| BackgroundLung cancer has become one of the major threats to human for its high morbidity and mortality. The risk factors that can increase lung cancer include smoking and environmental pollution etc. In recent years, the incidence rate of lung cancer in male is descending for the restriction of smoking, while the number of female patients is rising at the same time.According to the classification by WHO, lung cancer was divided into non-small-cell lung cancer (NSCLC) and small cell lung cancer (SCLC) on the basis of treatment effect, prognosis and biological property. As much as 80-85% of the histological type of lung cancer is NSCLC. The present studies show that the 5-year survival rate of lung cancer is not optimistic. Therefore, much work was needed to investigate effective treatment for prolonging the survival time of NSCLC patients.Basic research and clinical diagnosis and treatment of lung cancer have greatly changed. The studies of driver genes and targeted therapy have brought great improvement to lung cancer treatment. Driver genes can drive tumor formation and maintain tumor cell proliferation, and the targeted therapy can effectively control the tumor. The overall survival (OS) of advanced NSCLC was significantly different according to oncogenic drivers and genotype-directed therapy:the median survival was 3.5 years for patients with an oncogenic driver and genotype-directed therapy compared with 2.4 years for patients with oncogenic driver who did not receive genotype-directed therapy.Patients with epidermal growth factor receptor (EGFR) mutation are more likely to be female, never/light smoker and adenocarcinoma. The presence of EGFR mutation was a robust predictor of improved progression-free survival with EGFR tyrosine kinase inhibitors (TKIs), like gefitinib and erlotinib. EGFR-TKIs can greatly control the development of tumor with EGFR sensitive mutations (19del/L858R), and it can also prolong patients’progression-free survival (PFS) and OS.Anaplastic lymphoma kinase (ALK) rearrangement was another driver gene in NSCLC. ALK was located on chromosome 2p23, which could inversed and fused with NPM (nucleophosmin) on chromosome 5, leading to the activation of ALK kinase. So far, the activation of ALK gene have been reported in human hematologic and solid malignancies including anaplastic large-cell lymphoma, myofibrobalstic tumors as well as NSCLC, suggesting that ALK-mediated signaling might play a part in the development or progression of these tumors. Activation of the ALK gene included rearrangements, mutation and amplification. Mano proposed that tumors earring abnormal ALK as an essential growth driver were collectively termed as "ALKoma", and tumors earring ALK aberration can be greatly inhibited by ALK targeted therapy. A fusion gene between echinoderm microtubule-associated protein like 4 (EML4) and ALK led to an in-frame fusion protein with oncogenic activity in vitro. To date, multiple variants of EML4-ALK have been reported. Various ALK variants encoded the same intracellular tyrosine kinase domain of ALK, but with different truncations of EML4. Crizotinib has demonstrated an objective response rate (ORR) of approximately 60% patients with NSCLC harboring ALK rearrangements, but not all the ALK-positive patients could benefit from criztinib treatment. There are three main menthods in detecting ALK rearrangements, which are fluorescent in situ hybridization (FISH), reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). All those methods has its pros and cons, and the best method in detecting ALK rearrangements is still under investigation.Hence, this study aimed to investigate the consistency of FISH and rapid amplification of cDNA ends (RACE)-coupled polymerase chain reaction (PCR) in ALK rearrangements assay, and to evaluate the sensitivity and specificity of RACE-PCR when ALK FISH assay was used as the standard method. We also aimed to evaluate ALK variants, the percentage of ALK positive cells by FISH, treatment line of crizotinib usage on the efficacy of crizotinib in ALK rearranged NSCLC.Chapter 1 Comparisons between FISH and RACE-PCR in ALK rearrangement assayObjectiveTo investigate the consistency of FISH and RACE-PCR in ALK rearrangements assay.MethodsConsequent patients diagnosed as lung cancer and have detected ALK rearrangements by FISH or rapid-amplification of cDNA ends (RACE) polymerase chain reaction (PCR) at Guangdong lung cancer institute between January 2011 and March 2015 were enrolled in this retrospective analysis. All the patients had written informed consent for the genetic analysis. The inclusion criteria were as following:1) pathologically confirmed lung cancer; 2) ALK rearrangement was detected by FISH and RACE-PCR concurrently.All analysis was done with SPSS 22.0 software, Chi-square test (Kappa) were used to detect the consistency of FISH and RACE-PCR in ALK rearrangements assay. All statistical tests were two-sided and P< 0.05 was deemed to be statistically significant.ResultsA total of 88 patients with lung cancer underwent ALK rearrangements assay by FISH and RACE-PCR concurrently. Among them,60 cases (68.2%) were ALK-positive confirmed by the two methods,20 cases (22.7%) were ALK-negative confirmed by the two methods. FISH and RACE-PCR exhibited high consistency (Kappa=0.771, P<0.001). When ALK FISH assay was used as the standard method, the sensitivity and specificity of RACE-PCR was 90.9% and 90.9%, respectively.ConclusionFISH and RACE-PCR exhibited high consistency in detecting ALK rearrangements. When ALK FISH assay was used as the standard method, RACE-PCR demonstrated high sensitivity and specificity.Chapter 2 The predictive effect of ALK variants on the efficacy of crizotinib in ALK rearranged NSCLCObjectiveTo evaluated the correlation between ALK variants and the efficacy of crizotinib.MethodConsequent patients diagnosed as lung cancer and underwent ALK rearrangements assay at Guangdong lung cancer institute between December 2010 and March 2015 were enrolled in this retrospective analysis. All the patients had written informed consent for the genetic analysis. The inclusion criteria were as following:1) pathologically confirmed NSCLC; 2) ALK rearrangments confirmed by FISH, RACE-PCR or IHC; 3) at least one assessable target lesion was need; 4) with detailed treatment information; 5) received crizotinib treatment. For all of the patients, medical records were reviewed to extract data on their clinicopathological characteristics, treatment histories and survival data.Patients with sufficient frozen tissue for ALK RACE-coupled PCR sequencing were enrolled. Patients were categorized into three subgroups according to the frequency of ALK variants.Objective responses were assessed according to Response Evaluation Criteria In Solid Tumors (RECIST), version 1.1. PFS was calculated from the first day of crizotinib treatment to first radiological evidence of disease progression or death for any reason. Overall survival (OS) was measured from the date of metastatic NSCLC diagnosis to death resulting from any cause. The censored data was defined as the last follow-up or missing.All analysis was done with SPSS 22.0 software. Chi-square or Fisher’s exact test was used to detect difference of response rate of crizotinib. Survival curves were constructed with the Kaplan-Meier method and log-rank test was used to assess the differences between curves. All statistical tests were two-sided and P< 0.05 was deemed to be statistically significant.Results1. The frequency of ALK variants and characteristics of patients at baselineA total of 120 patients with NSCLC received crizotinib treatment were enrolled. We tested 68 ALK-positive cases by RACE-coupled PCR sequencing. ALK fusion genes were detected in 61 out of 68 cases (89.7%), and 7 cases (10.3%) were ALK negative by RACE-PCR. The most common variants were V1 and V3a/b, accounting for 36.1%(22/61) and 29.5%(18/61), respectively. In addition, a novel variant EML4-ALK E17; ins89A20 was identified. Patients were categorized into three subgroups, patients with EML4-ALK V1, EML4-ALK V3a/b and patients with other rare ALK variants. The characteristics at baseline like age, gender, smoking history, ECOG status, pathology and EGFR mutation status of the patients were well balanced among the three subgroups.2. The correlation between ALK variants and the response rate of crizotinibWe evaluated the response rate in the 61 patients with specific ALK variants. The ORR and disease control rate (DCR) was 68.9% and 91.8%, respectively. No statistically significant difference existed among the three subgroups in term of ORR and DCR (P=0.214; P=0.512)3. The correlation of the ALK variants and the PFS of crizotinibAt the time of data cutoff, the median PFS of crizotinib in all those cases was 10.0m (95% CI,6.1-14.0). The PFS of patients with EML4-ALK V1, EML4-ALK V3a/b and patients with other rare ALK variants was 11.0m (95% CI,5.6-16.5), 10.9m (95% CI,5.9-15.8) and 7.4m (95% CI,3.2-11.6), respectively. The log-rank test showed that there was no statistically difference among the three subgroups (χ2=0.459,P=0.795). Patients were also divided into two groups according to their ALK variants, patients with common ALK variants (EML4-ALK V1 and EML4-ALK V3a/b, n=40) and other rare ALK variants (n=21). The PFS of patients with common ALK variants and other rare ALK variants was 11.0m (95% CI,7.1-15.0) and 7.4m (95% CI,3.2-11.6), respectively. The data showed that the PFS of common ALK variants was longer than rare ALK variants, but the difference has no statistical significance(x2=0.445, P=0.505).Conclusion1. The most common variants were EML4-ALKV1 and EML4-ALK V3a/b.2. ALK variants has no correlation with the response rate and PFS of crizotinib.3. Crizotinib exhibited anti-tumor effect in patients with any kind of ALK variants.Chapter 3 The predictive effect of the percentage of ALK positive cells by FISH on the efficacy of crizotinib in ALK rearranged NSCLCObjectiveTo evaluate the correlation between the percentage of ALK positive cells by FISH and the efficacy of crizotinib.MethodPatients enrolled in section two were screened. ALK rearrangments was confirmed by FISH in 114 cases, and 106 cases had detailed ALK positive cells percentages. Patients were divided into two groups according the 50% cut-off value. All analysis was done with SPSS 22.0 software. The mean percentage of ALK positive cells by FISH was calculated. Chi-square or Fisher’s exact test was used to test difference of response rate of crizotinib. Survival curves were constructed with the Kaplan-Meier method and log-rank test was used to assess the differences between curves. All statistical tests were two-sided and P< 0.05 was deemed to be statistically significant.Results1. The distribution of the percentage of ALK positive cells by FISHAmong the 106 case, the mean of the percentage of ALK positive cells by FISH was 60.8%(Range:16%-98%). When chose 50% as the cut-off value, the percentage of patient with more than 50% and less than 50% were 71.7%(76/106) and 28.3%(30/106).2. The correlation of the percentage of ALK positive cells by FISH and the response rate of crizotinibWe evaluated the response rate in all 106 patients with detailed percentage of ALK positive cells. The ORR and DCR was 69.8% and 90.6%, respectively. When 50% was used as the cut-off value, no statistically significant difference existed between the two groups in term of ORR and DCR (P=0.658;P=0.142). Kendall’s tau_b test was used to investigate the correlation between the percentage of ALK positive cells by FISH and the best response of crizotinib, the data showed that those two variables are poorly correlated (rk=0.231,P=0.003).3. The correlation of the percentage of ALK positive cells by FISH and the PFS of crizotinibAt the time of data cutoff, the median PFS of crizotinib in all those cases was 8.5m (95% CI,6.2-10.7). The log-rank test showed that there was no statistically difference among the two groups when 50% as the cut-off value (χ2=2.946,P=0.086). Spearman test was used to investigate the correlation between the percentage of ALK positive cells by FISH and the PFS of crizotinib, the data showed that those two variables are poorly correlated (rs=0.235, P=0.015).Conclusion1. The percentage of positive cells within overall ALK FISH positive tumors covers a broad range.2. The percentage of ALK positive cells by FISH was weakly correlated with the best response of crizotinib.3.The percentage of ALK positive cells by FISH was weakly correlated with the PFS of crizotinib.Chapter 4 The correlation between the treatment line of crizotinib and the efficacy of crizotinib in ALK rearranged NSCLCObjectiveTo evaluated the correlation between the treatment line of crizotinib and the efficacy of crizotinib.MethodWe divided patients into two groups according to their treatment history of crizotinib. In total,68 patients were from clinical trials, and 52 patients received crizotinib in routine practice.All analysis was done with SPSS 22.0 software. Chi-square or Fisher’s exact test was used to detect differences of clinicopathological characteristics and the response rate of crizotinib. Survival curves were constructed with the Kaplan-Meier method and log-rank test was used to assess the differences between curves. A Cox regression model was used to calculate hazard ratio (HR) and its 95%confidence interval (CI), while a logistic regression model was used to assess the independent predictive factors for relative risk (RR). All statistical tests were two-sided and P<0.05 was deemed to be statistically significant.Results1. Characteristics of patients at baselineA total of 120 patients with NSCLC received crizotinib treatment were enrolled in this section. Among them, the median age of those patients was 48 years old, most of the patients were younger than 65 (90.8%), never-smoker (78.3%), the ECOG status 0-1 (91.7%), adenocarcinoma (96.7%).6 patients (5.0%) with concomitant EGFR mutation and ALK rearrangements.45 patients (37.5%,45/120) received crizotinib as first-line treatment. A total of 68 (56.7%) patients enrolled in PROFILE clinical trials and received crizotinib treatment.2. The correlation of the treatment line of crizotinib usage and the response rate of crizotinibThe ORR and DCR of these 120 patients were 69.2% and 90.0%, respectively. The ORR and DCR of crizotinib was 77.8% and 95.6% respectively in first-line setting; 65.3% and 91.8% respectively in second-line setting. Comparison on ORR and DCR between those two groups showed no statistically significant difference (P=0.182; P=0.679). When crizotinib was used in second and futher-line setting, the ORR and DCR was 64.0% and 86.7% respectively, the ORR and DCR did not differ between the first-line and non-first line (P=0.114; P=0.207). In clinical practice, patients tended to benefit more from first-line crizotinib treatment.3.The correlation of the treatment line of crizotinib usage and the PFS of crizotinibThe PFS of crizotinib was 8.5m (95% CI,6.8-10.1) for all the patients. The PFS of crizotinib when it used in first-line setting, second-line setting and second and further-line setting was 10.5m (95% CI,8.6-12.4),8.3m (95% CI,4.7-12.0),7.3m (95% CI,5.7-8.8), respectively. The median PFS in first-line was significantly longer than that in second-line (χ2=5.427, P=0.020). Additionally, the median PFS in first-line was longer than in second and further-line, the difference was marginally significant (χ2=3.243, P=0.072).When the 68 patients enrolled in PROFILE clinical trials were analyzed separately, the PFS of crizotinib was 8.5m (95% CI,5.6-11.3). The PFS of crizotinib in first-line setting, second-line setting and second and further-line setting was 11.2m (95%CI,4.8-17.6),8.2m (95%CI,1.7-14.7) and 7.0m (95%CI,2.1-11.9), respectively. The log-rank test showed that there was a statistically difference between first and second line crizotinib in terms of PFS (χ2=6.238, P=0.013); while the comparison between first and non-first line usage did not showed significant difference (χ2=2.465, P=0.116).4. Overall survivalThe OS of all the patients was 34.9m (95% CI,28.4-41.5) calculated from the date of metastatic NSCLC diagnosis.Conclusion1. EML4-ALK rearrangements were associated with younger patients, never smoker and adenocarcinoma.2. The response rate of crizotinib did not differ among different lines of crizotinib usage.3. The PFS of crizotinib used in first-line setting was longer than it used in second-line, and we recommend crizotinib as a first-line choice in advanced ALK-positive NSCLC patients. |
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