ALK Fusion Gene Research Articles

Một số bất thường gen ở bệnh nhân ung thư phổi không tế bào nhỏ tại Bệnh viện Đại học Y Hà Nội phát hiện bằng phương pháp giải trình tự gen thế hệ mới

This study aimed to evaluate the rate of genetic mutations in patients with non- small-cell lung cancer by Next-Generation Sequencing at Hanoi Medical university hospital from 2022 to 2023 and to assess the relationship between genetic mutations and pathological characteristics in non-small-cell lung cancer. In 90 non-small-cell lung cancer patients in the study, we found EGFR gene mutations in 41.1% of cases; KRAS gene mutation in 11.1%; ALK gene fusion in 4.4%; PIK3CA gene mutation in 2.2%; BRAF gene mutation in 1.1%; MET gene mutation in 1.1% and ROS1 gene fusion in 1.1%. We found that EGFR mutations were more commonly detected in female, non-smokers and having adenocarcinoma while KRAS mutations were more commonly detected in male.

An observational study on the efficacy of targeted therapy for pulmonary sarcomatoid carcinoma

BackgroundPulmonary sarcomatoid carcinoma is a rare tumor that is resistant to cytotoxic agents. This observational study aimed to evaluate the detection rate of driver gene alteration and the efficacy of targeted therapy for pulmonary sarcomatoid carcinoma.MethodsWe established a database of patients with pulmonary sarcomatoid carcinoma and their clinical information, including EGFR mutation, ALK fusion gene, ROS1 fusion gene, BRAF mutation, and MET exon 14 skipping mutation. The present study retrieved and analyzed the data of patients with pulmonary sarcomatoid carcinoma in whom driver gene alterations were evaluated, and the survival duration after the initiation of treatment with targeted therapy was examined.ResultsA total of 44 patients were included in the present study. The EGFR mutation, ALK fusion gene, and MET exon 14 skipping mutation were detected in 2/43 patients (4.7%), 2/34 patients (5.9%), and 2/16 patients (12.5%), respectively. The ROS1 fusion gene (0/18 patients) and BRAF mutation (0/15 patients) were not detected. Female patients (P = 0.063, Fisher’s exact test) and patients without smoking history (P = 0.025, Fisher’s exact test) were the dominant groups in which any driver mutation was detected. Five patients with driver gene alterations were treated with targeted therapy. Progression-free survival (PFS) was 1.3 months and 1.6 months in 2 of the patients treated with gefitinib. Two patients with the ALK fusion gene showed 2.1 and 14.0 months of PFS from the initiation of treatment with crizotinib, and a patient with the MET exon 14 skipping mutation showed 9.7 months of PFS from the initiation of treatment with tepotinib.ConclusionThe EGFR mutation, ALK fusion gene, and MET exon 14 skipping mutation were detected in patients with pulmonary sarcomatoid carcinoma in clinical practice, and some patients achieved long survival times after receiving targeted therapy. Further investigation is necessary to evaluate the efficacy of targeted therapy for pulmonary sarcomatoid carcinoma.

Performances of the Idylla GeneFusion Assay: contribution to a rapid diagnosis of targetable gene fusions in tumour samples

AimsWe aimed to evaluate the performances of the Idylla GeneFusion Assay (IGFA) designed to detect, in a single, rapid and fully automated assay, ALK, ROS1, RET, NTRK1, NTRK2 and NTRK3...

A phase 1/2, open label study of amivantamab in combination with tyrosine kinase inhibitors among participants with advanced NSCLC harboring ALK, ROS1, and RET gene fusions.

TPS8661 Background: Patients with ALK, ROS1, or RET rearranged NSCLC demonstrate dramatic and sustained responses to tyrosine kinase inhibitors (TKI). Unfortunately, acquired resistance remains a major clinical challenge. The EGFR and MET pathways have been reported as important escape mechanisms for cancer cells treated with highly potent TKIs. Combining amivantamab with ALK, ROS1, or RET TKIs upon radiographic progression represents a rational combination strategy in addressing TKI mediated resistance. Methods: This is an open-label, multicenter, Phase 1b / 2 study of amivantamab in combination with TKIs among participants with advanced NSCLC harboring ALK, ROS1, and RETgene fusions progressing on FDA-approved TKIs for their respective oncogene. Participants must be on their TKI at the same dose for at least 3 months prior to enrolling in the study. A pre-treatment biopsy is required to rule out neuroendocrine transformation and for translational objectives. Local confirmation of an ALK, ROS1, or RET gene fusion through tissue or circulating tumor DNA (ctDNA) assessment is allowed. Plasma collection for ctDNA assessments will occur prior to treatment, on C3D1, and post-treatment. This study will have both a safety lead-in cohort and a dose expansion cohort. A safety lead-in cohort will be treated with amivantamab at the RP2D in combination with their ALK, ROS1, or RET TKI. The primary endpoint will be dose-limiting toxicity (DLT) within the first cycle of dosing at a given dose level. A Bayesian optimal interval (BOIN) design will be employed as a safety lead-in using a dose de-escalation design to find the maximum tolerated dose (MTD) of amivantamab in combination with a standard fixed dose of common ALK, ROS1, and RET TKIs. The RP2D dose for amivantamab was previously set at 1050mg (for patients < 80kg) and 1400mg (for patients ≥ 80kg). Patients will be treated in cohorts of size 3, and the maximum sample size for the dose escalation portion of the trial is 12 patients. The dose expansion cohort will evaluate the safety and efficacy of amivantamab (at the RP2D dose) in combination with standard FDA-approved doses of ALK, ROS1, and RET TKIs. The primary endpoint will be objective response rate (ORR). All patients enrolled at the RP2D level will be included in the phase II efficacy analysis, including patients enrolled in the safety lead-in cohort. A total of 16 to 35 evaluable patients, depending on the safety of the tested treatments in the dose escalation phase, are expected to be accrued to this study. Relevant exclusion criteria include: (1) prior EGFR directed therapy, (2) prior receipt of a MET monoclonal antibody or MET-ADC, (3) crizotinib use as immediate prior line of therapy, (4) small cell transformation, and (5) active, untreated, CNS metastases. The study has initiated enrollment without DLT and is ongoing. Clinical trial information: NCT05845671 .

Comparative Performance Analysis of Idylla and Archer in the Detection of Gene Fusions in Spitzoid Melanocytic Tumors

Melanocytic neoplasms with spitzoid histomorphology are often difficult to classify without identifying genetic drivers such as kinase fusions. Traditional diagnostic methods, such as immunohistochemistry, can yield inconclusive results, and advanced techniques such as the Archer fusion assay are often inaccessible and costly. The Idylla GeneFusion Assay might offer a rapid and cost-effective alternative. This study compared Idylla and Archer in identifying ALK, pan-NTRK, RET, and ROS1 gene fusions. Of the 147 samples where next-generation sequencing did not detect genetic drivers, 89 (60.5%) meeting the tissue requirements were further analyzed using Idylla (Cohort A). Idylla demonstrated a sensitivity of 75% and a specificity of 100% in detecting these fusions. Additionally, among 27 randomly selected cases (Cohort B) that failed to meet the inclusion criteria, Idylla maintained the same levels of sensitivity and specificity. Our findings also show that Idylla can be effectively conducted with isolated RNA, broadening its applicability beyond tissue samples. Although the Idylla assay may not replace more comprehensive molecular assays such as Archer, it could serve as a valuable initial screening tool in diagnosing spitzoid melanocytic tumors.

ALK fusion genes in a large cohort of thyroid carcinomas

ALK fusion genes in a large cohort of thyroid carcinomas

Rare differential diagnosis of an osteolytic lesion of the mandible in a young adult

We report ararely occurring hematologic neoplasm in ayoung adult. Hematologic neoplasms were first described in 2008 and are now included in both accepted tumor classification systems, i.e., International Consensus Classification and World Health Organization. This hematologic neoplasm shows acharacteristic ALK positivity in immunohistochemical examination and correspondingly, ALK fusion genes in the molecular analysis. Pathologists should be aware of this entity, particularly as it is challenging to differentiate from other more frequent neoplasms of the same disease group or mesenchymal neoplasm with ALK aberration.

Abstract 6457: The real-world data of comprehensive genomic profiling tests with tissue specimens from lung cancer patients in Japan

Abstract Introduction: Molecularly targeted therapy improves prognosis of lung cancer patients with specific genomic alterations, and thus it is important to identify actionable genomic alterations leading to appropriate therapy. In Japan, comprehensive genomic profiling (CGP) tests have been covered by insurance since June 2019. 99.7% of the CGP data under insurance coverage have been registered with the nationwide database: Center for Cancer Genomics and Advanced Therapeutics (C-CAT). C-CAT data include CGP test results and clinical information such as companion diagnostic results for EGFR, ALK, ROS1, and BRAF as well as therapeutic history. The aim of this study is to evaluate the usefulness of CGP tests for identifying actionable genomic alterations in real-world lung cancer patients. Methods: Among the 3,240 lung cancer patients registered in the C-CAT database by September 2023, 2,076 non-small cell lung cancer cases were tested using tissue specimens and analyzed retrospectively. The analysis was based on the levels of evidence defined by C-CAT. Level A indicated approved drugs for the particular cancer type, while level C indicated approved drugs or presence of positive clinical trials regardless of cancer type. Level F indicated that the genomic alteration was an oncogenic marker. Results: The cases were divided into two groups: 335 cases with known genomic alterations with positive results from companion diagnostic tests or use of specific inhibitors and 1,741 cases without. Genomic alterations with level A, A to C, and A to F were identified in 430 cases (24.7%), 1174 cases (67.4%), and 1718 cases (98.7%) with unknown genomic alterations, respectively. Genomic alterations with level A included rearrangement of RET (43 of 1741 cases, 2.5%), ALK (14 cases, 0.8%), ROS1 (8 cases, 0.5%), and NTRK1 (2 cases, 0.1%), and mutations of EGFR (124 cases, 7.1%), MET (38 cases, 2.2%), ERBB2 (122 cases, 7.0%), KRAS G12C (66 cases, 3.8%), and BRAF V600E (15 cases, 0.9%). Genomic alterations with level C included mutations in KRAS (185 cases, 10.6%), PIK3CA (99 cases, 5.7%), BRAF (55 cases, 3.2%), and PTEN (32 cases, 1.8%) and amplifications in ERBB2 (147 cases, 8.4%) and MET (68 cases, 3.9%). Among cases with known genomic alterations, ALK and ROS1 fusion genes were not detected in 11 of 49 (22.4%) and 17 of 46 (37.0%), while EGFR and BRAF mutation was not detected in 11 of 218 (5.0%) and 3 of 4 cases (75.0%), respectively. The detection rate of fusion genes was lower than that of mutated genes (70.5% vs 93.7%, p<0.001). Conclusion: In non-small cell lung cancer patients with unknown driver genes, driver oncogenes with corresponding approved therapy were found in a quarter of the patients. CGP tests offer the possibility of increasing the opportunities of receiving new therapies. CGP tests may have a lower detection rate for fusion genes compared with other mutations. Citation Format: Koki Fujii, Momoko Morishita, Michiko Ueki, Hiroaki Ikushima, Hideaki Isago, Kousuke Watanabe, Katsutoshi Oda, Hidenori Kage. The real-world data of comprehensive genomic profiling tests with tissue specimens from lung cancer patients in Japan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6457.

Distinction of ALK fusion gene- and EGFR mutation-positive lung cancer with tumor markers.

It is difficult to predict gene mutations individually based on clinical background alone. Tumor markers may help to predict each gene mutation. Identifying tumor markers that can predict gene mutation will facilitate timely genetic testing and intervention. We selected 134 cases of advanced or recurrent ALK-positive and 172 cases of advanced or recurrent EGFR-positive lung cancer from our clinical database. The cutoff values for the tumor markers were defined as 5.0 ng/mL or higher for carcinoembryonic antigen (CEA) and 3.5 ng/mL or higher for soluble fragment of cytokeratin 19 (CYFRA21-1) in accordance with the institutional standards. A positive CYFRA21-1:CEA ratio was defined as 0.7 or higher. The CEA-positivity rate was 49% for ALK-positive lung cancers and 73% for EGFR-positive lung cancers, which was significantly different (p < 0.001). The CYFRA21-1 positivity rate was significantly higher in ALK-positive lung cancer (36%) compared with EGFR-positive lung cancer (23%) (p = 0.034). The median CYFRA21-1:CEA ratio was 0.395 for the ALK group, which was significantly higher compared with 0.098 for the EGFR group (p < 0.001). These trends were similar when excluding histology other than adenocarcinoma. The median time-to-treatment failure (TTF) for initial tyrosine kinase inhibitor (TKI) therapy was 308 days for the high CYFRA21-1:CEA ratio group and 617 days for the low CYFRA21-1:CEA ratio group for ALK-positive lung cancer (p = 0.100). A higher proportion of patients with ALK-positive lung cancer were CYFRA21-1 positive and had higher CYFRA21-1:CEA ratios compared with EGFR-positive lung cancer patients.

Current status of molecular diagnostics for lung cancer.

The management of lung cancer (LC) requires the analysis of a diverse spectrum of molecular targets, including kinase activating mutations in EGFR, ERBB2 (HER2), BRAF and MET oncogenes, KRAS G12C substitutions, and ALK, ROS1, RET and NTRK1-3 gene fusions. Administration of immune checkpoint inhibitors (ICIs) is based on the immunohistochemical (IHC) analysis of PD-L1 expression and determination of tumor mutation burden (TMB). Clinical characteristics of the patients, particularly age, gender and smoking history, significantly influence the probability of finding the above targets: for example, LC in young patients is characterized by high frequency of kinase gene rearrangements, while heavy smokers often have KRAS G12C mutations and/or high TMB. Proper selection of first-line therapy influences overall treatment outcomes, therefore, the majority of these tests need to be completed within no more than 10 working days. Activating events in MAPK signaling pathway are mutually exclusive, hence, fast single-gene testing remains an option for some laboratories. RNA next-generation sequencing (NGS) is capable of detecting the entire repertoire of druggable gene alterations, therefore it is gradually becoming a dominating technology in LC molecular diagnosis.

10167-MET-8 CLINICAL CHARACTERISTICS AND TREATMENT STRATEGY OF CANCER CASES PRESENTING WITH CENTRAL NERVOUS SYSTEM SYMPTOMS

Abstract Metastatic brain tumors were previously reported with prognoses of approximately 6 months. The accuracy of diagnostic imaging and advances in drug therapy have improved the treatment outcomes of various types of cancer. On the other hand, patients with advanced cancer who are detected with neurological symptoms caused by metastatic brain tumors usually face difficulty in deciding on a treatment strategy. In this study, we reported the results of cancer patients who were first detected with central nervous system symptoms in the last 10 years and treated at our hospital.There were 14 patients treated at our hospital from 2013 to 2022 with CNS symptoms as the initial symptom. The mean age was 65.5 years, and the most common primary cancer was lung cancer in 11 cases (9 NSCLC and 2 SCLC). The OS examined in the 11 lung cancer cases was 14 months. Among NSCLC, only 2 cases had EGFR mutation and no cases had ALK fusion gene. The mean Lung-molGPA was 0.89 in 9 patients with NSCLC and 0.75 in 2 patients with SCLC, and the expected survival time was 6.9 months and 2.8 months.With advances in drug therapy, median survival for patients with NSCLC brain metastases has been reported to range from 7 months to 2 years for patients with EGFR mutations and 4 years for patients with ALK fusion gene. Thus, it is necessary to consider treatment strategies according to primary tumor, histology, and genetic information, rather than considering metastatic brain tumors as a single entity.The results of this study suggest that aggressive therapeutic intervention may prolong the survival period expected at the time of initial diagnosis, even in cancer patients who are found to have metastatic brain tumors.

Usefulness of an RNA extraction-free test for the multiplexed detection of ALK, ROS1, and RET Gene Fusions in Real Life FFPE Specimens of Non-Small Cell Lung Cancers

ABSTRACT Background ALK, ROS1 and RET rearrangements occur, respectively, in 5%, 2%, and 1% non-small cell lung cancers (NSCLC). ALK and ROS1 fusion proteins detection by immunohistochemistry (IHC) has been validated for rapid patient screening, but ROS1 fusions need to be confirmed by another technique and no RET IHC test is available for clinical use. Research design and methods We report herein the usefulness of the HTG EdgeSeq Assay, an RNA extraction-free test combining a quantitative nuclease protection assay with NGS, for the detection of ALK, ROS1 and RET fusions from ‘real-life’ small NSCLC samples. A total of 203 FFPE samples were collected from 11 centers. They included 143 rearranged NSCLC (87 ALK, 39 ROS1, 17 RET) and 60 ALK-ROS1-RET negative controls. Results The assay had a specificity of 98% and a sensitivity for ALK, ROS1 and RET fusions of 80%, 94% and 100% respectively. Among the 19 HTG-assay false negative samples, the preanalytical conditions were identified as the major factors impacting the assay efficiency. Conclusions Overall, the HTG EdgeSeq assay offers comparable sensitivities and specificity than other RNA sequencing techniques, with the advantage that it can be used on very small and old samples collected multicentrically.

MIG6 Mediates Adaptive and Acquired Resistance to ALK/ROS1 Fusion Kinase Inhibition through EGFR Bypass Signaling.

Despite the initial benefit from tyrosine kinase inhibitors (TKI) targeting oncogenic ALK and ROS1 gene fusions in non-small cell lung cancer, complete responses are rare and resistance ultimately emerges from residual tumor cells. Although several acquired resistance mechanisms have been reported at the time of disease progression, adaptative resistance mechanisms that contribute to residual diseases before the outgrowth of tumor cells with acquired resistance are less clear. For the patients who have progressed after TKI treatments, but do not demonstrate ALK/ROS1 kinase mutations, there is a lack of biomarkers to guide effective treatments. Herein, we found that phosphorylation of MIG6, encoded by the ERRFI1 gene, was downregulated by ALK/ROS1 inhibitors as were mRNA levels, thus potentiating EGFR activity to support cell survival as an adaptive resistance mechanism. MIG6 downregulation was sustained following chronic exposure to ALK/ROS1 inhibitors to support the establishment of acquired resistance. A higher ratio of EGFR to MIG6 expression was found in ALK TKI-treated and ALK TKI-resistant tumors and correlated with the poor responsiveness to ALK/ROS1 inhibition in patient-derived cell lines. Furthermore, we identified and validated a MIG6 EGFR-binding domain truncation mutation in mediating resistance to ROS1 inhibitors but sensitivity to EGFR inhibitors. A MIG6 deletion was also found in a patient after progressing to ROS1 inhibition. Collectively, this study identifies MIG6 as a novel regulator for EGFR-mediated adaptive and acquired resistance to ALK/ROS1 inhibitors and suggests EGFR to MIG6 ratios and MIG6-damaging alterations as biomarkers to predict responsiveness to ALK/ROS1 and EGFR inhibitors.

ALK, ROS1, RET and NTRK1-3 Gene Fusions in Colorectal and Non-Colorectal Microsatellite-Unstable Cancers.

This study aimed to conduct a comprehensive analysis of actionable gene rearrangements in tumors with microsatellite instability (MSI). The detection of translocations involved tests for 5'/3'-end expression imbalance, variant-specific PCR and RNA-based next generation sequencing (NGS). Gene fusions were detected in 58/471 (12.3%) colorectal carcinomas (CRCs), 4/69 (5.8%) gastric cancers (GCs) and 3/65 (4.6%) endometrial cancers (ECs) (ALK: 8; RET: 12; NTRK1: 24; NTRK2: 2; NTRK3: 19), while none of these alterations were observed in five cervical carcinomas (CCs), four pancreatic cancers (PanCs), three cholangiocarcinomas (ChCs) and two ovarian cancers (OCs). The highest frequency of gene rearrangements was seen in KRAS/NRAS/BRAF wild-type colorectal carcinomas (53/204 (26%)). Surprisingly, as many as 5/267 (1.9%) KRAS/NRAS/BRAF-mutated CRCs also carried tyrosine kinase fusions. Droplet digital PCR (ddPCR) analysis of the fraction of KRAS/NRAS/BRAF mutated gene copies in kinase-rearranged tumors indicated that there was simultaneous co-occurrence of two activating events in cancer cells, but not genetic mosaicism. CRC patients aged above 50 years had a strikingly higher frequency of translocations as compared to younger subjects (56/365 (15.3%) vs. 2/106 (1.9%), p = 0.002), and this difference was particularly pronounced for tumors with normal KRAS/NRAS/BRAF status (52/150 (34.7%) vs. 1/54 (1.9%), p = 0.001). There were no instances of MSI in 56 non-colorectal tumors carrying ALK, ROS1, RET or NTRK1 rearrangements. An analysis of tyrosine kinase gene translocations is particularly feasible in KRAS/NRAS/BRAF wild-type microsatellite-unstable CRCs, although other categories of tumors with MSI also demonstrate moderate occurrence of these events.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: ALK-positive anaplastic large cell lymphomas (ALCL) are characterized by rearrangements involving the ALK gene on chromosome 2. The translocation t(2;5) (p23;q35) resulting in the NPM1::ALK gene fusion is detected in the majority of ALCL. In 10%–20% of ALCL, ALK is fused to various other partner genes. Compared to NPM1::ALK, variant ALK-fusion proteins are not expressed in the nucleus. The frequency, distribution, and possible prognostic significance of the different variant ALK partner genes have not been analyzed in population-based studies so far. We investigated all patients with ALK-positive ALCL with exclusive cytoplasmic ALK-expression diagnosed in a uniformly treated cohort of ALCL patients in the NHL-BFM study group. Patients and Methods: Between 2000 and 2017, 312 children and adolescents were diagnosed with an ALK-positive ALCL in Austria, Germany, Switzerland and the Czech Republic, and included in the ALCL 99 trial or the NHL-BFM registry 2012. Reference pathology demonstrated exclusive cytoplasmic ALK-staining in 49 tumors. Formalin-fixed-paraffin-embedded or frozen tumors of 43/49 patients were available for molecular analyses. Tumor-DNA was analyzed by an ALK-specific genomic capture high throughput sequencing assay. ALK-partner gene-specific RT-PCR assays were used to identify the respective partner genes when tumor cDNA was available. Results: 41 of 43 available tumors with exclusive cytoplasmic ALK-staining could be analyzed. 13 were NPM1::ALK positive and 28 tumors had a variant ALK partner. TPM3::ALK (n = 9, 32%) and ATIC::ALK (n = 8, 29%) were the most common variant partner genes. XPOI could be identified as a novel ALK fusion partner. The patients´ characteristics of ALCL expressing Variant::ALK were comparable to those with NPM1::ALK fusions. The five-year event-free survival (EFS) and overall survival were comparable between the 284 NPM1::ALK and the group of 28 different Variant::ALK-expressing patients. However, none of the 9 patients with TPM3::ALK positive ALCL relapsed, compared to 5 relapses among 8 patients with ATIC::ALK positive ALCL. Conclusions: In our population-based cohort of ALK-positive ALCL-patients, less than 10% carried variant ALK-fusion partners, 60% of which were TPM3 or ATIC. In patients with exclusive cytoplasmic ALK staining patterns, NPM1::ALK positivity should be excluded by molecular analysis. The tendency of a different relapse risk between ATIC::ALK and TPM3::ALK -positive ALCL-patients suggests that the fusion partner might confer tumor aggressiveness or drug resistance. The research was funded by: KNACK DEN KREBS Fördergemeinschaft Kinderkrebs-Zentrum Hamburg e.V. and HAMBURGER STIFTUNG ZUR FÖRDERUNG DER KREBSBEKÄMPFUNG Keyword: diagnostic and prognostic biomarkers No conflicts of interests pertinent to the abstract.

Correlation between ALK, ROS1 Biomarkers and EGFR Oncogene Mutations in Lung Tumours: Our Observations in an Apex Oncopathology Laboratory

Introduction: Analysis of Anaplastic lymphoma receptor tyrosine kinase gene (ALK), Repressor of Silencing 1 (ROS1) gene are determined by immunohistochemistry (IHC) and it is an easily applicable, cost-effective assay for potential treatment with crizotinib. Mutations of Epidermal growth factor receptor (EGFR) genes are evaluated by IHC/Multiplex RT-PCR. The purpose of this study is to assess the frequencies of ALK, ROS1 and their association with EGFR fusion gene mutations in a spectrum of lung tumours. Materials and methods: A total of 202 cases of lung tumours reported at our Center for Oncopathology from September 1st 2020 to 31st August 2021, were retrospectively analyzed for ALK, ROS1 and EGFR fusion genes based on Immunohistochemistry (IHC) and Multiplex PCR findings. ALK was tested using D5F3 clone, and ROS1 was analyzed using Cell Signalling’s D4D6 clone on the Ventana immunohistochemistry platform. EGFR status was analyzed using EGFR mutation test V2 real-time multiplex PCR assay on Roche Cobas Z480. Results: 202 biopsy samples and cellblocks of fluid aspirates were analyzed. 175/202 were histologically and immunologically proved as Non-small cell lung carcinoma (Primary pulmonary adenocarcinoma) and its metastases. 09/199 (5.23%) were Positive for ALK IHC and 03/199 (1.74%) cases had equivocal results. 06/179 (3.85%) cases were Positive for ROS1 IHC and 03/179 (1.92%) cases had equivocal results. Other histo-morphological diagnoses i.e., adenosquamous, squamous, small cell, mucinous carcinoma etc (27 cases) were all ALK and ROS1 Negative. 188/202 tumours were analyzed for EGFR mutation status, which showed 70/188 (37.23%) had specific EGFR mutations. 118/188 (62.76%) cases were EGFR wildtype. Conclusion: We observed that age related incidence of EGFR mutations was highest in elderly females, of 61 to 70 years. ALK gene mutations occurred in 6.03% and ROS1 gene mutations occurred in 5.02% of lung tumours and their metastases. EGFR-mutations were associated with ROS1 mutated lung adenocarcinomas. There are no coexistent ALK-EGFR or ALK-ROS1 mutations. All ALK IHC positive pulmonary adenocarcinomas are ROS1 negative and are mutually exclusive.

Detection of multiple druggable mutations of lung cancer from cytology specimens by MINtS: An advanced medicine A trial.

Most multigene mutation tests require tissue specimens. However, cytological specimens are easily obtained in the clinical practice and provide high-quality DNA and RNA. We aimed to establish a test that utilizes cytological specimens and performed a multi-institutional study to investigate the performance of MINtS, a test based on next-generation sequencing. A standard procedure for specimen isolation was defined. The specimens were considered suitable for the test if >100 ng DNA and >50 ng RNA could be extracted from them. In total, 500 specimens from 19 institutions were investigated. MINtS detected druggable mutations in 63% (136 of 222) of adenocarcinomas. Discordant results between MINtS and the companion diagnostics were observed in 14 of 310 specimens for the EGFR gene, and 6 of 339 specimens for the ALK fusion genes. Confirmation by other companion diagnostics for the EGFR mutations or the clinical response to an ALK inhibitor all supported the results obtained by MINtS. MINtS along with the isolation procedure presented in the current study will be a platform to establish multigene mutation tests that utilize cytological specimens. UMIN000040415.

ERBB family fusions are recurrent and actionable oncogenic targets across cancer types.

Gene fusions involving receptor tyrosine kinases (RTKs) define an important class of genomic alterations with many successful targeted therapies now approved for ALK, ROS1, RET and NTRK gene fusions. Fusions involving the ERBB family of RTKs have been sporadically reported, but their frequency has not yet been comprehensively analyzed and functional characterization is lacking on many types of ERBB fusions. We analyzed tumor samples submitted to Caris Life Sciences (n=64,354), as well as the TCGA (n=10,967), MSK IMPACT (n=10,945) and AACR GENIE (n=96,324) databases for evidence of EGFR, ERBB2 and ERBB4 gene fusions. We also expressed several novel fusions in cancer cell lines and analyzed their response to EGFR and HER2 tyrosine kinase inhibitors (TKIs). In total, we identified 1,251 ERBB family fusions, representing an incidence of approximately 0.7% across all cancer types. EGFR, ERBB2, and ERBB4 fusions were most frequently found in glioblastoma, breast cancer and ovarian cancer, respectively. We modeled two novel types of EGFR and ERBB2 fusions, one with a tethered kinase domain and the other with a tethered adapter protein. Specifically, we expressed EGFR-ERBB4, EGFR-SHC1, ERBB2-GRB7 and ERBB2-SHC1, in cancer cell lines and demonstrated that they are oncogenic, regulate downstream signaling and are sensitive to small molecule inhibition with EGFR and HER2 TKIs. We found that ERBB fusions are recurrent mutations that occur across multiple cancer types. We also establish that adapter-tethered and kinase-tethered fusions are oncogenic and can be inhibited with EGFR or HER2 inhibitors. We further propose a nomenclature system to categorize these fusions into several functional classes.

Application of the WHO Classification of Thoracic Tumors (2021) grading system in invasive pulmonary adenocarcinoma and its correlation with the targeted genes' variations

Objective: To investigate the applicability of the 2021 WHO classification of thoracic tumors' new grading system for invasive pulmonary adenocarcinoma (IPA) with different clinical stages and its correlation with the characteristics of targeted genes' variation. Methods: A total of 2 467 patients with surgically resected primary IPA in Shanghai Pulmonary Hospital, Shanghai, China from September to December 2020 were retrospectively analyzed. Eligible cases were graded using the new grading system of IPA of the 2021 WHO classification of thoracic tumors. The clinicopathological data and targeted-gene abnormality were collected. The utility of new grading system of IPA in different clinical stages was investigated. The correlation of clinicopathological features and targeted-gene abnormality in different grades of IPA were compared. Results: All 2 311 cases of IPA were included. There were 2 046 cases of stage Ⅰ IPA (88.5%), 169 cases of stage Ⅱ (7.3%), and 96 cases of stage Ⅲ (4.2%). According to the new classification system of IPA, 186 cases (9.1%), 1 413 cases (69.1%) and 447 cases (21.8%) of stage-Ⅰ adenocarcinoma were classified as Grade 1, Grade 2 and Grade 3, respectively. However, there were no Grade 1 adenocarcinomas in stages Ⅱ and Ⅲ cases. Among stage-Ⅱ and Ⅲ IPA cases, there were 38 Grade 2 cases (22.5%) and 131 Grade 3 cases (77.5%), and 3 Grade 2 cases (3.1%) and 93 Grade 3 cases (96.9%), respectively. In stage-Ⅰ cases, no tumor cells spreading through airspace (STAS), vascular invasion or pleural invasion was found in Grade 1 of IPA, while the positive rates of STAS in Grade 2 and 3 IPA cases were 11.3% (159/1 413) and 73.2% (327/447), respectively. There was a significant difference among the three grades (P<0.01). Similarly, the rates of vascular and pleural invasion in Grade 3 IPA cases were 21.3% (95/447) and 75.8% (339/447), respectively, which were significantly higher than those of 1.3% (19/1 413) and 3.0% (42/1 413) in Grade 2 (P<0.01). EGFR mutational rates in Grades 1, 2 and 3 IPA were 65.7% (94/143), 76.4% (984/1 288) and 51.3% (216/421), respectively. The differences among the three grades were statistically significant (P<0.01). No fusion genes were detected in Grade 1 IPA, while the positive rates of ROS1 and ALK fusion genes in Grade 3 were 2.4% (10/421) and 8.3% (35/421), respectively, which were significantly higher than that of 0.5% (7/1 288) and 1.6% (20/1 288) in Grade 2 (P<0.01). In stage-Ⅱ cases, only EGFR mutation rate in Grade 2 adenocarcinoma (31/37, 83.8%) was higher than that in Grade 3 adenocarcinoma (71/123, 57.7%; P<0.01). However, the correlation between the new grade system of IPA and the distribution characteristics of targeted-gene variation cannot be evaluated in stage Ⅲ cases. Conclusions: The new grading system for IPA is mainly applicable to clinical stage-Ⅰ patients. Tumor grades of IPA are strongly correlated with the high-risk factors of prognosis and the distribution features of therapeutic targets. It is of great significance and clinical value to manage postoperative patients with early-stage IPA.

Clinicopathological and genetic analyses of pulmonary enteric adenocarcinoma

AimsPulmonary enteric adenocarcinoma (PEAC) is a rare variant of pulmonary adenocarcinoma. Due to its rarity, few pathological and molecular studies have been performed on PEAC. We herein conducted clinicopathological, immunohistochemical...