Anaplastic Lymphoma Kinase Fusion Partner Research Articles

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.

Alk-rearranged lung adenocarcinoma: From molecular genetics to therapeutic targeting.

Anaplastic Lymphoma Kinase (ALK) is a potent oncogenic driver of lung adenocarcinoma (LUAD). ALK is constitutively activated by gene fusion events between the ALK and other gene fusion partners in about 2-3% of LUADs, characterized by few other gene alterations. ALK-fusions are a druggable target through potent pharmacological inhibitors of tyrosine kinase activity. Thus, several ALK-TKIs (Tyrosine Kinase Inhibitors) of first-, second- and third-generation have been developed that improved the outcomes of ALK-rearranged LUADs when used as first- or second-line agents. However, resistance mechanisms greatly limit the durability of the therapeutic effects elicited by these TKIs. The molecular mechanisms responsible for these resistance mechanisms have been in part elucidated, but overcoming acquired resistance to ALK-derived therapy remains a great challenge. Some new therapeutic strategies under investigation aim to induce long-term remission in ALK-fusion positive LUADs.

Survival outcomes in ALK-positive NSCLC patients (p) treated 1st line brigatinib and impact of the ALK fusion variant.

e21015 Background: There is limited data on brigatinib (a next-generation anaplastic lymphoma kinase (ALK) inhibitor) efficacy according to the ALK fusion variant. Here we investigate the prevalence of ALK fusion variants and evaluate treatment outcomes in ALK-positive NSCLC treated with first-line brigatinib. Methods: This is an open-label, non-randomized, phase II, exploratory, multi-center clinical trial. Stage IV, ALK-positive NSCLC p were treated with brigatinib 90mg for the first 7 days (D 1-7 at cycle 1) and then 180 mg daily thereafter for QW4 cycles of duration (28 days ±3days). Brigatinib was administered until loss of clinical benefit, unacceptable toxicity, p or physician decision to discontinue, or death. Response to treatment was evaluated as per RECIST v1.1. ALK rearrangement was identified locally by immunohistochemistry and confirmed by fluorescence in situ hybridization. In addition, baseline tissue samples were analyzed by NGS using the Oncomine™ Focus Assay in a centralized laboratory. Results: Between May 2020 and October 2021, 33 p were enrolled from eight different institutions. One p was considered not valid because there no tissue or plasma sample was available at baseline. Therefore, 32 p were finally considered for the study. Baseline characteristics were as follows: median age (range): 53 (35-86) years; female, n = 15 (47%); ECOG PS of 0 n = 6 (19%), 1 n = 23 (72%), 2 n = 3 (9%); never smoker, n = 14 (44%). All tumors were adenocarcinoma. The average number of metastases per p was four. In total, seven (22%) p had brain metastases. A sample was valid for NGS analysis in 29 cases. ALK fusion partners were EML4 in 28 cases (97%) and HIP1 in one case (3%). The ALK fusion variant 1 was the most frequently detected (n = 13, 45%), followed by variant 3 (n = 9; 31%), and variant 2 (n = 4; 14%). The EML4(6)-ALK(19) variant was detected in 2 cases, and variant 8 was detected in one case. The median follow-up time was 18 months. During the study, 13 deaths were recorded, and progressive disease (PD) was observed in 15 p. With a data maturity of 87%, median progression-free survival (PFS) was 16.7 months (95%CI 6.2-NR), and median overall survival (OS) was NR (95%CI 12.4-NR). There were no significant differences in survival outcomes according to the type of variant. The same was true when grouping long (variants 1,2 and 8) and short (variant 3, EML4(6) - ALK(19)) variants. The p whose tumor harbored the HIP1(21)-ALK(20) variant was alive and with and in partial response at the time of data cutoff. On the other hand, a patient whose tumor harbored the short variant EML4(6) - ALK(19) along with the KRAS G12C mutation was diagnosed with disease progression after three months of treatment initiation and deceased after eight months from the start of treatment. Conclusions: In this interim analysis we were unable to demonstrate a significant difference in survival outcomes according to variant type. Clinical trial information: NCT04223596 .

Discovery of a miniaturized PROTAC with potent activity and high selectivity

The approved small-molecule inhibitors of anaplastic lymphoma kinase (ALK) have shown remarkable efficacy in some subset of cancer patients. However, the numerous ALK mutants or fusion partners are resistant to such drugs, greatly limiting their application in clinic. Despite the drug design strategy of proteolysis-targeting chimera (PROTAC) holds great potential to overcome drug resistance in theory, there are obvious disadvantages for the reported PROTACs that include high molecular weight, long linkers, difficult synthesis routes as well as insufficient evidence in activity for diverse ALK mutants. In this study, we designed and synthesized a miniaturized PROTAC of ALK named AP-1 following the principle of minimalist design. Two simple chemical units of ligands and a minimized linker with only two atoms were selected for synthesis of AP-1. At cellular level, AP-1 successfully degraded three types of ALK mutants including NPM-ALK, EML4-ALK and F1174L mutation ALK form with potent activity, high selectivity in ALK-positive cells. In xenograft mouse model, AP-1 showed the stronger antitumor efficacy than ceritinib as well as ALK degraders reported in literatures. AP-1 with an extremely simple PROTAC structure can be served as an effective candidate drug for therapy of various types of ALK-positive cancers. And the design principle of AP-1 has a good guiding significance for overcoming the disadvantages such as excessive molecular weight and poor solubility of PROTAC.

A Novel CASC15-ALK and TFG-ROS1 Fusion Observed in Uterine Inflammatory Myofibroblastic Tumor.

The majority of inflammatory myofibroblastic tumors (IMTs) in the gynecologic tract occur in the uterine corpus and harbor anaplastic lymphoma kinase ( ALK ) rearrangement. Herein, we report 1 uterine IMT case with a novel fusion involving ALK and 1 uterine IMT case with ROS1 rearrangement. The ages of the patients were 56 and 57yr, respectively. The tumor size was 10.0 and 8.0cm, respectively. Both patients had stage IB disease. Histologically, the 2 IMT cases had classic morphologic features and predominantly comprised bland spindle cells with hypercellular (fascicular/storiform) and hypocellular (myxoid rich) areas admixed with variably prominent lymphoplasmacytic infiltration. Immunohistochemically, the ALK -rearranged case was positive for ALK , and the ROS1 -rearranged case was positive for ROS1 . Both cases were diffusely positive for desmin. The tumor cells were variably positive for estrogen receptor (1/2 cases, 50.0%) and progesterone receptor (1/2 cases, 50.0%). Targeted RNA sequencing revealed one case each with either a novel CASC15-ALK or TFG-ROS 1 fusion. We identified a novel ALK fusion partner CASC15 in IMT and described the first uterine IMT with a TFG-ROS1 fusion. This study improves our understanding of molecular events in IMT.

ALK fusion typing and response to crizotinib in ALK+ lung cancer-naive patients.

e21008 Background: Anaplastic lymphoma kinase (ALK) is one of the major oncogenic driver genes in lung cancer, and fusion as the main mutation type, of which EML4 is the most common fusion ligand. With the development of next-generation sequencing (NGS), some other rare fusion ligands and complex fusions have been discovered. The prognosis of patients with EML4-ALK fusion, non EML4-ALK fusion and multiple ALK fusion was different. Methods: We retrospectively analyzed NGS (including tissue, blood, or other body fluid samples) in 61 patients with ALK[+] lung cancer, including 59 patients with NSCLC and 2 patients with SCLC. Grouping was determined according to whether it was EML4-ALK fusion and multiple fusion. Results: In our data, the partner fused to the ALK gene are diverse, including EML4, KIF5B, TSN and others. Among them, EML4 was the most common ligand in ALK[+] NSCLC, accounting for 93.2% (55/59). Among the different fusion sites of EML4-ALK, V1 (E13:A20) and V3 (E6:A20) mutations were the most common, accounting for 45.5% (25/55) and 41.8% (23/55), respectively. Several novel ALK fusion partners were discovered in this study, including MCFD2, LINC01251, MVP17, CREG2, LINCO1498 and so on. In addition, the incidence of intergenic sequence-ALK fusions was 16.4% (10/61), such as ANXA4-ALK(A[intergenic]:A20), CREG2-ALK (C[intergenic]:A20), HCN1-ALK(H[intergenic]:A20), TSN-ALK(T[intergenic]:A20), RSAD2-ALK(R [intergenic]:A20) and so on. 19.0% (12/61) of the patient had multiple fusion, of which only one patient had multiple fusion of EML4-ALK (V2+V5'+V3a/b), and the remaining patients had co-fusion of EML4-ALK and non EML4-ALK (Table). V3 were more likely to coexist with non EML4-ALK fusions than V1 or other variants (P = 0.047). Multiple fusions were more likely to occur with non EML4-ALK fusions (P < 0.000). Compared with EML4-ALK fusion, mPFS treated with crizotinib in non EML4-ALK fusion were significantly shorter (18.2 vs. 8.5 months, P = 0.035). In the context of crizotinib treatment, multiple fusions were associated with worse mPFS compared with single fusions (13.8 vs. 8.4 months, P = 0.008). In addition, For SCLC patients with ALK fusion (n = 2), the PFS of crizotinib treatment were 8.0 and 30.6 months, respectively. Conclusions: Due to the heterogeneity of tumor development resulting in the diversity of ALK fusions, non EML4-ALK fusions and multiple fusions imply a poorer response to crizotinib. ALK gene status prior to ALK-TKI therapy helps predict drug efficacy and patient prognosis.[Table: see text]

Novel HIVEP1-ALK fusion in a patient with lung adenocarcinoma demonstrating sensitivity to alectinib: a case report

BackgroundAnaplastic lymphoma kinase (ALK) fusion is an important oncogenic driver in non-small cell lung cancer (NSCLC). Reports on the intergenic region (IGR) as an ALK fusion partner are rare. Here, we report the case of a patient with advanced NSCLC harboring a human immunodeficiency virus type I enhancer binding protein 1 (HIVEP1)-ALK fusion that responded effectively to alectinib.Case DescriptionA 60-year-old non-smoking male was referred with a 3-month history of productive cough secondary to lung adenocarcinoma metastatic to mediastinal lymph nodes, brain, liver, and bone (T2N3M1c, stage IVB). Next-generation sequencing identified an IGR (upstream HIVEP1-) ALK fusion, and immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) results were consistent with an ALK-positive tumor. The patient was subsequently started on alectinib, with no obvious adverse reaction. After 1 month of therapy, the patient achieved significantly remission of the clinical symptoms and had led to an ongoing partial response (PR) lasting >33 months.ConclusionsOur experience highlights the efficacy of alectinib in a patient with HIVEP1-ALK fusion positive NSCLC with multiple metastases including brain disease, and the need for multiple genetic testing methods to verify the oncogenicity of ALK fusions prior to treatment. It could provide useful guidance for the treatment of similar cases in the future.

Development of a novel ALK rearrangement screening test for non-small cell lung cancers.

Approximately 5–7% of non–small cell lung cancer (NSCLC) cases harbor an anaplastic lymphoma kinase (ALK) fusion gene and may benefit from ALK inhibitor therapy. To detect ALK fusion genes, we developed a novel test using reverse transcription polymerase chain reaction (RT-PCR) for the ALK kinase domain (KD). Since ALK expression is mostly silenced in the adult with the exception of neuronal tissue, the normal lung tissue, mesothelial lining, and inflammatory cells are devoid of ALK transcript, making ALK KD RT-PCR an ideal surrogate test for ALK fusion transcripts in lung or pleural effusion. The test was designed with a short PCR product (197 bp) to work for both malignant pleural effusion (MPE) and formalin-fixed, paraffin-embedded (FFPE) NSCLC samples. Using ALK IHC as a reference, the sensitivity of the test was 100% for both MPE and FFPE. The specificity was 97.6% for MPE and 97.4% for FFPE. Two false positive cases were found. One was a metastatic brain lesion which should be avoided in the future due to intrinsic ALK expression in the neuronal tissue. The other one resulted from ALK gene amplification. Due to potential false positivity, subsequent confirmation tests such as fluorescence in situ hybridization or multiplex PCR would be preferable. Nevertheless, the test is simple and inexpensive with no false negativity, making it a desirable screening test. It also offers an advantage over multiplex RT-PCR with the capability to detect novel ALK fusions. Indeed through the screening test, we found a novel ALK fusion partner (sperm antigen with calponin homology and coiled-coil domains 1 like gene, SPECC1L) with increased sensitivity to crizotinib in vitro. In summary, a novel RNA-based ALK KD analysis was developed for ALK rearrangement screening in MPE and FFPE specimens of NSCLC. This simple inexpensive test can be implemented as routine diagnostics.

Alpha-T: An innovative decentralized (home-based) phase 2 trial of alectinib in ALK-positive (ALK+) solid tumors in a histology-agnostic setting.

TPS3155 Background: Anaplastic lymphoma kinase ( ALK) fusions are found in 3–8% of non-small cell lung cancers (NSCLC) and ̃0.2% of other tumor types. Alectinib is a selective, CNS-active ALK tyrosine kinase inhibitor approved as first-line treatment for adults with advanced ALK fusion-positive ( ALK-fp) NSCLC based on the phase 3 ALEX trial (NCT02075840; PMID 28586279). Preliminary evidence supports investigation of alectinib in a tumor-agnostic setting. ALK inhibitors have shown efficacy in ALK-fp tumor types other than NSCLC (PMIDs 29685646; 28977547; 30591488), with alectinib showing efficacy irrespective of ALK fusion partner and against ALK-activating mutations (PMIDs 29642598; 29559559). Methods: Alpha-T is a phase 2 open-label, single-arm trial (NCT04644315) with an innovative home-based remote design, currently enrolling adults with histologically confirmed, locally advanced/metastatic solid tumors (except lung cancer and cancers of unknown primary) harboring ALK fusions or selected mutations ( ALK-mut; R1275Q, F1245C, F1174X). The decentralized design permits enrollment regardless of location, allows most assessments to be home-based and maintains the relationship between patients (pts) and their treating oncologist. Key inclusion criteria: no alternative or unsuitable to receive standard therapy; ECOG PS 0–2; adequate hematologic, renal and hepatic functions; no prior ALK inhibitor; measurable solid tumor (RECIST 1.1) or primary brain tumor (RANO); asymptomatic or stable CNS metastases permitted. Pts are identified via the Foundation Medicine Inc. (FMI) Precision Enrollment service. For any solid tumor identified as ALK+ (fusion/selected mutation) by next-generation sequencing in tissue/blood (F1 CDx/F1L CDx, FMI), the ordering oncologist is given trial details and can liaise with a Science 37 investigator. Pts receive 600mg oral alectinib twice a day with food until radiological PD, unacceptable toxicity, withdrawal of consent or death. Home-based assessments (eg, physical examination, blood sampling, questionnaire completion) are conducted in-person by a mobile nurse at baseline and every 4 weeks, with remote support from the Science 37 investigator via their telemedicine platform. Tumor assessments at screening and every 8 weeks are performed at a local radiology facility. Primary endpoint: confirmed ORR by investigator in pts with ALK-fp tumors (RECIST 1.1). Secondary endpoints: ORR by independent review facility (IRF); DoR and PFS; intracranial ORR and DoR (IRF); OS and safety. Descriptive analyses are planned for pts with ALK-mut tumors and primary brain tumors. Pharmacokinetics and biomarkers will be evaluated. Patient-reported outcomes will be assessed via the EORTC QLQ-C30 and EuroQoL EQ-5D-5L questionnaires. Target enrollment is 50 pts with ALK-fp tumors evaluable by RECIST. As of 9 Feb 2021, 1 pt is in screening. Clinical trial information: NCT04644315.

Anaplastic lymphoma kinase (ALK) partners identified by next-generation sequencing in Chinese patients with solid tumors.

3555 Background: Anaplastic lymphoma kinase (ALK) rearrangement is a validated therapeutic driver gene in non-small cell lung cancer (NSCLC). More than 30 different fusion partner genes of ALK in NSCLC have been reportedand most of these ALK fusions respond well to ALK inhibitors crizotinib. With the development of next-generation sequencing (NGS), more novel partners for ALK rearrangement have been identified. Here, we aimed to report the landscape of ALK rearrangement in Chinese patients with solid tumors. Methods: Tissue or blood samples were subjected to NGS in a College of American Pathologists-certified and Clinical Laboratory Improvement Amendments-accredited lab for ALK arrangement. Results: In total, we profiled more than 40,000 patients, among which 72 cases with 52 ALK fusion partner, harboring 17 reported partners and 35 novel partners. The average ALK rearrangement patients' age was 53 years (range, 17-76 years). Among all the ALK fusion cases (n = 72), lung cancer were the largest proportion with 77.8% (n = 56), colorectal cancer accounted for, 5.5% (n = 4), liver cancer accounted for 4.2% (n = 3), biliary cancer, melanoma, carcinosarcoma and inflammatory myofibroblastic tumor accounted for 2.8% (n = 2) respectively, and only one case (n = 1) was malignant peritoneal mesothelioma. The most common ALK fusion partners were KIF5B (n = 6), DCTN1 (n = 5) and STRN (n = 5). In 38 cases, 35 novel ALK fusion partners were discovered. The novel CLIP4-ALK, EHBP1-ALK, PLB1-ALK occurred twice in 6 patients, which were two lung cancer patients with CLIP4-ALK fusion, two lung cancer patients with PLB1-ALK fusion, one hepatic cellular cancer patients with EHBP1-ALK, and one melanoma patients with EHBP1-ALK. There were two special lung cancer cases with two ALK fusions. One case detected the novel LRIG1-ALK fusion and novel PLB1-ALK fusion, the other case detected novel GLI3-ALK fusion and reported HIP1-ALK fusion. Conclusions: Novel ALK fusions are detected in patients with not only NSCLC but also other solid tumors. NGS fusion assay is an optional method for screening novel fusions.

Association of Programmed Death-Ligand 1 Expression with Fusion Variants and Clinical Outcomes in Patients with Anaplastic Lymphoma Kinase-Positive Lung Adenocarcinoma Receiving Crizotinib.

Programmed death-ligand 1 (PD-L1) expression is associated with clinical outcomes of epidermal growth factor receptor (EGFR) mutant lung adenocarcinoma (ADC) treated with tyrosine kinase inhibitors (TKIs). However, whether PD-L1 expression plays a role in anaplastic lymphoma kinase (ALK)-positive lung ADC is unknown. We aimed to evaluate the impact of PD-L1 in patients with ALK-positive lung ADC receiving crizotinib. PD-L1 expression was identified by immunohistochemistry (IHC). Reverse transcriptase-polymerase chain reaction was used for ALK variant detection, and immunofluorescence-based multiplex staining was applied for exploring immune cells in tumor microenvironments. A total of 78 patients with ALK-positive advanced ADC were enrolled in our study, of whom 52 received crizotinib. Compared with EGFR/ALK wild-type tumors, PD-L1 expression was lower in ALK-positive ADC. ALK fusion variants were identified in 32 patients, and those with variant 3 and 5 (short variants) had higher PD-L1 expression than those with other variants. The crizotinib objective response rate (ORR) and progression-free survival (PFS) was better in tumors with negative PD-L1 expression (ORR/PFS in PD-L1 0% vs. 1%-49% vs. 50%-100%: 60.7%/11.8 months vs. 38.5%/6.5 months vs. 36.4%/4.0 months, p = .007/.022). The multivariate Cox proportional hazards model revealed that PD-L1 0% (vs. ≥1%) was an independent factor for longer PFS (adjusted hazard ratio 0.322, 95% confidence interval 0.160-0.650, p = .002). Multiplex IHC in three cases showed a varied extent of immune cell infiltrations in tumors with different PD-L1 expression. Positive PD-L1 expression was associated with unfavorable clinical outcomes in patients with ALK-positive lung ADC receiving crizotinib. Not all lung adenocarcinoma with sensitizing driver mutations experienced durable responses to small-molecule tyrosine kinase inhibitors (TKIs). Similar to the negative impact of programmed death-ligand 1 (PD-L1) in epidermal growth factor receptor mutant tumors treated with TKIs, this study demonstrated that positive PD-L1 expression was also associated with worse response rate and shorter progression-free survival of anaplastic lymphoma kinase (ALK)-positive adenocarcinoma treated with crizotinib. Among different ALK fusion partners, tumors with short variants (V3 and V5) had higher PD-L1 compared with long variants (V1, V2, and V6). Testing PD-L1 before initiating crizotinib for ALK-positive lung cancer could be a simple method to provide important prognostic information.

The Genomic Characteristics of ALK Fusion Positive Tumors in Chinese NSCLC Patients.

Anaplastic lymphoma kinase (ALK) fusion events account for ~3–7% genetic alterations in patients with non-small cell lung cancer (NSCLC). In this study, we identified the ALK fusion patterns and a novel ALK fusion partner in 44 ALK positive NSCLC patients using a customized HapOncoCDx panel, and identified ALK fusion partners. The most common partner is EML4, forming the variant 1 (v1, E13:A20, 18/44), variant 2 (v2, E20:A20, 5/44), and variant 3 (v3, E6:A20, 13/44). Moreover, we detected a new ALK fusion partner HMBOX1. At the mutation level, TP53 is the most frequently mutated gene (24%), followed by ALK (12%) and STED2 (12%). The median tumor mutation burden (TMB) of these samples is 2.29 mutations/Mb, ranging from 0.76 mut/Mb to 16.79 muts/Mb. We further elaborately portrayed the TP53 mutation sites on the peptide sequence of the encoded protein by lollipop. The mutational signature and copy number alterations (CNAs) of the samples were also analyzed. The CNA events were found in 13 (13/44) patients, and the most commonly amplified genes were MDM2 (n = 4/13) and TERT (n = 4/13). Together, these results may guide personalized clinical management of patients with ALK fusion in the era of precision medicine.

Molecular and clinical analysis of Chinese patients with anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer.

Anaplastic lymphoma kinase (ALK) fusions have been recognized as a therapeutic target in non‐small cell lung cancer (NSCLC). However, molecular signatures and clinical characteristics of the Chinese population with ALK‐rearranged NSCLC are not well elucidated. In the present study, we carried out targeted next‐generation sequencing on tissue and plasma ctDNA samples in 1688 patients with NSCLC. Overall, ALK fusions were detected in 70 patients (4.1%), and the frequencies of ALK fusions detected in tissue and plasma samples were 5.1% and 3.3%, respectively. Additionally, the prevalence of breakpoint locations for EML4‐ALK fusions in ctDNA was significantly correlated with that in tumor tissues (R 2 = .91, P = .045). According to age, the incidence rates of ALK fusions among young (age <45 years), middle‐aged (between 45 and 70 years) and elderly (>70 years) patients were significantly different (P < .001). In 70 ALK‐rearranged cases, coexistence of epidermal growth factor receptor (EGFR) alterations and ALK fusions was detected in 12 cases (17.1%) and EGFR mutations tended to coexist with non‐EML4‐ALK rearrangements. Notably, novel ALK fusion partners, including TRIM66,SWAP70,WNK3,ERC1,TCF12 and FBN1 were identified in the present study. Among EML4‐ALK fusion variants, patients with variant V1 were younger than patients with variant V3 (P = .023), and TP53 mutations were more frequently concurrent with variant V3 compared with variant V1 (P = .009). In conclusion, these findings provide new insights into the molecular‐clinical profiles of patients with ALK‐rearranged NSCLC that may improve the treatment strategy of this population.

Implementation of functional precision medicine for anaplastic lymphoma kinase-rearranged non-small lung cancer

Anaplastic lymphoma kinase (ALK)-rearranged kinase drives non-small cell lung cancer (NSCLC) in 3–7% of patients and treatment consists of a broad range of approved inhibitors which are administered sequentially in case of resistance developing invariably after approximately 1 year of therapy. Besides the former standard tyrosine kinase inhibitor (TKI) crizotinib, the second-line ALK inhibitors alectinib, ceritinib, brigatinib, as well as the third-line lorlatinib are approved for the treatment of ALK-positive NSCLC patients. The main challenge is to find individual schemes of ALK inhibitors therapy which provide the best benefit for the patients. A host of ALK fusion partners, rearrangement variants and ALK mutations prevent a direct correlation of ALK alterations and sensitivity to specific TKIs within the framework of genomic precision medicine. However, recurrent ALK-positive NSCLC is an aggressive disease resulting in accumulation of tumor cells in pleural effusions which may be collected for in vitro sensitivity testing in a manner designated as functional precision medicine. Provided that these cells are present in sufficient numbers, they can be exposed to all possible drug candidates and their chemosensitivity tested in short-term proliferation assays. In addition to ALK-directed TKIs, cytotoxic drugs and inhibitors of non-target bypassing pathways may be included in the tests. In contrast to other trials in genome-guided precision medicine, a range of suitable therapeutics is available for modified ALK proteins. In summary, ALK-positive NSCLC with pleural effusions offer a unique model to develop, test and validate concepts of functional precision medicine.

Use of ALK Immunohistochemistry for Optimal Therapeutic Strategy of Pulmonary Large-cell Neuroendocrine Carcinoma and Identification of a Novel KIF5B-ALK Fusion Oncokinase.

Patients with adenocarcinoma of the lung are routinely screened for anaplastic lymphoma kinase (ALK) rearrangement because they can be treated by ALK-specific targeted therapy. The clinical and molecular characteristics of large-cell neuroendocrine carcinoma (LCNEC) associated with ALK rearrangement are still unclear. Herein, we assessed the ALK status in a series of patients with LCNEC by testing methods commonly used for adenocarcinoma. ALK expression was first examined by immunohistochemistry. For a positively stained tumor, molecular analyses were then conducted. The ALK fusion partner found in a patient with ALK rearrangement was further identified by direct DNA sequencing. Patient clinicopathological features were also analyzed, focusing on the ALK rearrangement-positive case. Immunohistochemistry of seven patients identified strong ALK expression in one case of stage IV LCNEC. Molecular analysis identified a novel rearranged gene resulting from the fusion of kinesin family member 5B (KIF5B) exon 17 to ALK exon 20. The patient was treated with ALK-specific inhibitors, crizotinib and later, alectinib, and has remained alive for more than 24 months without disease progression. Three of the remaining six patients without ALK rearrangement had stage IV cancer and received cytotoxic chemotherapies. Their average overall survival was 5.4 months. To our knowledge, this is the first report of a KIF5B-ALK fusion gene in LCNEC. The patient was successfully treated with ALK inhibitors, suggesting that sensitivity to ALK inhibitor may define a specific LCNEC subtype. We propose that screening for ALK rearrangement in patients with LCNEC may assist in selecting potential candidates for targeted therapy.

ALK Fusion Partners Impact Response to ALK Inhibition: Differential Effects on Sensitivity, Cellular Phenotypes, and Biochemical Properties.

Oncogenic tyrosine kinase fusions involving the anaplastic lymphoma kinase (ALK) are detected in numerous tumor types. Although more than 30 distinct 5' fusion partner genes have been reported, treatment of ALK-rearranged cancers is decided without regard to which 5' partner is present. There is little data addressing how the 5' partner affects the biology of the fusion or responsiveness to ALK tyrosine kinase inhibitors (TKI). On the basis of the hypothesis that the 5' partner influences the intrinsic properties of the fusion protein, cellular functions that impact oncogenic potential, and sensitivity to ALK TKIs, clonal 3T3 cell lines stably expressing seven different ALK fusion variants were generated. Biochemical and cellular assays were used to assess the efficacy of various ALK TKIs in clinical use, transformative phenotypes, and biochemical properties of each fusion. All seven ALK fusions induced focus formation and colonies in soft agar, albeit to varying degrees. IC50s were calculated for different ALK TKIs (crizotinib, ensartinib, alectinib, lorlatinib) and consistent differences (5-10 fold) in drug sensitivity were noted across the seven ALK fusions tested. Finally, biochemical analyses revealed negative correlations between kinase activity and protein stability. These results demonstrate that the 5' fusion partner plays an important biological role that affects sensitivity to ALK TKIs.Implications: This study shows that the 5' ALK fusion partner influences ALK TKI drug sensitivity. As many other kinase fusions are found in numerous cancers, often with overlapping fusion partners, these studies have ramifications for other kinase-driven malignancies. Mol Cancer Res; 16(11); 1724-36. ©2018 AACR.

Uncommon ALK fusion partners in advanced ALK-positive non-small-cell lung cancer.

8561Background: The variants affect the efficacy of crizotinib in echinoderm microtubule protein-like-4 (EML4)- anaplastic lymphoma kinase (ALK) fusion-positive non–small-cell lung cancers (NSCLCs)...

Non-Canonical Thinking for Targeting ALK-Fusion Onco-Proteins in Lung Cancer.

Anaplastic lymphoma kinase (ALK) gene rearrangements have been identified in lung cancer at 3–7% frequency, thus representing an important subset of genetic lesions that drive oncogenesis in this disease. Despite the availability of multiple FDA-approved small molecule inhibitors targeting ALK fusion proteins, drug resistance to ALK kinase inhibitors is a common problem in clinic. Thus, there is an unmet need to deepen the current understanding of genomic characteristics of ALK rearrangements and to develop novel therapeutic strategies that can overcome ALK inhibitor resistance. In this review, we present the genomic landscape of ALK fusions in the context of co-occurring mutations with other cancer-related genes, pointing to the central role of genetic epistasis (gene-gene interactions) in ALK-driven advanced-stage lung cancer. We discuss the possibility of targeting druggable domains within ALK fusion partners in addition to available strategies inhibiting the ALK kinase domain directly. Finally, we examine the potential of targeting ALK fusion-specific neoantigens in combination with other treatments, a strategy that could open a new avenue for the improved treatment of ALK positive lung cancer patients.

Molecular breakdown: a comprehensive view of anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer.

Most anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancers (NSCLCs) show good clinical response to ALK inhibitors. However, some ALK-rearranged NSCLC patients show various primary responses with unknown reasons. Previous studies focused on the clinical aspects of ALK fusions in small cohorts, or were conducted in vitro and/or in vivo to investigate the function of ALK. One of the suggested theories describes how echinoderm microtubule-associated protein-like 4 (EML4)-ALK variants play a role towards different sensitivities in ALK inhibitors. Until now, there has been no integrated comprehensive study that dissects ALK at the molecular level in a large scale. Here, we report the largest extensive molecular analysis of 158 ALK-rearranged NSCLCs and have investigated these findings in a cell line construct experiment. We discovered that NSCLCs with EML4-ALK short forms (variant 3/others) had more advanced stage and frequent metastases than cases with the long forms (variant 1/others) (p = 0.057, p < 0.05). In vitro experiments revealed that EML4-ALK short forms show lower sensitivity to ALK inhibitors than do long forms. Clinical analysis also showed a trend for the short forms showing worse PFS. Interestingly, we found that breakpoints of ALK are evenly distributed mainly in intron 19 and almost all of them undergo a non-homologous end-joining repair to generate ALK fusions. We also discovered four novel somatic ALK mutations in NSCLC (T1151R, R1192P, A1280V, and L1535Q) that confer primary resistance; all of them showed strong resistance to ALK inhibitors, as G1202R does. Through targeted deep sequencing, we discovered three novel ALK fusion partners (GCC2, LMO7, and PHACTR1), and different ALK fusion partners showed different intracellular localization. With our findings that the EML4-ALK variants, new ALK somatic mutations, and novel ALK-fusion partners may affect sensitivity to ALK inhibitors, we stress the importance of targeted therapy to take the ALK molecular profiling into consideration. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Anaplastic lymphoma kinase-positive anaplastic large cell lymphoma with the variant RNF213-, ATIC- and TPM3-ALK fusions is characterized by copy number gain of the rearranged ALK gene

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma is characterized by 2p23/ALK aberrations, including the classic t(2;5)(p23;q35)/NPM1-ALK rearrangement present in ~80% of cases and several variant t(2p23/ALK) occurring in the remaining cases. The ALK fusion partners play a key role in the constitutive activation of the chimeric protein and its subcellular localization. Using various molecular technologies, we have characterized ALK fusions in eight recently diagnosed anaplastic large cell lymphoma cases with cytoplasmic-only ALK expression. The identified partner genes included EEF1G (one case), RNF213/ALO17 (one case), ATIC (four cases) and TPM3 (two cases). Notably, all cases showed copy number gain of the rearranged ALK gene, which is never observed in NPM1-ALK-positive lymphomas. We hypothesized that this could be due to lower expression levels and/or lower oncogenic potential of the variant ALK fusions. Indeed, all partner genes, except EEF1G, showed lower expression in normal and malignant T cells, in comparison with NPM1. In addition, we investigated the transformation potential of endogenous Npm1-Alk and Atic-Alk fusions generated by clustered regularly interspaced short palindromic repeats/Cas9 genome editing in Ba/F3 cells. We found that Npm1-Alk has a stronger transformation potential than Atic-Alk, and we observed a subclonal gain of Atic-Alk after a longer culture period, which was not observed for Npm1-Alk. Taken together, our data illustrate that lymphomas driven by the variant ATIC-ALK fusion (and likely by RNF213-ALK and TPM3-ALK), but not the classic NPM1-ALK, require an increased dosage of the ALK hybrid gene to compensate for the relatively low and insufficient expression and signaling properties of the chimeric gene.