Case Report: ALK rearranged locally advanced lung adenocarcinoma showing inconsistent radiographic findings and pathological responses during neoadjuvant alectinib therapy.

Alectinib has been approved as first-line treatment for anaplastic lymphoma kinase (ALK)-positive non-small cell lung carcinoma. Oncologists are also exploring the possibility of applying alectinib in the perioperative period. Here, we present a patient with locally advanced lung adenocarcinoma associated with EML4-ALK fusion mutation, who received neoadjuvant chemotherapy and alectinib treatment, and then underwent thoracoscopic left lower lung lobectomy. The patient initially received eight chemotherapy cycles and achieved partial remission. After eight cycles of chemotherapy, the lymph nodes in the hilar region again enlarged. The patient was then switched to 4 months of alectinib therapy, but no significant lesion changes were detected on imaging during this period. This raised the question of whether the patient developed alectinib resistance. The pathological findings of the postoperative lung lobe specimens indicated extensive necrosis in the tumor area with no residual tumor cells and massive chronic inflammatory cell infiltration around the tumor area, confirming inconsistency between the imaging findings and pathological results. Multi-point tumor specimen sampling was postoperatively performed. Tumor immune-related gene expression was detected in the sample with the help of the PanCancer IO360™ panel based on the nCounter platform. This is a rare case of a patient who was treated with neoadjuvant alectinib and had paradoxical radiographic findings and pathological responses. The possibility that intratumoral immune heterogeneity was responsible for this phenomenon has been discussed. Based on the findings, it is argued that the pathological response should be an important basis for assessing the effectiveness of neoadjuvant alectinib therapy.

[A Case of Non-small Cell Lung Cancer Treated with Three ALK Inhibitors 
and Chemotherapy].

The echinoderm microtubule associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) were fractured and fused to become EML4-ALK. Most of these EML4-ALK-positive non-small cell lung cancer patients respond well to the ALK inhibitor. Many patients can benefit from drug target therapy for a long time, and some patients can achieve long-term survival of more than 7 years under the optimized treatment mode. This patient has lung adenocarcinoma positive for EML4-ALK fusion gene, but the treatment outcome is obviously different from that of other patients with lung cancer positive for EML4-ALK fusion gene. After the first to third generations of ALK inhibitor targeted therapy and chemotherapy, the disease progresses rapidly, the drug resistance time is short, the survival time is short, and the benefit is limited. The patient received targeted therapy of Crizotinib, Ceritinib and Lorlatinib successively from July 15, 2019, followed by two chemotherapy courses of Bevacizumab combined with Pemetrexed and Carboplatin. The patient died on September 10, 2020, with a survival of 15 months. At the same time, the treatment showed common adverse reactions of ALK inhibitors. This paper analyzed the therapeutic effect and treatment dilemma of this patient, and provided an exploration direction for the treatment of patients with EML4-ALK fusion gene positive lung cancer.
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A Case of Non-small Cell Lung Cancer Treated with Three ALK Inhibitors and Chemotherapy / 中国肺癌杂志

The echinoderm microtubule associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) were fractured and fused to become EML4-ALK. Most of these EML4-ALK-positive non-small cell lung cancer patients respond well to the ALK inhibitor. Many patients can benefit from drug target therapy for a long time, and some patients can achieve long-term survival of more than 7 years under the optimized treatment mode. This patient has lung adenocarcinoma positive for EML4-ALK fusion gene, but the treatment outcome is obviously different from that of other patients with lung cancer positive for EML4-ALK fusion gene. After the first to third generations of ALK inhibitor targeted therapy and chemotherapy, the disease progresses rapidly, the drug resistance time is short, the survival time is short, and the benefit is limited. The patient received targeted therapy of Crizotinib, Ceritinib and Lorlatinib successively from July 15, 2019, followed by two chemotherapy courses of Bevacizumab combined with Pemetrexed and Carboplatin. The patient died on September 10, 2020, with a survival of 15 months. At the same time, the treatment showed common adverse reactions of ALK inhibitors. This paper analyzed the therapeutic effect and treatment dilemma of this patient, and provided an exploration direction for the treatment of patients with EML4-ALK fusion gene positive lung cancer.
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EML4-ALK, a potential therapeutic target that responds to alectinib in ovarian cancer.

Ovarian cancer is prone to recurrence and chemotherapy resistance. Ovarian tumours of some patients have been positive for anaplastic lymphoma kinase fusion gene expression (ALK+). Preclinical studies indicate that anaplastic lymphoma kinase inhibitor can suppress the growth of ovarian cancer cells and transplantation tumours. Here, we present a patient with metastatic ALK+ high-grade serous ovarian cancer that testing positive for EML4-ALK (microtubule-associated protein-like 4 gene, fused to the anaplastic lymphoma kinase gene), experienced dramatic benefit after administration of the anaplastic lymphoma kinase inhibitor alectinib. This is the first clinical evidence that treatment with alectinib may provide a personalized maximum benefit for patients with high-grade serous ovarian cancer who are positive for EML4-ALK.

Comparison of EML4-ALK fusion gene positive rate in different detection methods and samples of non-small cell lung cancer.

Objective: To evaluate differences of EML4-ALK positive rates in tissues samples between immunohistochemistry, reverse transcriptase polymerase chain reaction and the next-generation sequencing method. Besides, to compare the differences of EML4-ALK positive rates in blood samples and tissue samples by next-generation sequencing. The results provide a basis for the selection of a suitable EML4-ALK fusion gene detection method. Methods: Immunohistochemistry analysis of EML4-ALK in tumors was performed on samples from 2631 patients with non-small cell lung cancer. The mutation of EML4-ALK in the tissue samples of 399 patients with non-small cell lung cancer was detected by reverse transcription polymerase chain reaction. Next-generation sequencing was used to detect the mutation of EML4-ALK in 1505 non-small cell lung cancer patients, including 1208 tissue samples and 297 blood samples. Results: The positive incidence of EML4-ALK by immunohistochemistry was 7.11% (187/2631). Histologically, 9.51% (170/1787) of the samples were lung adenocarcinomas, and 2.01% (17/844) were squamous cell carcinomas. The positive rate of EML4-ALK was 8.52% (34/399) in 399 patients with non-small cell lung cancer, as detected by reverse transcription polymerase chain reaction; the mutation rate of adenocarcinoma was 11.62% (33/284), and the mutation rate of squamous cell carcinoma was 0.86% (1/115). In 1208 patients with non-small cell lung cancer with tissue samples, the positive rate of EML4-ALK was 4.88% (59/1208), as determined by next-generation sequencing, the mutation rate of adenocarcinoma was 5.84% (58/994), and the mutation rate of squamous cell carcinoma was 0.47% (1/214). The positive rate of EML4-ALK detected by reverse transcription polymerase chain reaction was higher than that detected by immunohistochemistry. Compared with the next-generation sequencing results, the positive rates of EML4-ALK detected by immunohistochemistry and reverse transcription polymerase chain reaction were higher, and the differences were significant (p<0.05). In blood samples from 297 patients with non-small cell lung cancer, the positive rate of EML4-ALK detected by next-generation sequencing was 3.70% (11/297), the mutation rate of adenocarcinoma was 3.82% (10/262), and the mutation rate of squamous cell carcinoma was 2.86% (1/35). The EML4-ALK positive rate of the tissue samples was thus higher than that of the blood biopsy samples. Conclusion: Among the three methods for detecting EML4-ALK, reverse transcription polymerase chain reaction has the highest positive rate, followed by immunohistochemistry, and next-generation sequencing has the lowest positive rate. The positive detection rate of EML4-ALK in tissue samples by next-generation sequencing was higher than that in blood samples.

Validation of prognostic impact of number of extrathoracic metastases according to the eighth TNM classification: a single-institution retrospective study in Japan.

BACKGROUND: In the eighth edition of the TNM classification of lung cancer, the M1b and M1c descriptors are newly defined by the number of extrathoracic metastases. To verify the prognostic value of these descriptors in Japan, we reclassified our cases and re-evaluated prognosis in M1b and M1c patients. METHODS: All non-small cell lung cancer (NSCLC) patients with extrathoracic metastases who visited Saitama Medical Center from 2010 to 2016 were evaluated, divided according to the eighth edition of the TNM classification criteria into two groups (M1b, patients with single extrathoracic metastasis, and M1c, patients with multiple extrathoracic metastases), and followed up until December 31, 2017. Survival time analysis was performed using the Kaplan-Meier method, and between-group differences in overall survival time (OS) were evaluated by the log-rank test. RESULTS: A total of 231 NSCLC patients were divided into 57 patients with M1b and 174 with M1c. Median OS was 15.2 months (95% confidence interval [CI]: 9.3-19.9) and 7.3 months (95% CI 5.7-10.7) for M1b and M1c, respectively, with no significant between-group difference (P = 0.239). However, after excluding patients with epidermal growth factor receptor (EGFR) mutation or echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK) fusion gene, median OS was 12.9 months (95% CI 7.2-19.9) for M1b and 5.4 months (95% CI 3.8-6.3) for M1c, respectively, showing a significant difference (P = 0.029). CONCLUSIONS: The effect of therapy directed toward EGFR mutation or EML4-ALK fusion gene might obscure the significant prognostic difference between M1b and M1c.

Bypass signaling pathway activation mediates resistance of EML4-ALK fusion gene positive lung cancer cell line H3122 to alectinib / 中国病理生理杂志

AIM:To detect the changes of active status of bypass signaling pathways in EML4-ALK positive lung cancer cell line H3122 treated with alectinib, hepatocyte growth factor (HGF), epidermal growth factor (EGF) and transforming growth factor-α (TGF-α), and to explore the potential mechanisms.METHODS:EML4-ALK positive cell line H3122 was treated with increasing concentrations of alectinib or/and induced by HGF, EGF and TGF-α.The cell viability was measured by CCK-8 assay.The cell apoptosis was analyzed by flow cytometry.The protein levels and phosphorylation status of ALK, c-Met and EGFR, and the downstream molecules AKT, ERK, p-AKT and p-ERK were examined by Western blot.RESULTS:The viability of the H3122 cells was inhibited by alectinib in a dose-dependent manner after administrated for 72 h, and the IC50 value was 0.042 μmol/L.The concentration-growth curves of the H3122 cells shifted to the right after induced by HGF, EGF and TGF-α.After treatment with alectinib at 0.05 μmol/L for 48 h, the apoptotic rate of H3122 cells was (20.12±1.36)%, while the apoptotic rates of the cells in the groups of alectinib combined with HGF, EGF or TGF-α were (7.85±1.03)%, (5.60±0.79)% and (4.58±1.00)%, respectively.Those values were remarkably lower than those in alectinib single treatment group (P<0.05).Alectinib inhibited the protein levels of p-ALK and its downstream signaling pathway molecules, while HGF significantly up-regulated the protein levels of p-Met and its downstream p-AKT and p-ERK.Besides, EGF and TGF-α remarkablely up-regulated the protein levels of p-EGFR and its downstream p-AKT and p-ERK.Combined treatment with crizotinib and 17-DMAG successfully inhibited the viability of the H3122 cells even in the presence of the HGF and EGFR ligands, respectively.CONCLUSION:Bypass signaling pathways are activated by HGF, EGF and TGF-α in EML4-ALK positive lung cancer cell line H3122, which may be linked to alectinib resistance.

Statistical analysis of EGFR mutation and EML4-ALK gene fusion in non-small cell lung cancer patients of Zhongshan City / 国际检验医学杂志

Objective To detect the mutation of epidermal growth factor receptor(EGFR) gene,fusion of echinoderms microtubule associated protein sample-4 and gradual change of lymphoma kinase(EML4-ALK) gene,as well as describe their relationship with the clinicopathological features in patients with non-small cell lung cancer(NSCLC) from Zhongshan city of Guangdong province.Methods Mutations of EGFR gene and EML4-ALK fusion gene in 753 NSCLC patients from Zhongshan People's hospital were detected by ARMS real-time PCR.To study the relationship between the mutation and clinical features and explore the significance of EGFR gene mutation and EML4-ALK fusion in NSCLC.Results The EGFR mutation rate of 753 NSCLC patients is 43.16％(325/753),with highest mutation rate in 19 and 21 exons,43.08％(140/325) and 47.38％ (154/325) respectively,and the main mutation in 21 exon is L858R mutation.EGFR mutation is more common in female/non-smoking patients,or patients with adenocarcinoma/adenosquqmous carcinoma/adenocarcinoma metastasis(P＜0.05),but not relates with the age of patients(P＞0.05).The EML4-ALK fusion gene of 110 patients whose EGFR mutation were checked were simultaneously detected,showing a 9.09 ％ (10/110) mutation rate,and the mutation rate in type 1(80％) is significantly higher than type 2(10％) and 3(10％).Patients with EML4-ALK gene mutation tend to be younger(P＜0.05),but the EML4-ALK gene mutation rates show no significant differences in groups classified by gender,smoking history or pathological classification(P＞0.05).EGFR gene mutation and EML4-ALK fusion were detected in one patient simultaneously.Conclusion The EGFR mutation rate of patients with NSCLC in Zhongshan city is consistent with results reported in domestic and foreign literatures.Detections of EGFR gene mutation and EML4-ALK fusion are necessary test items,providing important evidence in molecular targeting therapy in NSCLS.

Statistical analysis of EGFR mutation and EML4-ALK gene fusion in non-small cell lung cancer patients of Zhongshan City / 国际检验医学杂志

Objective To detect the mutation of epidermal growth factor receptor(EGFR) gene,fusion of echinoderms microtubule associated protein sample-4 and gradual change of lymphoma kinase(EML4-ALK) gene,as well as describe their relationship with the clinicopathological features in patients with non-small cell lung cancer(NSCLC) from Zhongshan city of Guangdong province.Methods Mutations of EGFR gene and EML4-ALK fusion gene in 753 NSCLC patients from Zhongshan People's hospital were detected by ARMS real-time PCR.To study the relationship between the mutation and clinical features and explore the significance of EGFR gene mutation and EML4-ALK fusion in NSCLC.Results The EGFR mutation rate of 753 NSCLC patients is 43.16％(325/753),with highest mutation rate in 19 and 21 exons,43.08％(140/325) and 47.38％ (154/325) respectively,and the main mutation in 21 exon is L858R mutation.EGFR mutation is more common in female/non-smoking patients,or patients with adenocarcinoma/adenosquqmous carcinoma/adenocarcinoma metastasis(P＜0.05),but not relates with the age of patients(P＞0.05).The EML4-ALK fusion gene of 110 patients whose EGFR mutation were checked were simultaneously detected,showing a 9.09 ％ (10/110) mutation rate,and the mutation rate in type 1(80％) is significantly higher than type 2(10％) and 3(10％).Patients with EML4-ALK gene mutation tend to be younger(P＜0.05),but the EML4-ALK gene mutation rates show no significant differences in groups classified by gender,smoking history or pathological classification(P＞0.05).EGFR gene mutation and EML4-ALK fusion were detected in one patient simultaneously.Conclusion The EGFR mutation rate of patients with NSCLC in Zhongshan city is consistent with results reported in domestic and foreign literatures.Detections of EGFR gene mutation and EML4-ALK fusion are necessary test items,providing important evidence in molecular targeting therapy in NSCLS.

Detection of EML4-ALK fusion gene and features associated with EGFR mutations in Chinese patients with non-small-cell lung cancer.

PURPOSE: Echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) and epidermal growth factor receptor (EGFR) define specific molecular subsets of lung cancer with distinct clinical features. We aimed at revealing the clinical features of EML4-ALK fusion gene and EGFR mutation in non-small-cell lung cancer (NSCLC). METHODS: We enrolled 694 Chinese patients with NSCLC for analysis. EML4-ALK fusion gene was analyzed by real-time polymerase chain reaction, and EGFR mutations were analyzed by amplified refractory mutation system. RESULTS: Among the 694 patients, 60 (8.65%) patients had EML4-ALK fusions. In continuity correction χ (2) test analysis, EML4-ALK fusion gene was correlated with sex, age, smoking status, and histology, but no significant association was observed between EML4-ALK fusion gene and clinical stage. A total of 147 (21.18%) patients had EGFR mutations. In concordance with previous reports, EGFR mutation was correlated with age, smoking status, histology, and clinical stage, whereas patient age was not significantly associated with EGFR mutation. Meanwhile, to our surprise, six (0.86%) patients had coexisting EML4-ALK fusions and EGFR mutations. CONCLUSION: EML4-ALK fusion gene defines a new molecular subset in patients with NSCLC. Six patients who harbored both EML4-ALK fusion genes and EGFR mutations were identified in our study. The EGFR mutations and the EML4-ALK fusion genes are coexistent.

Effective enrichment strategy for EML4-ALK fusion gene screening in patients with non-small cell lung cancer.

BACKGROUND: A novel fusion gene that comprises the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) genes was recently identified in non-small cell lung cancer (NSCLC), particularly in adenocarcinoma. A specific ALK inhibitor has been shown to exert anti-tumor effects in NSCLC with the EML4-ALK fusion gene. Previous reports suggested an EML4-ALK incidence of approximately 5% in a pan-NSCLC population, with an increased frequency in younger patients, but an appropriate strategy for further selecting patients with the EML4-ALK fusion gene remains unknown. METHODS: Patients, 55 years of age or younger, who were diagnosed with NSCLC without typical squamous cell carcinoma features at our institute were retrospectively evaluated. The tumor specimens were examined by immunohistochemistry for the EML4-ALK fusion gene and by polymerase chain reaction for epidermal growth factor receptor (EGFR) mutations. RESULTS: Between January 2004 and September 2011, the EML4-ALK fusion gene was detected in 19.6% (9/46) of patients. The fusion gene incidence increased to 31% (9/29) when patients with EGFR mutations were excluded. The EML4-ALK fusion gene was further detected in 2 cases of undifferentiated cell carcinoma. CONCLUSIONS: EML4-ALK fusion gene examinations could be more effectively performed by selecting young NSCLC patients without EGFR mutations, whereas selection on the basis of a non-smoking or adenocarcinoma history, as reported in previous studies, may not correctly identify the patient groups with potential EML4-ALK fusion gene.

Targeting ALK in patients with advanced non small cell lung cancer: biology, diagnostic and therapeutic options.

The discovery of EML4-ALK fusion gene in a subgroup of patients with lung adenocarcinoma led to the development of a new class of agents, the ALK inhibitors, and dramatically improved the clinical outcome of these patients. The striking results from clinical trials with crizotinib, the first ALK inhibitor evaluated, allowed the accelerated approval of crizotinib from the USA Food and Drug Administration (FDA). Despite the high initial results, patients acquire resistance to crizotinib, and different next generation ALK kinase inhibitors have been developed. In the current review, we will analyze the biology of EML4-ALK gene, the acquired resistance mechanisms to crizotinib, the therapeutic strategies, currently under evaluation, designed to overcome crizotinib resistance, and the open issues that need to be addressed in order to improve outcome in ALK+ Non Small Cell Lung Cancer (NSCLC) patients.

Role of mTOR signaling pathway in crizotinib-induced apoptosis of EML4-ALK fusion gene-positive lung adenocarcinoma cell line H2228 / 中国病理生理杂志

AIM:To investigate the mammalian target of rapamycin ( mTOR) signaling pathway as the center playing a role in the crizotinib-induced apoptosis of non-small cell lung cancer (NSCLC) cell line H2228, which represents positive echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene. METHODS:H2228 cells were processed according to different purposes .Fluorescence quantitative PCR is used to ob-serve the gene states .MTT assay is used to detect the cell inhibition rates .The cell apoptosis and cell cycle were analyzed by flow cytometry .The expression and activation levels of the key proteins in the mTOR signaling pathway were determined by Western blotting .RESULTS:Crizotinib promoted the apoptosis of H 2228 cells in a time-and dose-dependent manner . Crizotinib blocked the H2228 cells staying at the G1 phase.In apoptotic H2228 cells processed with crizotinib, the activa-tion level of mTOR was decreased , and the activation levels of the key proteins in upstream and downstream of mTOR path -way were both declined .The expression level of the fusion protein EML 4-ALK variant 3 was not affected , but its active form of p-ALK was significantly suppressed .CONCLUSION:mTOR signaling pathway has a certain relationship with the crizotinib-induced apoptosis of lung cancer cell H 2228, which represents positive EML4-ALK fusion gene.

Progress of EML4-ALK fusion gene non-small cell lung cancer inhibitor / 国际肿瘤学杂志

EML4-ALK fusion gene is a new molecular type of non-small cell lung cancer,which is formed by inversion of two gene segments on chromosome 2.Up to now,9 subtypes have been found and all of them have deteriorated and carcinogenic abilities. The detection rate of this fusion gene is much higher in patients who have adenocarcinoma,never or seldom smoke and lack EGFR/KRAS mutation.This fusion gene codes ALK tyrosine kinase,that abnormally phosphorylates downstream products through signaling pathways,leading to canceration.Therefore,the specific inhibitors which target this fusion gene could inhibit its expression and have good treatment effect.At present,the relevent inhibitors which are undergoing clinical trials are PF02341066 and TAE684.