The Expression of TP63 as a Biomarker of Early Recurrence in Resected Esophageal Squamous Cell Carcinoma after Neoadjuvant Chemoradiotherapy.

Esophageal cancer ranks among the ten most common cancers worldwide. Despite the adoption of neoadjuvant concurrent chemoradiotherapy (nCCRT) followed by surgery as the standard treatment approach in recent years, the local recurrence rate remains high. In this study, we employed RNA-seq to investigate distinctive gene expression profiles in esophageal squamous cell carcinoma (ESCC) with or without recurrence following a standard treatment course. Our findings indicate that recurrent ESCC exhibits heightened keratinizing and epidermis development activity compared to non-recurrent ESCC. We identified TP63 as a potential candidate for distinguishing clinical outcomes. Furthermore, immunohistochemistry confirmed the trend of TP63 overexpression in ESCC recurrence. Patients with elevated TP63 expression had poorer overall survival and lower 3-year recurrence-free survival. This study underscores the potential of TP63 as a biomarker for detecting cancer recurrence and suggests its role in guiding future treatment options.

Chemoresistance in acute myeloid leukemia: An alternative single-cell RNA sequencing approach.

Our previous study demonstrated that myc, mitochondrial oxidative phosphorylation, mTOR, and stemness are independently responsible for chemoresistance in acute myeloid leukemia (AML) cells. This study aimed to identify potential mechanisms of chemoresistance of the "7 + 3" induction in AML by using a single-cell RNA sequencing (scRNA-seq) approach. In the present study, 13 untreated patients with de novo AML were enrolled and stratified into two groups: complete remission (CR; n = 8) and non-CR (n = 5). Single-cell RNA sequencing was used to analyze genetic profiles of 28,950 AML cells from these patients; results were validated using a previously published bulk RNA-seq dataset. Our study results showed chemoresistant AML cells had premature accumulation during early hematopoiesis. Hematopoietic stem cell-like cells from the non-CR group expressed more leukemic stem cell markers (CD9, CD82, IL3RA, and IL1RAP) than those from the CR group. Chemoresistant progenitor cells had impaired myeloid differentiation owing to early arrest of hematopoiesis. Notably, AML cells analyzed by scRNA-seq and bulk RNA-seq harbored a comparable myeloid lineage cell fraction, which internally validated our results. Using the TCGA database, our analysis demonstrated that patients with AML with higher expression of chemoresistant genetic markers (IL3RA and IL1RAP) had a worse overall survival (p < 0.01 for IL3RA; p < 0.05 for IL1RAP). In conclusion, AML cells responsive and resistant to the "7 + 3" induction were derived from a diverse cancerous hematopoietic stem cell population, as indicated by the specific genetic biomarkers obtained using scRNA-seq approach. Furthermore, arrest of hematopoiesis was shown to occur earlier in chemoresistant AML cells, furthering the current understanding of chemoresistance in AML.

AGR2 expression as a predictive biomarker for therapy response in esophageal squamous cell carcinoma.

Despite multidisciplinary therapy, the prognosis is poor for esophageal squamous cell carcinoma (ESCC). In the locally advanced stage, neoadjuvant chemoradiotherapy (nCRT) followed by surgery could provide survival benefits to some patients. Here, we aimed to identify for tumor therapy response a biomarker based on RNA sequencing. We collected endoscopic biopsies of 32 ESCC patients, who were divided according to nCRT response, into two groups: the complete response group (n = 13) and the non-complete response group (n = 19). RNA-sequencing data showed that 464 genes were differentially expressed. Increased in non-complete response group, 4 genes increased expressions were AGR2 (anterior gradient 2), GADD45B (growth arrest and DNA damage inducible beta), PPP1R15A (protein phosphatase 1 regulatory subunit 15A) and LRG1 (leucine rich alpha-2-glycoprotein 1). The areas under the curve (AUC) of the AGR2 gene was 0.671 according to read counts of RNA-seq and therapy response of nCRT. In vitro study showed that apoptosis cell was significantly increased in the AGR2-knockdown TE-2 cell line treated with cisplatin and 5-Fluorouracil (5-FU), when compared with si-control. Results suggest that in ESCC, the AGR2 gene is a promising and predictive gene marker for the response to anti-tumor therapy.

Quality of life and symptom distress after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy.

BACKGROUND: Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS/ HIPEC) for peritoneal surface malignancy can effectively control the disease, however it is also associated with adverse effects which may affect quality of life (QoL). AIM: To investigate early perioperative QoL after CRS/HIPEC, which has not been discussed in Taiwan. METHODS: This single institution, observational cohort study enrolled patients who received CRS/HIPEC. We assessed QoL using the Taiwanese version of the MD Anderson Symptom Inventory (MDASI-T) and European Organization Research and Treatment of Cancer Core Quality of Life Questionnaire (EORTC QLQ-C30). Participants completed the questionnaires before CRS/HIPEC (S1), at the first outpatient follow-up (S2), and 3 mo after CRS/HIPEC (S3). RESULTS: Fifty-eight patients were analyzed. There was no significant perioperative difference in global health status. Significant changes in physical and role functioning scores decreased at S2, and fatigue and pain scores increased at S2 but returned to baseline at S3. Multiple regression analysis showed that age and performance status were significantly correlated with QoL. In the MDASI-T questionnaire, distress/feeling upset and lack of appetite had the highest scores at S1, compared to fatigue and distress/feeling upset at S2, and fatigue and lack of appetite at S3. The leading interference items were working at S1 and S2 and activity at S3. MDASI-T scores were significantly negatively correlated with the EORTC QLQ-C30 results. CONCLUSION: QoL and symptom severity improved or returned to baseline in most categories within 3 mo after CRS/HIPEC. Our findings can help with preoperative consultation and perioperative care.

Lipid Droplets in Lung Cancers Are Crucial for the Cell Growth and Starvation Survival.

For rapid and unlimited cell growth and proliferation, cancer cells require large quantities of nutrients. Many metabolic pathways and nutrient uptake systems are frequently reprogrammed and upregulated to meet the demand from cancer cells, including the demand for lipids. The lipids for most adult normal cells are mainly acquired from the circulatory system. Whether different cancer cells adopt identical mechanisms to ensure sufficient lipid supply, and whether the lipid demand and supply meet each other, remains unclear, and was investigated in lung cancer cells. Results showed that, despite frequent upregulation in de novo lipogenesis and the lipid transporter system, different lung cancer cells adopt different proteins to acquire sufficient lipids, and the lipid supply frequently exceeds the demand, as significant amounts of lipids stored in the lipid droplets could be found within lung cancer cells. Lipid droplet surface protein, PLIN3, was found frequently overexpressed since the early stage in lung cancer tissues. Although the expression is not significantly associated with a specific gender, age, histology type, disease stage, and smoking habit, the frequently elevated expression of PLIN3 protein indicates the importance of lipid droplets for lung cancer. These lipid droplets are not only for nutrient storage, but are also crucial for tumor growth and proliferation, as well as survival in starvation. These results suggest that manipulation of lipid droplet formation or TG storage in lung cancer cells could potentially decrease the progression of lung cancer. Further exploration of lipid biology in lung cancer could help design novel treatment strategies.

Leveraging advances in immunopathology and artificial intelligence to analyze in vitro tumor models in composition and space.

Cancer is the leading cause of death worldwide. Unfortunately, efforts to understand this disease are confounded by the complex, heterogenous tumor microenvironment (TME). Better understanding of the TME could lead to novel diagnostic, prognostic, and therapeutic discoveries. One way to achieve this involves in vitro tumor models that recapitulate the in vivo TME composition and spatial arrangement. Here, we review the potential of harnessing in vitro tumor models and artificial intelligence to delineate the TME. This includes (i) identification of novel features, (ii) investigation of higher-order relationships, and (iii) analysis and interpretation of multiomics data in a (iv) holistic, objective, reproducible, and efficient manner, which surpasses previous methods of TME analysis. We also discuss limitations of this approach, namely inadequate datasets, indeterminate biological correlations, ethical concerns, and logistical constraints; finally, we speculate on future avenues of research that could overcome these limitations, ultimately translating to improved clinical outcomes.

Adding Value of MRI over CT in Predicting Peritoneal Cancer Index and Completeness of Cytoreduction.

BACKGROUND: This study aimed to investigate the adding value of MRI over CT for preoperative cytoreductive surgery with hyperthermic intraperitoneal chemotherapies (CRS/HIPEC). METHODS: Imaging and intraoperative peritoneal cancer index (PCI) were calculated in 62 patients with peritoneal metastasis. Predictive models for the completeness of cytoreductive score using PCI data were established using decision tree algorithms. RESULTS: In gastric cancer patients, a large discrepancy and poor agreement was appreciated between CT and surgical PCI, and a nonsignificant difference was noted between MRI and surgical PCI. In colon cancer patients, a better agreement and higher correlation with a smaller error was observed in PCI score using MRI than in that using CT. However, the addition of MRI to CT was limited for appendiceal and ovarian cancer patients. For predicting incomplete cytoreduction, CT models yielded inadequate accuracy while MRI models were more accurate with fair discrimination ability. CONCLUSIONS: CT was suitable for estimating PCI and surgery outcome in appendiceal and ovarian cancer patients, while further MRI in addition to CT was recommended for colon and gastric cancer patients. However, for classifying patients with peritoneal carcinomatosis into complete and incomplete cytoreduction, MRI was more effective than CT.

Risk factors of acute renal impairment after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy.

BACKGROUND: Acute renal impairment (ARI) is a major complication after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) for cancer patients with peritoneal metastases. This study aimed to investigate the incidence and identify the risk factors of post-HIPEC creatinine increased. METHODS: From April 2015 to December 2019, demographic and perioperative data of 169 patients undergoing CRS/HIPEC with a preoperative creatinine level <1.5 mg/dL were retrospectively reviewed. Renal impairment was defined according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 5.0. The risk factors of creatinine increased were analyzed using univariate and multiple logistic regression analyses. RESULTS: Among the 169 enrolled patients, 21 (12.4%) had postoperative creatinine increased (ARI group) and 148 (87.6%) did not (non-ARI group). Significantly more of the ARI group received a cisplatin HIPEC regimen than the non-ARI group (71.4 vs. 37.8%, p = 0.004). Multiple logistic regression analysis revealed that the patients who received a cisplatin HIPEC regimen (adjusted odds ratio [AOR] = 11.38, p < 0.001) and peritoneal dialysis solution as HIPEC perfusate (AOR = 7.07, p = 0.002) were more likely to develop post-HIPEC creatinine increased. CONCLUSIONS: Identifying the risk factors of post-HIPEC creatinine increased can help to improve patient selection, a dose of HIPEC regimens modification and perioperative care. We also identified the detrimental renal effect of peritoneal dialysis solution as HIPEC perfusate. More prospective studies are warranted to confirm these findings.

A 16-Year Study on Incidence and Progression of Diseased Sphenoethmoidal (Onodi) Cell.

Traumatic operative injury of the optic nerve in an endoscopic sinus surgery may cause immediate or delayed blindness. It should be cautioned when operating in a sphenoethmoidal cell, or known as Onodi cell, with contact or bulge of the optic canal. It remains unclear how frequent progression to visual loss occurs and how long it progresses to visual loss because of a diseased sphenoethmoidal cell. Research to discuss these questions is expected to help decision making to treat diseased sphenoethmoidal cells. From July 2001 to June 2017, 216 patients received conservative endoscopic sinus surgery without opening a diseased sphenoethmoidal cell. We used their computed tomography images of paranasal sinuses to identify diseased sphenoethmoidal cells that could be associated with progression to visual loss. Among the 216 patients, 52.3% had at least one sphenoethmoidal cell, and 14.8% developed at least one diseased sphenoethmoidal cell. One patient developed acute visual loss 4412 days after the first computed tomography. Our results show that over half of the patients have a sphenoethmoidal cell but suggest a rare incidence of a diseased sphenoethmoidal cell progressing to visual loss during the follow-up period.

Age-Specific Breast Density Changes in Taiwanese Women: A Cross-Sectional Study.

Breast density is a risk factor for breast cancer. This study explored distribution of mammographic density quantitatively and qualitatively in a wide age range of Taiwanese women. Subjects with negative and benign mammographic findings were included. According to the Breast Imaging Reporting and Data System, the proportion of extremely dense breasts declined from 58.0% in women < 30 years to 1.9% in women > 74 years. More than 80% of mammograms in women < 55 years old were classified as extremely or heterogeneously dense, while the proportion of dense breasts was still high in women aged 60-64 years (59.3%). The absolute dense area of the breast declined from 35.8% in women < 30 years to 18.5% in women > 74 years. The correlation between breast density and age was significant, with and without controlling for the effect of body composition (p < 0.001), implying that the relationship between breast density and age was not wholly related to body composition. In conclusion, the higher breast density in Taiwanese women aged 60-64 years was comparable to that of Western women aged 40-44 years in the literature. This suggests that breast cancer screening using mammography may be more challenging for Asian women than for Western women of the same age.

Molecular profiling of thymoma with myasthenia gravis: Risk factors of developing myasthenia gravis in thymoma patients.

OBJECTIVE: Thymoma is a rare epithelial tumor arising from the thymus in the anterior mediastinum. Nearly 50% of patients with thymoma develop myasthenia gravis, which is an indication of a poor long-term prognosis. Here, we identified specific and effective molecular markers for predicting in the development of myasthenia gravis patients with thymoma. MATERIAL AND METHODS: We investigated molecular profiling based on RNA-sequencing (RNA-seq) for myasthenia gravis development in patients with thymoma. RNA was extracted from 34 patients with thymoma, 16 of whom had myasthenic and 18 of whom did not, and transcriptome profiles were analyzed through next-generation sequencing. RESULTS: We discovered 140 differential expressed genes associated with myasthenia gravis in thymoma patients. The four genes, hypoxia-inducible factor 3 alpha (HIF3A), insulin-like growth factor-binding protein 1, pyruvate dehydrogenase kinase, and Krüppel-like factor 15 were differentially expressed in patients with thymoma who has myasthenia gravis and were validated by quantitative polymerase chain reaction. HIF3A expression was significantly higher in patients with myasthenia gravis than in those without. CONCLUSION: HIF3A is aberrantly expressed in patient with thymoma who has myasthenia gravis and may be involved in the development of myasthenia gravis in thymoma patient.

Demographic factors affecting parental attitudes to clinical research in pediatric surgery: A pilot study.

PURPOSE: The purpose of this study was to evaluate demographic factors impacting parental attitudes to clinical research in pediatric surgery. METHODS: A prospective survey administered to caregivers accompanying children (pediatric surgical outpatient clinic or day surgery) using convenience sampling (September-November 2017) using a previously published survey with Likert scale was performed. Questions included demographics, parental willingness to enroll children in specified types of research, and beliefs regarding conduct of research. RESULTS: Eighty-four parents were surveyed (100 approached). No demographic factors significantly predicted research participation involving sample collection (urine, saliva, blood) or research requiring follow-up. However, mothers were less likely to agree to studies using common medications (p = 0.049) or common surgical procedures (p = 0.013) and less likely to agree to randomization involving surgery (assigning to common surgical procedure, p = 0.013; surgery vs no surgery, p = 0.031). University graduates were less likely to agree to randomization to surgery vs no surgery (p = 0.02). Beliefs regarding conduct of research were similar, except that non-university graduates were more likely to believe that privacy would be compromised (p = 0.003). Boys were deemed less likely to be too sick for participation (p = 0.03) and more likely to want to participate (p = 0.03). CONCLUSION: Behavioral and attitude differences in caregivers can inform strategies for recruitment among researchers. Impact of caregiver and child gender on responses requires further evaluation. TYPE OF STUDY: Treatment study. LEVEL OF EVIDENCE: Level II.

Cytoreductive surgery with hyperthermic intraperitoneal chemotherapy for peritoneal malignancy: preliminary results of a multi-disciplinary teamwork model in Asia.

BACKGROUND: Cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) is an emerging surgical procedure for peritoneal carcinomatosis (PC). CRS/HIPEC is a complicated treatment that requires multi-disciplinary teamwork (MDT), which may be lacking when establishing a CRS/HIPEC programme. Herein, we report our preliminary treatment outcomes with the early implementation of an MDT model for CRS/HIPEC. METHODS: From April 2015 to December 2016, 45 patients with a diagnosis of PC who received CRS/HIPEC were reviewed retrospectively in a single institution in Taiwan. RESULTS: Among the 45 patients, CRS was mainly performed by laparotomy (n = 42), and only three patients with limited PC underwent laparoscopic CRS. The first 13 patients received treatment before the MDT had been established (group 1), and the other 32 patients were treated after the MDT had been established (group 2). The highest peri-HIPEC body temperature in group 2 was significantly lower than that in group 1 (36.8 °C vs. 37.5 °C, p < 0.001). Overall, eight patients experienced major complications. The trend of a lower major complication rate was observed after the MDT model had been implemented (30.7% in group 1 vs. 12.4% in group 2, p = 0.202). Pre-CRS/HIPEC abdominal pain significantly increased the risk of post-operative major complications (p = 0.017). CONCLUSIONS: Our experience suggests that the early implementation of an MDT model when establishing a CRS/HIPEC programme at a single institution may result in a higher complete cytoreduction rate and lower major complication rate, and also shorten the learning curve of this complicated procedure.

Use of Myometrium as an Internal Reference for Endometrial and Cervical Cancer on Multiphase Contrast-Enhanced MRI.

BACKGROUND: Myometrial smooth muscle is normally within the field of view for the gynecological imaging. This study aimed to investigate the use of normal myometrium as an internal reference for endometrial and cervical cancer during multiphase contrast-enhanced magnetic resonance imaging (MCE-MRI) and to explore whether this information regarding tumor enhancement relative to the myometrium could be used to discriminate between endometrial and cervical cancer. METHODS: MRI images, before and after contrast enhancement, were analyzed in newly diagnosed cervical (n = 18) and endometrial cancer (n = 19) patients. Signal intensities (SIs) from tumor tissue and non-neoplastic myometrium were measured using imaging software. RESULTS: The relative signal for cervical cancer was approximately 30% higher than that of endometrial cancer after contrast administration. The area under receiver operating characteristic curve for SI, relative signal enhancement, and tumor to myometrium contrast ratio (as used to discriminate between cervical cancer and endometrial cancer) was 0.7807, 0.7456 and 0.7895, respectively. There was no difference in SI of the normal myometrium between endometrial and cervical cancer patients prior to and after contrast administration. Using non-tumorous myometrium as an internal reference, the tumor to myometrium contrast ratio was significantly higher in tumor tissue from cervical cancer compared with that from endometrial cancer at 25 s post contrast enhancement (p = 0.0016), with an optimal sensitivity of 72.22% and specificity of 84.21%. CONCLUSION: With SI normalized to baseline or normal myometrium, tumor tissue from cervical cancer patients showed significant hyperintensity compared with that of tumor tissue from endometrial cancer patients after contrast enhancement, yielding acceptable performance. The use of the myometrium as an internal reference may provide an alternative method to analyze MCE-MRI data.

GLK/MAP4K3 overexpression associates with recurrence risk for non-small cell lung cancer.

Lung cancer is the leading cause of cancer death worldwide. Non-small cell lung cancer (NSCLC) accounts for 85% of total lung cancers; 40% to 60% of NSCLC patients die of cancer recurrence after cancer resection. Since GLK (also named MAP4K3) induces activation of NF-κB, which contributes to tumor progression, we investigated the role of GLK in NSCLC. GLK protein levels of 190 samples from pulmonary tissue arrays and 58 pulmonary resection samples from stage I to stage III NSCLC patients were studied using immunohistochemistry or immunoblotting. High levels of GLK proteins were detected in pulmonary tissues from NSCLC patients. Elevated GLK protein levels were correlated with increased recurrence risks and poor recurrence-free survival rates in NSCLC patients after adjusting for pathologic stage, smoking status, alcohol status, and EGFR levels. Thus, GLK is a novel prognostic biomarker for NSCLC recurrence.

Epidermal growth factor activates telomerase activity by direct binding of Ets-2 to hTERT promoter in lung cancer cells.

Growth signals are directly or indirectly involved in telomerase regulation. In this study, we investigated molecular mechanisms of the effect of EGF (epidermal growth factor) on regulating hTERT (human telomerase reverse transcriptase) expression. To elucidate specific transcription factors involved in EGF-stimulated hTERT transcription in A549 and H1299 lung cancer cells, transcription factors drives hTERT promoter activity, such as Myc, Mad, and Ets-2, was evaluated on luciferase reporter assay. The upregulation of hTERT promoter by Ets-2 and Myc were abolished by Mad. Using DAPA (DNA affinity precipitation assay), Ets-2 binding to SNP (T) was stronger than Ets-2 binding to SNP (C) at -245 bp upstream of the transcription start site within the core promoter of hTERT. Ets-2 silence by siRNA decreased hTERT expression at mRNA and protein levels. The regulation of hTERT promoter by EGF/Ets-2 was diminished via the EGFR kinase signal pathway-specific inhibitors AG1478 and Iressa. Inhibitors of Erk and Akt inhibited Ets-2-activated hTERT promoter activity. These data suggested that Ets-2, a genuine cancer-specific transcription factor, is actively involved in EGFR kinase-induced hTERT overexpression pathway in lung cancer cells. Blockage of this pathway may contribute to targeted gene therapy in lung cancer.

Electrical characterization of single cells using polysilicon wire ion sensor in an isolation window.

A polysilicon wire (PSW) sensor can detect the H(+) ion density (pH value) of the medium coated on its surface, and different cells produce different extracellular acidification and hence different H(+) ion densities. Based on this, we used a PSW sensor in combination with a mold-cast polydimethylsiloxane (PDMS) isolation window to detect the adhesion, apoptosis and extracellular acidification of single normal cells and single cancer cells. Single living human normal cells WI38, MRC5, and BEAS-2B as well as non-small-cell lung cancer (NSCLC) cells A549, H1299, and CH27 were cultivated separately inside the isolation window. The current flowing through the PSW channel was measured. From the PSW channel current change as a function of time, we determined the cell adhesion time by observing the time required for the current change to saturate, since a stable extracellular ion density was established after the cells were completely adhered to the PSW surface. The apoptosis of cells can also be determined when the channel current change drops to zero. We found that all the NSCLC cells had a higher channel current change and hence a lower pH value than the normal cells anytime after they were seeded. The corresponding average pH values were 5.86 for A549, 6.00 for H1299, 6.20 for CH27, 6.90 for BEAS-2B, 6.96for MRC5, and 7.02 for WI38, respectively, after the cells were completely adhered to the PSW surface. Our results show that NSCLC cells have a stronger cell-substrate adhesion and a higher extracellular acidification rate than normal cells.

Expression of glutathione S-transferase M2 in stage I/II non-small cell lung cancer and alleviation of DNA damage exposure to benzo[a]pyrene.

Glutathione S-transferases (GSTs) are a family of inducible enzymes that are important in carcinogen detoxification. GST-Mu class is showing the high activity towards most polycyclic aromatic hydrocarbon (PAH) epoxide. Our objective is to clarify the expression of GST-M2 in non-small-cell lung carcinoma (NSCLC) patients and to determine the role of GST-M2 in protecting against DNA damage. We detected changes in GST-M2 expression at mRNA levels with a panel of lung cell lines and clinical samples of malignant and paired adjacent non-malignant tissues from 50 patients with stage I or II non-small-cell lung carcinoma using real-time RT-PCR. Comet assay and gamma-H2AX were used to clarify whether DNA damaged was protected by GST-M2. Our data demonstrate that the expression of GST-M2 in tumor tissues is significantly lower than in paired adjacent non-malignant tissues (p=0.016). Loss of GST-M2 is closely associated with age, gender, T value, N value and cell differentiation (p<0.05) in early stage I/II patients. Downregulation of GST-M2 is mediated through aberrant hypermethylation in lung cancer cell lines. Protection against B[a]P-induced DNA damage by GST-M2 in lung cancer cells was detected by Comet assay and gamma-H2AX. In conclusion, DNA hypermethylation altered and reduced GST-M2 expression that resulted in susceptible to benzo[a]pyrene (B[a]P) induced DNA damage. It implies that GST-M2 reduction occurs prior to tumorigenesis.

Modulation of telomere shelterin by TRF1 [corrected] and TRF2 interacts with telomerase to maintain the telomere length in non-small cell lung cancer.

Our previous report demonstrated good correlations between the expressions of h-TERT and its associated genes, such as c-Myc, TRF1 and TRF2. To observe the interaction between telomerase activity and expression of its associated genes in regulation of the telomere restriction fragment length (TRFL) in non-small cell lung cancer (NSCLC), 79 NSCLC specimens were examined. Telomerase activity, h-TERT, TRF1 and TRF2 genes expression were observed in 60.8, 66.7, 74.7, and 83.5% of the tumour tissues, respectively. The TRFL were shorter in both tumour tissues and telomerase positive tissues, as compared to their counterparts. The t/n-TRFLR (tumour-to-normal TRFL ratio) was also lower in telomerase positive tissues. When telomerase was negative, the t/n-TRFLR was lower in both TRF1 positive and TFR2 positive. However, when telomerase was positive, the t/n-TRFLR was only lower in the TFR2 positive group. When t/n-TRFLR level was equal to or less than 75%, the majority of the specimens became TRF1 and TRF2 positive. To explain these findings, our hypothesis is that when the TRF length becomes shorter during tumour progression, the tumour cells can sustain a better tolerance to shorter telomere with the help of both TRF1 and TRF2, but without immediate activation of the telomerase. However, when the TRF length reaches a critical level, changing the telomere shelterin by persistent expression of the TRF2, which in combination with telomerase activation reverses the telomere shortening.