Anaplastic Lymphoma Kinase Fusion Research Articles

Exploring ALK fusion in colorectal cancer: a case series and comprehensive analysis

Anaplastic lymphoma kinase (ALK) fusion-positive colorectal cancer (CRC) is a rare and chemotherapy-refractory subtype that lacks established and effective treatment strategies. Additionally, the efficacy and safety of ALK inhibitors (ALKi) in CRC remain undetermined. Herein, we examined a series of ALK-positive CRC patients who underwent various lines of ALKi treatment. Notably, we detected an ALK 1196M resistance mutation in a CRC patient who received multiple lines of chemotherapy and ALKi treatment. Importantly, we found that Brigatinib and Lorlatinib demonstrated some efficacy in managing this patient, although the observed effectiveness was not as pronounced as in non-small cell lung cancer cases. Furthermore, based on our preliminary analyses, we surmise that ALK-positive CRC patients are likely to exhibit inner resistance to Cetuximab. Taken together, our findings have important implications for the treatment of ALK-positive CRC patients.

EMA-Positive Superficial ALK–Rearranged Myxoid Spindle Cell Neoplasm Masquerading as Perineurioma/Hybrid Nerve Sheath Tumor

Abstract: Superficial anaplastic lymphoma kinase (ALK)–rearranged myxoid spindle cell neoplasm (SAMS) is a recently described entity which coexpresses ALK, CD34, and commonly S100. These neoplasms are characterized morphologically by concentric spindle cell whorls and cords and are commonly set in an abundant myxoid to myxocollagenous stroma, thus mimicking perineurioma or hybrid nerve sheath tumor. EMA immunostain has been reported to be negative in SAMS which helps in excluding the latter entities. Herein, we report the first EMA-positive SAMS of the right leg in a 37-year-old female patient masquerading as perineurioma/hybrid nerve sheath tumor. The tumor morphologically was comprised of spindle cells arranged in loose whorls and short fascicles set in myxoid to collagenous stroma and coexpressed CD34 and EMA, reminiscent of perineurioma. S100 showed focal staining. ALK immunostain was subsequently performed and was positive. ALK gene rearrangement was identified by fluorescence in situ hybridization break-apart assay and was further confirmed by next-generation sequencing–based RNA sequencing demonstrating FLNA::ALK fusion, thus supporting the diagnosis of SAMS. In conclusion, EMA can be expressed in SAMS, thus posing as a diagnostic pitfall. ALK immunostain and molecular studies are essential for confirming the diagnosis of SAMS and excluding potential mimickers, particularly perineurioma or hybrid nerve sheath tumor.

Inflammatory Myofibroblastic Tumor of the Esophagus and Stomach Successfully Treated With ALK Inhibitor in a Pediatric Patient: A Case Report and Concise Review of Literature.

An inflammatory myofibroblastic tumor (IMT) is a rare mesenchymal neoplasm of borderline malignant potential. Nearly half of all IMTs have rearrangement of anaplastic lymphoma kinase (ALK) locus on chromosome 2p23 which can be treated with targeted therapy. Herein, we describe an unusual presentation of IMT involving an anatomical region rarely implicated in this disease process. A 15-year-old male patient came to the ER with dysphagia and coffee ground emesis. On esophagogastroscopy, a nodular luminal obstructing 30 × 50 mm mass in the lower esophagus was found, which was continuous with a large, partially circumferential gastric mass extending from the mid-body to the proximal antrum. Biopsies from esophageal and gastric masses revealed submucosal lesions composed of cytologically bland spindle and epithelioid cells, intermingled with inflammatory infiltrate, for which several immunohistochemical (IHC) stains were performed. The molecular study demonstrated ATIC::ALK fusion. Based on morphological, IHC, and molecular study findings, the diagnosis of ALK-positive IMT was rendered. Because surgical excision was deemed infeasible, the patient was started on ALK-inhibiting therapy with crizotinib. The patient responded well with no evidence of residual or recurrent disease on follow-up imaging or surveillance esophagogastroduodenoscopy. Crizotinib was ultimately discontinued after 10 months of therapy, and the patient continues to undergo surveillance imaging for monitoring of disease burden.

Clinicopathologic Features and Cytologic Correlation of ALK-Rearranged Papillary Thyroid Carcinoma: A Series of Eight Cases.

Anaplastic lymphoma kinase (ALK) gene fusions are rare in papillary thyroid carcinoma (PTC) but may serve as a therapeutic target. This study aims to evaluate the preoperative cytologic findings and clinicopathologic features of a series of eight ALK-rearranged PTCs from our pathology archives and consultations. All cases were confirmed by ALK D5F3 immunohistochemistry and six with additional targeted RNA-based next-generation sequencing (NGS). The original fine-needle aspiration (FNA) cytology diagnosis included the Bethesda System (TBS) category II in three (37.5%), TBS III in two (25%), TBS V in two (25%), and TBS VI in one (12.5%). Six cases had available FNA cytology and were reviewed. The cytologic features showed microfollicular architecture as well aslimited or reducednuclear elongation and chromatin alterations in all six. Nuclear grooves and pseudoinclusions were absent in two cases, rarely or focally noted in three, and frequently found in one. Two cases initially diagnosed asTBS II, showing microfollicular architecture without well-developed nuclear features, were revised to TBS III (with architectural atypia only). For histologic correlations, four were infiltrative follicular variant PTCs, three as classic subtype PTC with predominant follicular growth, and one as solid/trabecular subtype PTC. All eight cases demonstrated reducedPTC nuclear features with respect to nuclear elongation and chromatin alterations compared to thosetypically identified in "BRAF-like" PTCs. The NGS testing revealed EML4::ALK fusion in three, STRN::ALK fusion in two, and ITSN2::ALK fusion in one. In conclusion, although ALK-rearranged PTCs have been associated with neutral gene expression profile from a BRAF-RAS scoring perspective, the "RAS-like" nuclear features were more commonly identified in this series, resulting in frequent indeterminate diagnosis of preoperative FNA.

Development of crizotinib-associated renal cyst in a non-small cell lung cancer patient with ALK fusion: a case report and review of the literature

BackgroundCrizotinib, an oral first-generation tyrosine kinase inhibitor (TKI), is superior to systemic chemotherapy for the treatment of non-small cell lung cancer (NSCLC) with positive rearrangement of anaplastic lymphoma kinase (ALK). However, an increased incidence of renal and hepatic cysts has been reported in the patients on crizotinib treatment.Case presentationHere, we describe a case of a 71-year-old Chinese women developed multiple cystic lesions in kidney and liver during crizotinib treatment for the primary and metastatic NSCLC. The renal and hepatic cysts were noted by CT scan 3 months after crizotinib treatment, which were spontaneously and significantly regressed after stopping crizotinib.ConclusionsBased on literature review and our experience in this case report, we concluded that crizotinib-associated renal cyst (CARCs) has features of malignancy and abscess in radiographic imaging, and thus, pathological confirmation is necessary to avoid inappropriate treatment decision. In addition, to benefit the patients with progress-free survival (PFS), switching from crizotinib to alectinib is recommended for the treatment of NSCLC patients who developed CARCs.

[Chinese expert consensus on iruplinalkib for the treatment of locally advanced or metastatic ALK-positive non-small cell lung cancer (2024 edition)].

Anaplastic lymphoma kinase (ALK) fusion gene is one of the most common driver gene in non-small cell lung cancer (NSCLC). Epidemiological data showed that ALK gene fusion is detected in 9.06% of Chinese advanced NSCLC patients. ALK-tyrosine kinase inhibitors (TKIs) have become the standard treatment for advanced NSCLC patients with ALK gene fusion. Seven different ALK-TKIs have been approved by the National Medical Products Administration (NMPA) of China, including crizotinib, ceritinib, alectinib, ensartinib, brigatinib, lorlatinib, and iruplinalkib. Iruplinalkib is a novel new-generation ALK-TKI independently developed in China. On June 27, 2023, the NMPA approved iruplinalkib for the treatment of locally advanced or metastatic ALK-positive NSCLC patients whose disease has progressed after previous treatment with crizotinib or who are intolerant to crizotinib. On January 16, 2024, the NMPA approved iruplinalkib for the first-line treatment of locally advanced or metastatic ALK-positive NSCLC patients. In order to better understand the efficacy and safety of iruplinalkib, and facilitate more rationally clinical application of iruplinalkib, the Medical Oncology Branch of China International Exchange and Promotive Association for Medical and Health Care and the Chinese Association for Clinical Oncologists co-organized experts to compile the "Chinese expert consensus on iruplinalkib for the treatment of locally advanced or metastatic ALK-positive non-small cell lung cancer (2024 edition)".

Abstract 987: Novel ALK mutations in EML4::ALK+ NSCLC resistant to TKIs identified by liquid biopsy

Abstract The identification of Anaplastic Lymphoma Kinase (ALK) chromosomal translocations led to the development of effective targeted therapies in a subset of Non-Small Cell Lung Cancer (NSCLC) patients. Several drugs are currently in clinical practice for advanced ALK+ NSCLC. However, resistance remains a challenge in these patients. The possibility to analyze circulating tumor DNA (ctDNA) with non-invasive methods can greatly improve prognostic capabilities, by allowing early detection of relapse and of drug-resistant mutations from peripheral blood. We report a series of 15 consecutive relapsed NSCLC patients (12 ALK+, 3 ROS1+) treated at the Fondazione IRCCS-San Gerardo dei Tintori (Monza, Italy) that were investigated by liquid biopsy at tyrosine kinase inhibitor (TKI) failure, through amplicon deep sequencing of the ALK/ROS1 kinase domains. Median DNA yield was 4.8 ng per ml of plasma and mean fragment size was 176 bp. Mutational hotspot coding regions of ALK and ROS1 genes were amplified by high-fidelity PCR and sequenced. Mean coverage of called variants was 24,687x. The threshold for mutation calling was set at 0.6%, after correcting for mapping quality, minimum coverage, and strand bias. Analysis of background signal on healthy control DNA identified 0.0024 mismatches per mapped base. Overall, plasma ctDNA analysis detected an ALK mutation in 7/18 relapse samples from 7/15 patients. No ROS1 mutations were found in ROS1 fusion-positive patients. Among 12 patients carrying an EML4::ALK fusion, 10 variants potentially driving resistance were found. Along with mutations that were previously identified (L1196M/G1202R, E1154K, F1174L), novel ALK variants were detected such as L1198R, T1211I, C1235R, C1237Y, L1196P. Mutations were characterized for sensitivity to ALK inhibitors in a BaF3-EML4::ALK cell model, using cell proliferation assays and western blotting to evaluate the effects on ALK phosphorylation. C1237Y and L1196P mutants appeared to resist all TKIs; hence, we further tested their sensitivity to novel investigational drugs, zotizalkib and repotrectinib. Both mutants demonstrated resistance, suggesting that these two mutations confer broad resistance to TKIs. Molecular modelling of C1237Y and L1196P mutants was run to elucidate the mechanisms of resistance. The ADP-C1237Y complex showed a much smaller root-mean-square deviation than ADP-WT, indicating a higher affinity/activity of the mutant. L1196 interacts hydrophobically with a methyl group of lorlatinib, P1196 is predicted to lose such interaction, while ATP binding is not affected. The presence of C1237Y and L1196P variants, is a relevant finding due to their resistance, even after the exposure to the fourth-generation inhibitor zotizalkib. The clonal selection of these mutations, made by second/third generation ALK TKIs may lead to a complete refractory status, which would then render TKIs useless in a sequential therapeutic strategy. Citation Format: Federica Malighetti, Matteo Villa, Elisa Sala, Geeta Sharma, Giulia Arosio, Maria Gemelli, Chiara Manfroni, Diletta Fontana, Nicoletta Cordani, Raffaella Meneveri, Alfonso Zambon, Rocco Piazza, Fabio Pagni, Diego Cortinovis, Luca Mologni. Novel ALK mutations in EML4::ALK+ NSCLC resistant to TKIs identified by liquid biopsy [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 987.

Abstract 2024: Deep and durable responses to ALK-targeted therapy require CD8+ T cells

Abstract Despite major advances in therapies for lung cancer, it remains the leading cause of cancer related deaths in the United States. Tyrosine kinase inhibitors (TKI) designed to inhibit oncogenic anaplastic lymphoma kinase (ALK) fusions have transformed the treatment of patients with ALK+ NSCLC and many survive for several years on TKIs. While most patients experience a decrease in tumor burden on initial alectinib therapy, for many, tumor shrinkage is incomplete and persistent, residual disease remains. Critically, the degree of tumor shrinkage and amount of residual disease that persists despite TKI associates with overall survival. Multiple cancer-cell intrinsic mechanisms of persistence despite TKI therapy have been reported, but they do not fully account for the heterogeneity of this initial tumor response, suggesting other determinants are present beyond inhibiting the primary oncogene. However, extrinsic mediators of persistent disease, such as immune cells within the tumor microenvironment (TME), have not been rigorously studied in ALK+ NSCLC as they are considered immunologically “cold” tumors. Using EA-1 and EA-2, two distinct murine ALK+ adenocarcinoma cells lines derived from immunocompetent C57BL/6J mice, we have demonstrated that ALK+ NSCLC response to alectinib requires the adaptive immune system. C57BL/6J mice bearing orthotopic EA-1 tumors experience tumor shrinkage but have persistent, residual disease on CT imaging; those bearing EA-2 tumors have no residual disease. In this model system, when alectinib treatment stops, no EA-2 tumors recur but all animals bearing EA-1 tumors experience tumor outgrowth. TME interrogations by multispectral tissue imaging demonstrated more CD8+ T cells in the EA-2 TME compared to EA-1 and no difference in CD4+ T cells. Flow cytometry revealed a significant increase in activated CD8+ T cells and CD86+ CD103+ dendritic cells within the TME of EA-2 tumors compared to EA-1. When implanted into nude mice, EA-1 and EA-2 both experience initial disease shrinkage on alectinib, but EA-2 tumors now exhibit residual disease on imaging. Notably for both cell lines, tumor outgrowth occurs in the nude mice despite continued TKI therapy. Using knock out animal models, we established that CD8+ T cells, but not CD4+ T cells, were required for response to alectinib. In animals lacking CD8+ T cells, EA-2 tumors shrink with alectinib therapy but are not eliminated as in the wild type controls. Notably, despite continued alectinib therapy, tumors grew out in the CD8+ knock out animals. In the EA-2 model, CD4+ T cells we not required for response to alectinib, and CD4+ knock out animals did not experience tumor outgrowth when alectinib was held. Taken together, these data suggest that the adaptive immune system, specifically CD8+ T cells, are required for a deep and durable response to molecularly targeted therapy for ALK+ NSCLC. Citation Format: Greg Reis, Stevie Phelabaum, Andre Navarro, Emily Kleczko, Lynn Heasley, Raphael Nemenoff, Erin L. Schenk. Deep and durable responses to ALK-targeted therapy require CD8+ T cells [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 2024.

Identification of gene variation feature for targeted therapy of non-small cell lung cancer through combined method of DNA and RNA sequencing

Next generation sequencing (NGS) is typically used to reveal tumor gene variation feature for targeted therapy of various types of human cancers, including non-small cell lung cancer (NSCLC). Here, we report the role and potential applicable value of combining DNA and RNA sequencing in gene variation detection in NSCLC. 386 NSCLC patients with stage II-IV were enrolled and detected using NGS sequencing of DNA and RNA panels that covered all well-documented target driver genes from the Chinese Society of Clinical Oncology (CSCO). The rate of epidermal growth factor receptor (EGFR) single nucleotide variation (SNV)/indel, mesenchymal-epithelial transition factor (MET) copy number variation (CNV) and anaplastic lymphoma kinase (ALK) fusion were 52.1%, 4.1% and 6.0% in the NSCLC cohort. The landscapes of SNV/indel, CNV and gene fusion in the cohort were depicted as well. Meanwhile, we assessed detection efficacy of DNA and RNA sequencing in gene fusion. Detected number and types of gene fusion using the RNA sequencing were better than those using the DNA sequencing. Gene fusion with intergenic region was only detected by DNA sequencing and MET exon 14 skipping (METΔex14) was more easily identified by RNA sequencing. Finally, we investigated clinical correlations of SNV/indel/CNV/fusion with clinicopathologic features in the NSCLC cohort. Taken together, RNA sequencing significantly complements deficiency of DNA sequencing for gene fusion, which cooperatively presents comprehensive and reliable gene variation features and facilitate the identification of potential drug targets for NSCLC patients.

2021 WHO Classification of Lung Cancer: Molecular Biology Research and Radiologic-Pathologic Correlation.

The 2021 World Health Organization (WHO) classification system for thoracic tumors (including lung cancer) contains several updates to the 2015 edition. Revisions for lung cancer include a new grading system for invasive nonmucinous adenocarcinoma that better reflects prognosis, reorganization of squamous cell carcinomas and neuroendocrine neoplasms, and description of some new entities. Moreover, remarkable advancements in our knowledge of genetic mutations and targeted therapies have led to a much greater emphasis on genetic testing than that in 2015. In 2015, guidelines recommended evaluation of only two driver mutations, ie, epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) fusions, in patients with nonsquamous non-small cell lung cancer. The 2021 guidelines recommend testing for numerous additional gene mutations for which targeted therapies are now available including ROS1, RET, NTRK1-3, KRAS, BRAF, and MET. The correlation of imaging features and genetic mutations is being studied. Testing for the immune biomarker programmed death ligand 1 is now recommended before starting first-line therapy in patients with metastatic non-small cell lung cancer. Because 70% of lung cancers are unresectable at patient presentation, diagnosis of lung cancer is usually based on small diagnostic samples (ie, biopsy specimens) rather than surgical resection specimens. The 2021 version emphasizes differences in the histopathologic interpretation of small diagnostic samples and resection specimens. Radiologists play a key role not only in evaluation of tumor and metastatic disease but also in identification of optimal biopsy targets. ©RSNA, 2024 Test Your Knowledge questions in the supplemental material and the slide presentation from the RSNA Annual Meeting are available for this article.

Concomitant double-fusion of PLEKHA7-ALK and INPP5D-ALK reveals favorable alectinib sensitivity in lung adenocarcinoma: a case report and literature review

Anaplastic lymphoma kinase (ALK) gene fusion is a classic driver mutation in non-small cell lung cancer (NSCLC); however, ALK double-fusion variants in NSCLC have rarely been reported. In this study, we reported a case with extremely uncommon ALK double-fusion variants. A 32-year-old female diagnosed with lung adenocarcinoma, who had developed multiple intrapulmonary and brain metastases, experienced worsening of her condition despite undergoing prior chemotherapy. Subsequent testing using next-generation sequencing (NGS) detected the presence of PLEKHA7-ALK and INPP5D-ALK double-fusion. The prescription of alectinib revealed potent efficacy and resulted in an increase in the survival rate. This case presented two uncommon and concomitant ALK fusion partners in NSCLC; more importantly, the INPP5D-ALK subtype has not been reported, therefore this study broadens the spectrum of ALK double-fusion variants and provides insight into the use of ALK inhibitors for the treatment of NSCLC in patients with double ALK fusions.

[China expert recommendations on anaplastic lymphoma kinase-tyrosine kinase inhibitors treatment for advanced non-small cell lung cancer (2024 edition)].

Anaplastic lymphoma kinase (ALK) fusion represents one of pivotal driver genes within the realm of non-small cell lung cancer (NSCLC). ALK-tyrosine kinase inhibitors (ALK-TKI) have demonstrated remarkable therapeutic efficacy for patients afflicted with ALK-positive NSCLC. As of June 27, 2023, seven ALK-TKI, including crizotinib, ceritinib, alectinib, ensartinib, brigatinib, lorlatinib, and iruplinalkib, have garnered approval from the China National Medical Products Administration (NMPA)(ranking according to the approval time for marketing by NMPA), providing individualized treatment modalities for ALK-positive NSCLC patients. To standardize the application of ALK-TKI, the Chinese Association for Clinical Oncologists and the Medical Oncology Branch of China International Exchange and Promotive Association for Medical and Health Care has organized experts to compile the " China expert recommendations on anaplastic lymphoma kinase-tyrosine kinase inhibitors treatment for advanced non-small cell lung cancer (2024 edition)". This treatment expert recommendation provides recommendations in four aspects, encompassing ALK fusion testing, ALK-TKI targeted therapy, ALK-TKI adverse events management, and patient post-treatment follow-up, thus serving as a valuable reference for the standardized treatment of Chinese advanced ALK fusion-positive NSCLC.

Expansion of targeted degradation by Gilteritinib-Warheaded PROTACs to ALK fusion proteins

Proteolysis targeting chimeras (PROTACs) induce the ubiquitination and subsequent proteasomal degradation of targeted proteins. Numerous PROTACs have emerged as promising drug candidates for various disease-related proteins. This study investigates PROTACs targeted to degrade anaplastic lymphoma kinase (ALK) fusion proteins, which are implicated in diseases such as anaplastic large cell lymphoma and non-small cell lung cancer. We recently reported the development of a gilteritinib-warheaded PROTAC to target and degrade the Fms-like tyrosine kinase 3 (FLT3) protein. Gilteritinib is a tyrosine kinase inhibitor that targets FLT3, and recent studies have revealed that it also functions as an ALK inhibitor. We conducted a structure–activity relationship (SAR) study and expanded the range of target proteins for gilteritinib-warheaded PROTACs to include echinoderm microtubule-associated protein-like 4 (EML4)-ALK and nucleophosmin (NPM)-ALK, in addition to FLT3. Our SAR study utilized three types of ligands for E3 ligase— inhibitor of apoptosis protein (IAP), cereblon (CRBN), and von Hippel-Lindau (VHL)— in the PROTAC designs and we observed varied efficacy in the degradation of target proteins. The CRBN-based PROTAC effectively reduced the protein expression of FLT3, EML4-ALK, and NPM-ALK. The IAP-based PROTAC reduced expression of both FLT3 and EML4-ALK proteins but not that of NPM-ALK, while the VHL-based PROTAC was ineffective against all target proteins. Several ALK-targeted PROTACs have already been developed using CRBN or VHL as E3 ligase, but this is the first report of an IAP-based ALK degrader. The length of the linker structure utilized in PROTAC also had a significant effect on their efficacy and activity. PROTACs formed with shorter linkers demonstrated an enhanced degradation activity to target proteins compared with those formed with longer linkers. These findings provide valuable insight for the development of effective PROTACs to target and degrade ALK fusion proteins.

Environment-Sensitive Fluorescent Probe Enables Assessment of Anaplastic Lymphoma Kinase Activity in Nonsmall Cell Lung Cancer.

Anaplastic lymphoma kinase (ALK) rearrangements have been identified as key oncogenic drivers of a subset of nonsmall cell lung cancer (NSCLC). The final chimeric protein of the fusion gene can be constitutively activated, which accounts for the growth and proliferation of ALK-rearranged tumors and thus strongly associates with cancer invasion and metastasis. Diagnostic tools enabling the visualization of ALK activity in a structure-function-based approach are highly desirable to determine ALK status and guide ALK tyrosine kinase inhibitor (ALK-TKI) treatment making. Here, we describe the design, synthesis, and application of a new environment-sensitive fluorescent probe HX16 by introducing an environment-sensitive fluorophore 4-sulfonamidebenzoxadiazole to visualize ALK activity in living cancer cells and tumor tissue slices (mouse model and human biopsy sample). HX16 is a multifunctional chemical tool based on the pharmacophore of ALK-TKI (ceritinib) and can specifically target the kinase domain of ALK with a high sensitivity. Using flow cytometry and confocal microscopy, HX16 enables visualization of ALK activity in various cancer cells with distinct ALK fusion genes, as well as xenograft mouse models. Importantly, HX16 was also applied to visualize ALK activity in a tumor biopsy from a NSCLC patient with ALK-echinoderm microtubule-associated protein-like-4 fusion gene for prediction of ALK-TKI sensitivity. These results demonstrate that strategically designed ALK-TKI-based probe allows the assessment of ALK activity in tumor tissues and hold promise as a useful diagnostic tool in predicting ALK-TKI therapy response.

Overcoming Central β-Sheet #6 (Cβ6) ALK Mutation (L1256F), TP53 Mutations and Short Forms of EML4-ALK v3/b and v5a/b Splice Variants are the Unmet Need That a Re-Imagined 5th-Generation (5G) ALK TKI Must Deliver.

Despite the development and approval of seven anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) spanning over three "generations" since the discovery of ALK fusion positive (ALK+) non-small cell lung cancer (NSCLC), there remains intrinsic and acquired resistances to these approved TKIs. Currently, a fourth-generation (4G) ALK TKI, NVL-655, is being developed to attack some of the unmet needs such as compound resistance mutations in cis. However, EML4-ALK variant 3 and TP53 mutations are intrinsic genomic alterations that negatively modulate efficacy of ALK TKIs. Potentially, in the shifting landscape where lorlatinib should be the first-line ALK TKI of choice based on the CROWN trial, the central β-sheet #6 (Cβ6) mutation ALK L1256F will be the potential acquired resistance mutation to lorlatinib which may be resistant to current ALK TKIs. Here we opine on what additional capacities a putative fifth-generation (5G) ALK TKI will need to possess if it can be achieved in one single molecule. We propose randomized trial schemas targeting some of the intrinsic resistance mechanisms that will lead to approval of a prototypic fifth-generation (5G) ALK TKI and actually be beneficial to ALK+ NSCLC patients rather than just design a positive pivotal superiority trial for the sole purpose of drug approval.

Lung adenocarcinoma with brain metastasis detected dual fusion of LOC399815-ALK and ALK-EML4 in combined treatment of Alectinib and CyberKnife: A case report.

The anaplastic lymphoma kinase (ALK) gene fusion occurs in approximately 3% to 7% of nonsmall cell lung cancer (NSCLC), in which occurs approximately 23% to 31% of brain metastasis patients in poor prognosis. ALK tyrosine kinase inhibitors have shown efficacy in treating ALK-positive (ALK+) NSCLC. More than 90 distinct subtypes of ALK fusions have been identified through sequencing technique and would lead to significant differences in clinical efficacy, it is necessary to guide clinical treatment effectively by gene detection. A 56-year-old nonsmoking female admitted to hospital due to cough, expectoration, and chest pain. Chest computed tomography revealed a space-occupying lesion in the upper left lobe (5.0 cm × 2.4 cm × 2.9 cm), multiple enlarged lymph nodes in mediastinum 3A and 5 (largest size 1.5 cm × 1.4 cm), and evidence of thoracic vertebral metastasis, brain magnetic resonance imaging also showed brain metastasis. Lung adenocarcinoma with brain metastasis. The patient initially received conventional first-line chemotherapy, which led to a deteriorated condition. Blood-base liquid biopsy by next-generation sequencing resulted in double ALK fusions, in which with a neo-partner of lncRNA (LOC399815-ALK). Following subsequent treatment with Alectinib and stereotactic radiotherapy (CyberKnife) was subsequently employed to manage the brain metastatic lesions, resulting in a substantial decreased in both the number and size of tumor lesions. The patient's response to therapy efficacy resulted in a substantial decreased in both the number and size of tumor lesions that assessed comprehensively evaluated through computed tomography imaging and ctDNA sequencing. Patient's condition has been under control for over 29 months. Liquid biopsy may reveal the rare fusion forms of ALK, precisely guiding personalized treatment, and providing a reference method for longitudinal monitoring and efficacy evaluation of ALK-tyrosine kinase inhibitors in NSCLC patients.

ALK F1174S mutation impairs ALK kinase activity in EML4-ALK variant 1 and sensitizes EML4-ALK variant 3 to crizotinib.

To assess the influence of F1174S mutation on kinase activity and drug sensitivity of the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) fusion (EML4-ALK) variants 1 and 3. We constructed mammalian expression plasmids of both wildtype and F1174 mutant EML4-ALK variants 1 and 3, and then characterized them with cell models by performing immunoblotting, neurite outgrowth assay, focus formation assay as well as protein stability assay. Drug sensitivity to ALK tyrosine kinase inhibitors was also compared between wildtype and F1174 mutant EML4-ALK fusions. In addition, we characterized the effect of different F1174 kinase domain mutations in the context of EML4-ALK fusions. In contrast to the oncogenic ALK-F1174S mutation that has been reported to be activating in the context of full-length ALK in neuroblastoma, EML4-ALK (F1174S) variant 1 exhibits impaired kinase activity leading to loss of oncogenicity. Furthermore, unlike the previously reported F1174C/L/V mutations, mutation of F1174 to S sensitizes EML4-ALK variants 3a and 3b to crizotinib. These findings highlight the complexity of drug selection when treating patients harboring resistance mutations and suggest that the F1174S mutation in EML4-ALK variant 1 is likely not a potent oncogenic driver. Additional oncogenic driver or other resistance mechanisms should be considered in the case of EML4-ALK variant 1 with F1174S mutation.

Successful Crizotinib-targeted Therapy of Pediatric Unresectable ERC1::ALK Fusion Sarcoma.

Anaplastic lymphoma kinase ( ALK )-fusion sarcomas are rare part of the emerging theoretically targetable tyrosine kinase RAS::MAPK pathway fusion myopericytic-ovoid sarcomas. We report our clinicopathologic and treatment experience with an ALK fusion sarcoma. A novel ELKS/RAB6-interacting/CAST family member 1 - unaligned ALK fusion infiltrative nonmetastatic low-grade sarcoma of the right hand of a 15-month-old male was treated with crizotinib, an ALK tyrosine kinase inhibitor as oral monotherapy, inducing complete radiographic and clinical resolution by 10 months and sustained response now over 12 months after elective discontinuation. Crizotinib can successfully be used to treat unresectable novel ALK fusion sarcomas.

Abstract B033: Patterns of testing and survival for EGFR mutation and ALK abnormalities in advanced lung cancer at John Peter Smith Hospital

Abstract Background: There is paucity of literature regarding outcomes of patients treated for non-small cell lung cancer (NSCLC) at safety-net hospitals (Muslim et al., 2022). John Peter Smith (JPS) hospital is a safety-net hospital serving the needs of underserved population in north Texas. We evaluated testing patterns, prevalence of Epidermal Growth Factor Receptor (EGFR) mutations and Anaplastic Lymphoma Kinase (ALK) fusions in advanced NSCLC at JPS, and outcome measures of 1-, 3- and 5-year overall survival (OS) as compared to patients without these alterations. Methods: We used data from the JPS Oncology Registry and abstracted information from medical records. Eligible patients were diagnosed with stages IIIB-IV NSCLC and treated at JPS from 1/1/2017 to 12/31/2021. Patients were grouped into EGFR+, ALK+, both EGFR- and ALK-, test not ordered/completed (NT). A Kaplan Meier curve was used to assess the difference in post diagnosis survival time across testing status. The log rank test was then performed to assess if the survival distributions were significantly different.  To further assess the impact of EGFR and ALK results on patient death three cox proportional hazard models were generated assessing 1-, 3- and 5-year OS. To establish a full model all items collected underwent univariate pre-filtering.  All available items were analyzed via a univariate model and items with a p-value of 0.2 or lower were analyzed in our multivariate model using SAS 9.4. Results: 220 patients were included for analysis, 183 (83%) were stage IV. 57% were males. Racial/ethnic distribution included 45% Non-Hispanic Whites (NHW), 32% Blacks, 13% Hispanic Whites (HW) and 10% Asians. 71% were tested for EGFR, ALK, or both.  21 patients (13.7%) were found to have EGFR mutations and 7 patients (4.9%) had ALK fusions. For EGFR, tissue (T) was tested in 134 and blood (B) in 44, both T and B in 23 with concordance in 20. For ALK, T was done in 129, B in 44, 23 had both, with concordance in 21. Testing was inconclusive in 3 (T) for EGFR and 5 (3 T and 2 B) for ALK. Lung was the most common site tested for EGFR with 90 patients followed by liver (8), lymph node (8), and bone (8). Females accounted for 67% of EGFR+ and 57% for ALK+. Asians accounted for 43% of EGFR+ and 28% of ALK+. Median OS in days for study population: 155, EGFR+: 845, ALK+: 1333, both-:162, NT: 105. At 3 years, EGFR+ (HR: 0.427; 95% CI: 0.222, 0.822; p-value: 0.0109) and ALK+ (HR: 0.191; 95% CI: 0.046, 0.795; p-value: 0.0229) showed a significant reduction in the hazard of death compared to both-. NT patients had 1.679 times higher hazard of death when compared to both- (HR: 1.679; 95% CI: 1.206, 2.338; p-value: 0.0021). Similar pattern was seen for EGFR+ for 1 and 5 year OS and for ALK+ at 5 year OS. Conclusions: Prevalence of EGFR and ALK alterations at JPS is similar to nationally reported statistics. Tissue testing is the gold standard for identifying these alterations. 29% of the patients were not tested. Median survival of EGFR+ or ALK+ patients is significantly longer than other cohorts. Citation Format: Vivek Athipatla, James Blackwell, Anna Diaz, Bassam Ghabach, Kalyani Narra. Patterns of testing and survival for EGFR mutation and ALK abnormalities in advanced lung cancer at John Peter Smith Hospital [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B033.

Clinical characteristics and progression of pre-/minimally invasive lung adenocarcinoma harboring ALK or RET rearrangements: a retrospective cohort study.

Patients harboring anaplastic lymphoma kinase (ALK) or rearranged during transfection (RET) rearrangements are usually diagnosed at a relatively late stage with nodal and distant metastasis, and rapid progression course of ALK/RET fusion-positive lung cancer were well-known. However, clinical characteristics and course of pre-/minimally invasive lung adenocarcinoma harboring ALK or RET fusions are poorly described. Identifying patients with gene fusions at early stage may offer surgical options that could cure those patients. We retrospectively included patients with surgically resected pre-/minimally invasive lung adenocarcinomas harboring epidermal growth factor receptor (EGFR) mutations or ALK/RET rearrangements, and further compared the patient clinical characteristics, nodule natural course, and survival outcomes. Radiological characteristics including ground-glass component, cystic airspace, pleural attachment, etc. were specially assessed for this study. EGFR (exons 18-22) was detected by Sanger sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the ALK/RET rearrangements. Lung cancer-specific survival (LCSS), relapse-free survival (RFS), and overall survival (OS) were all evaluated. Of 238 patients with pre-/minimally invasive lung adenocarcinomas, 226 patients had EGFR mutations, 7 patients had ALK fusions, and 5 patients had RET fusions. Average age at surgery was 45.3 years for ALK/RET-positive group and 52.6 years for EGFR-positive group (P=0.049). Radiologically, among the 12 patients with ALK/RET fusions, the majority of lesions (10/12) manifested as mixed ground-glass opacities (mGGOs), which was significantly more prevalent when compared with patients with EGFR mutations (83.4% vs. 24.3%, P<0.001). Moreover, a substantial proportion of cystic airspace was found in ALK/RET-positive group but not in EGFR-positive group (66.7% vs. 14.2%, P<0.001). Among four patients with ALK/RET fusions undergoing surveillance over 1 year before surgery, two of them developed rapid radiologic progression. The 5-year LCSS and RFS were 100%, 100% for ALK/RET-positive group, and 100%, 100% for EGFR-positive group, respectively. ALK/RET-positive pre-/minimally invasive lung adenocarcinomas were mostly characterized as mGGOs with cystic airspace developing rapid nodule progression, and no recurrence occurred during long-term follow-up after resection. This provides insights into proper curative surgery timing in the management of patients with gene fusions. However, these findings must be treated with caution and validated in future multi-center studies with larger sample size.