to observe the expression of A73 gene and its subcellular fraction. RESULTS: Except for BJAB cells, EBV W sequence existed in all samples. A73 transcribed in 79.63%(43/54)of NPC and 4%(1/25)of noncarcinoma nasopharynx biopsies between which the difference was significant, and no A73 mRNA was detected in all peripheral blood lymphocyte of NPC.A73 expressed at lower levels in B95.8 and Raji than in SUNE1. The constructed recombinant plasmid expressed stably in HEK293 cells, in which the fusion protein was found to be mainly cytoplasmic. CONCLUSION: EBV-A73 gene expresses widely in NPC tissue from Guangdong region and its encoding protein can stably express in cytoplasm of epithelial cell in vitro,suggesting A73 is highly correlated with the occurrence and development of NPC, and A73 gene may take part in the signal conduction related to epithelial oncogenesis as a signal molecule.

[Analysis of suppressive roles of BLU gene at 3p21.3 in nasopharyngeal carcinoma].

BACKGROUND & OBJECTIVE: Nonrandom allelic loss at chromosome 3p21.3 is a common and early event in nasopharyngeal carcinoma (NPC), which implicates the presence of tumor suppressor genes (TSGs) that may be involved in the pathogenesis of NPCs. BLU gene, containing a MYND domain and located at 3p21.3, has been considered as a NPC associated candidate tumor suppressor gene (TSG) due to the occurrence of loss of its expression and aberrant promoter hypermethylation in most NPCs. This study was designed to construct expression vectors containing either wild type BLU gene and its mutants and to analyze the effect of BLU gene on proliferation of NPC cells by transfection assays. METHODS: The full-length cDNA of BLU gene was amplified by RT-PCR. The expression vectors containing various BLU mutants were constructed by site-directed mutagenesis by overlapping PCR. These mutants include a MYND domain deletion mutant, a Ser402Phe and del405Cys, del406Ser mutant, and a Gly160Arg mutant. The wild type BLU gene and the MYND domain deletion mutant were transfected into NPC cell lines CNE1 and CNE2. The effect on apoptosis was determined by TUNEL assay. Cellular proliferation of the stably-transfected cells was examined with cell growth curve and by colony formation assays. Tumorigenicity in nude mice of CNE2 stably-transfected with BLU was investigated. RESULTS: No significant difference in apoptosis index (AI) was observed between cells transfected with wild type or MYND domain deleted BLU gene and cells transfected with plasmid controls. Exogenous expression of wild type BLU gene had no effect on growth rate and colony formation ability of CNE1 and CNE2. BLU gene showed no suppressor ability in CNE2 tumorigenicity. CONCLUSION: Although BLU gene was frequently altered in NPCs, its suppressor role in NPC cells proliferation was not evident. Thus, the possibility of BLU gene as a TSG involved in NPC development remained to be elucidated by further studies.