RET Rearrangements Research Articles

A functional sgRNA-CRISPR screening method for generating murine RET and NTRK1 rearranged oncogenes.

CRISPR/Cas9 gene editing represents a powerful tool for investigating fusion oncogenes in cancer biology. Successful experiments require that sgRNAs correctly associate with their target sequence and initiate double stranded breaks which are subsequently repaired by endogenous DNA repair systems yielding fusion chromosomes. Simple tests to ensure sgRNAs are functional are not generally available and often require single cell cloning to identify successful CRISPR-editing events. Here, we describe a novel method relying on acquisition of IL3-independence in Ba/F3 cells to identify sgRNA pairs that generate oncogenic gene rearrangements of the Ret and Ntrk1 tyrosine kinases. The rearrangements were confirmed with PCR, RT-PCR and sequencing and Ba/F3 cells harboring Ret or Ntrk1 rearrangements acquired sensitivity to RET and TRK inhibitors, respectively. Adenoviruses encoding Cas9 and sgRNA pairs inducing the Kif5b-Ret and Trim24-Ret rearrangements were intratracheally instilled into mice and yielded lung adenocarcinomas. A cell line (TR.1) established from a Trim24-Ret positive tumor exhibited high in vitro sensitivity to the RET inhibitors LOXO-292 and BLU-667 and orthotopic TR.1 cell-derived tumors underwent marked shrinkage upon LOXO-292 treatment. Thus, the method offers an efficient means to validate sgRNAs that successfully target their intended loci for the generation of novel, syngeneic murine oncogene-driven tumor models.

Improved Survival Outcomes in Patients With MET-Dysregulated Advanced NSCLC Treated With MET Inhibitors: Results of a Multinational Retrospective Chart Review

BackgroundWe evaluated the disease and patient characteristics, treatment, and MET testing patterns, predictive biomarkers and survival outcomes in patients with MET-dysregulated metastatic non–small-cell lung cancer (NSCLC) in a real-world setting. Patients and MethodsThis was a multinational, retrospective, noninterventional chart review study. Data from medical records of patients with advanced/metastatic EGFR wild-type, MET-dysregulated NSCLC (December 2017-September 2018) were abstracted into electronic data collection forms. ResultsOverall, 211 patient charts were included in this analysis; 157 patients had MET exon 14 skipping mutations (METex14; with or without concomitant MET amplification) and 54 had MET amplification only. All patients were tested for METex14, whereas MET amplification was evaluated in 168 patients. No overlap was reported between MET dysregulation and ALK, ROS1 or RET rearrangements, or HER2 exon 20 insertions. Overall, 56 of 211 patients (26.5%) received MET inhibitor (METi) therapy in any treatment-line setting (31.2% in the METex14 cohort; 13% in the MET-amplified only cohort). In the METex14 cohort, median OS in patients receiving METi was 25.4 months versus 10.7 months in patients who did not (HR [95% CI]: 0.532 [0.340-0.832]; P = .0055). In the MET-amplified only cohort, median OS was 20.6 months in patients treated with METi compared with 7.6 months in those without METi (HR [95% CI]: 0.388 [0.152-0.991]; P = .0479). ConclusionsMET alterations in NSCLC typically occur in the absence of other oncogenic driver mutations and are associated with poor survival outcomes. Notably, METi therapies are associated with improved survival outcomes in patients with MET-dysregulated NSCLC.

Mutational profiling of Chinese patients with thyroid cancer.

The incidence of thyroid cancer in China has rapidly increased in recent decades. As the genetic profiles of thyroid cancer vary dramatically between different geographical regions, a comprehensive genetic landscape of thyroid cancer in the Chinese population is urgently needed. We retrospectively included thyroid cancer patients from three Chinese medical centers between February 2015 and August 2020. To dissect the genomic profiling of these patients, we performed targeted next-generation sequencing on their tumor tissues using a 1,021-gene panel. A total of 458 Chinese patients with thyroid cancer were enrolled, including four malignant histological subtypes arising from follicular epithelial thyroid cells. BRAF driver mutations were identified in 76.0% of patients, followed by RET rearrangements (7.6%) and RAS driver mutations (4.1%). Tumors with more somatic mutations correlated with worse clinical characteristics, including older age at diagnosis, less differentiation of tumor, larger tumor size, lymph node metastasis and distal metastasis. Subclonal BRAF mutations occurred in 20% (6/30) of patients and were frequent in poorly differentiated or anaplastic tumors (33.3% [2/6] vs. 4.2% [1/24], P = 0.09) and those with distal metastasis (50.0% [2/4] vs. 8.7% [2/23], P = 0.09). Tumors with TERT promoter mutations had significantly more somatic mutations (average: 6.5 vs. 1.8, P < 0.001). Moreover, TERT promoter mutations were not associated with lymph node metastasis but significantly associated with older age at diagnosis and poorly differentiated or anaplastic tumors, regardless of their clonal architecture. Our results shed light on the molecular pathogenesis and clinical characteristics of thyroid cancer in the Chinese population. The number of somatic mutations, TERT promoter mutations, and the clonal architecture of BRAF mutations should be considered in the risk stratification of thyroid cancer.

Rapid and Cost-Efficient Detection of RET Rearrangements in a Large Consecutive Series of Lung Carcinomas.

RET-kinase-activating gene rearrangements occur in approximately 1-2% of non-small-cell lung carcinomas (NSCLCs). Their reliable detection requires next-generation sequencing (NGS), while conventional methods, such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) or variant-specific PCR, have significant limitations. We developed an assay that compares the level of RNA transcripts corresponding to 5'- and 3'-end portions of the RET gene; this test relies on the fact that RET translocations result in the upregulation of the kinase domain of the gene and, therefore, the 5'/3'-end expression imbalance. The present study included 16,106 consecutive NSCLC patients, 14,449 (89.7%) of whom passed cDNA quality control. The 5'/3'-end unbalanced RET expression was observed in 184 (1.3%) tumors, 169 of which had a sufficient amount of material for the identification of translocation variants. Variant-specific PCR revealed RET rearrangements in 155/169 (91.7%) tumors. RNA quality was sufficient for RNA-based NGS in 10 cases, 8 of which carried exceptionally rare or novel (HOOK1::RET and ZC3H7A::RET) RET translocations. We also applied variant-specific PCR for eight common RET rearrangements in 4680 tumors, which emerged negative upon the 5'/3'-end unbalanced expression test; 33 (0.7%) of these NSCLCs showed RET fusion. While the combination of the analysis of 5'/3'-end RET expression imbalance and variant-specific PCR allowed identification of RET translocations in approximately 2% of consecutive NSCLCs, this estimate approached 120/2361 (5.1%) in EGFR/KRAS/ALK/ROS1/BRAF/MET-negative carcinomas. RET-rearranged tumors obtained from females, but not males, had a decreased level of expression of thymidylate synthase (p < 0.00001), which is a known predictive marker of the efficacy of pemetrexed. The results of our study provide a viable alternative for RET testing in facilities that do not have access to NGS due to cost or technical limitations.

Amplification of wild-type RET and clinical response to selpercatinib for non–small-cell lung cancer (NSCLC).

9123 Background: RET rearrangements and RET kinase domain mutations represent targetable genomic alterations in various cancer types. However, the frequency and characteristics of RET amplification in cancer and its potential role as a targetable oncogenic driver are not well-characterized. Methods: In a pan-cancer cohort of patients at the Dana-Farber Cancer Institute (DFCI) whose tumors underwent next-generation sequencing (NGS), we evaluated the frequency of wild-type (wt) RET amplification (defined as ≥6 copies) in the absence of RET rearrangements or activating mutations. We validated our findings using merged data from the TCGA, GENIE, and China Pan-Cancer datasets. Results: The frequency of wt RET amplification across all cancers was 0.08% (28/34,463) in the DFCI cohort and 0.16% (145/91,466) in the validation cohort. In NSCLC, the frequency of RET amplification was 0.10% (5/4773) in the DFCI cohort and 0.13% (15/11,622) in the validation cohort. Other cancer types with RET amplification included hepatobiliary cancer, prostate cancer, and breast cancer, among others. For 22 RET-amplified cases with available copy number data , the median RET gene copy number was 7 (range 6-36). Among 20 RET-amplified NSCLCs, 10 had no other known driver mutations, and 10 had concurrent driver mutations (including MET exon 14 skipping, MET amplification, KRAS G12C/S/D, EGFR L858R, EGFR exon 20 insertion, and EGFR exon 19 deletion). In cases with available smoking status, 5 out of 6 patients with RET-amplified NSCLC had a history of tobacco use. A 69-year-old man with unresectable stage III NSCLC had disease recurrence after chemoradiation and durvalumab. Both the initial and recurrent biopsy samples showed focal high-level RET amplification (22 and 28 copies, respectively); genomic and RNA sequencing showed no other driver mutations or RET rearrangements. He experienced partial response to the selective RET inhibitor selpercatinib with decrease of both intracranial and systemic tumor burden. Conclusions: Amplification of wt RET represents a novel, targetable, rare molecular subset of NSCLC and other cancers.

Utility of ctDNA tumor fraction to inform negative liquid biopsy (LBx) results and need for tissue reflex in advanced non-small cell lung cancer (aNSCLC).

9076 Background: Negative LBx results can be challenging to interpret. Absence of a targetable alteration may accurately reflect the tumor genotype (true negative) or may represent a false negative due to insufficient ctDNA shed, concealing a targetable oncogenic driver. Reflex to tissue biopsy (TBx) profiling is thus advised, per FDA label, for negative LBx results. Here we investigate whether algorithmic quantification of ctDNA in a LBx sample can increase confidence in negative LBx results, reducing need for confirmatory TBx testing. Methods: This study used the nationwide (US-based) de-identified Flatiron Health-Foundation Medicine NSCLC clinico-genomic database (CGDB) from approximately 280 US cancer clinics (~800 sites of care), limited to patients (pts) with aNSCLC receiving any multi-gene LBx followed by TBx CGP. In parallel, we queried an institutional database of pts with both LBx (FoundationOneLiquid CDx, F1LCDx) and TBx (FoundationOneCDx, F1CDx) to calculate positive predictive agreement (PPA) and negative predictive value (NPV) of LBx compared to TBx for detection of 7 targetable NSCLC driver alterations: KRAS G12X, EGFR, MET exon 14, BRAF V600E mutations or ALK, ROS1, and RET rearrangements. Foundation Medicine’s ctDNA tumor fraction (TF) on F1LCDx is a composite algorithm prioritizing aneuploidy at higher levels to avoid germline signal and prioritizing variant allele frequency of canonical alterations at lower levels to maximize dynamic range. Results: Of 1,734 LBx sent prior to 1st-line therapy in pts with aNSCLC, 59% (1,024) were negative for an NCCN biomarker. For 465 pts with TBx after negative LBx (median 2.4 weeks from LBx to TBx result), 179 (38%) had a driver detected on reflex to TBx. In 45 receiving matched targeted therapy, median real-world PFS was 25 months. For pts starting therapy after reflex to TBx (n = 233), median time from LBx result to start of therapy was 4.7 weeks. To understand whether reflex to tissue may be avoided in some cases, LBx (F1LCDx) sensitivity was studied across 2,138 pairs in the institutional database. PPA for the 7 driver alterations was 59% overall (679/1,157) and improved to 96% (433/453) when limited to cases with TF ≥1%. Similarly, NPV was modest overall (67%) but improved when limited to LBx specimens with TF ≥1% (96%). Studying TF in the CGDB cohort, 33 of 85 pts (39%) with reflex TBx after negative LBx had a LBx TF ≥1% and, given high NPV for driver alterations, might have avoided reflex to confirmatory TBx. Conclusions: Elevated TF (≥1%) on F1LCDx informs assay sensitivity and identifies a subset of LBx results with high NPV where reflex to TBx, and the associated treatment delay, may be less valuable. Conversely, for pts with a negative LBx with low TF ( &lt; 1%) on F1LCDx, reflex to TBx is an important follow-on step to identify potentially missed driver alterations with potential for durable benefit on targeted therapy.

Biomarkers to predict the efficacy of immune checkpoint inhibitors in patients with non-small-cell lung cancer with actionable genetic alterations.

e21175 Background: Although the efficacy of immune checkpoint inhibitors (ICI) in patients with actionable genetic alterations (AGAs) in non-small-cell lung cancer (NSCLC) has been known to be modest, some patients demonstrated improved survival. Thus, this study aimed to find predictive biomarkers for ICI in patients with NSCLC with various AGAs and to identify the predictive biomarkers for ICI efficacy. Methods: We compared the progression-free survival (PFS) of 324 advanced NSCLC patients who received ICI monotherapy (as over second-line therapy) by different AGAs from January 2018 and July 2022 at the Samsung Medical Center. To identify predictive markers of ICI, we adjusted the impacts on PFS of ICI in smoking status, PD-L1 expression, TP53, KEAP, STK11 mutation status, and steroid use during ICI monotherapy. Results: The median age was 63.1 years (range, 27.8–84.8) and 53.1% of patients was male, and 46.3% of patients was an ex-/or current smoker. A total of 324 patients were included with the following AGAs: EGFR mutation (n = 149, 46.0%), ALK rearrangement, (n = 12, 3.7%), KRAS mutation (n = 72, 22.2%), HER2 mutation or amplification (n = 34, 10.5%), and MET ex14 skipping or amplification (n = 32, 9.9%), ROS1 rearrangement (n = 9, 2.8%), BRAF V600E (n = 9, 2.8%), and RET rearrangement (n = 7, 2.2%). The median PFS was 2.0 months (95% confidence interval, 1.8–2.2) in the total AGA group. The 6-month PFS rate was 25.8% (19.5–34.3) in the group with KRAS/ BRAF V600E/ MET/ HER2 and 12.8% (8.6–19.1) in the group with EGFR/ ALK/ ROS1/ RET (P &lt; 0.01). In the group with KRAS/ BRAF V600E/ MET/ HER2, KEAP, or STK11 wild-type (P = 0.03), PD-L1 (≥1%) (P &lt; 0.01) and steroid use during ICI monotherapy due to immune-related adverse events (IRAE) (P &lt; 0.01) were related to a favorable PFS of ICI; however, no statistical significance was found for TP53 mutation and smoking status for PFS of ICI. In the group with EGFR/ ALK/ ROS1/ RET, PD-L1(≥ 50%) was the only factor that was related to a favorable PFS, and not TP53 mutation status, KEAP, or STK11. Conclusions: This study showed a difference in PFS among each type of AGAs. Although there are limitations due to the small number of patients, the KRAS/ BRAF V600E/ MET/ HER2 group had a favorable PFS compared with the EGFR/ ALK/ ROS1/ RET group . KEAP/STK11/PD-L1 status and steroid use due to IRAE predicted favorable PFS of ICI in the KRAS/ BRAF V600E/ MET/ HER2 group .

Genomic profiling in advanced NSCLC in Italy: First results of NERoNE (NSCLC Emilia Romagna Next Generation Sequencing Evaluation) project.

e21077 Background: The NERoNE Project evaluates the use of Next Generation Sequencing (NGS) in molecular profiling of advanced NSCLC (aNSCLC) patients (pts) in three Oncology Units (OU) of Emilia Romagna Region aiming to clarify the impact of such extensive tool in characterizing different NSCLC subpopulations. Methods: Since January 2020 to December 2022, 970 sequential aNSCLC pts from three OU (AUSL-IRCCS of Reggio Emilia, Modena University Hospital and IRST Meldola) were profiled by NGS. Samples from Modena and Reggio Emilia were analyzed at Modena University Hospital Molecular Pathology Lab using Myriapod IL- 56G, Cancer Panel DNA, Cancer Panel RNA, Oncomine DX Target Test Assay, Oncomine Focus Assay. Samples from IRST Meldola were processed at its Molecular Diagnostic Unit by Myriapod NGS cancer panel DNA e RNA, Oncomine Focus Assay and Oncomine Comprehensive Assay v3.We considered molecular alterations with available target therapies in Italy: EGFR mutations (mut) (exon 18,19,20,21), KRAS mut, BRAF V600E mut, ALK, ROS1, RET and NTRK rearrangements, MET exon 14 skipping mut, ErbB2 mut, FGFR 1-3 mut. PDL1 expression was assessed by standard immunohistochemistry on 486 pts. Results: Among 970 pts, 501 with at least one mut (52%) were considered for analysis: 266 were female (53%), mean age was 69 ± 11 years (min-max: 33-94) and 473 pts (94%) had adenocarcinoma. Molecular features are listed in table 1. Distribution of mut among ages, categorized as &lt; 60, 60-70 and &gt; 70 y, was similar, EGFR activating mut were most prevalent in females; KRAS mut (G12C and non G12C) were most prevalent in males; 41 pts had co-mutations (8.2%): the most frequent co-mut were EGFR mut (27%). PDL1 expression was &lt; 1% 129 (27%), 1-49% 211 (43,4%) and ≥50% 146 pts (30%). The majority of pts with EGFR activating mut had PDL1 &lt; 50%. Conclusions: NERoNE evaluates data from NGS profiling of three OU mapping key actionable molecular alterations in a large population of aNSCLC also accounting for PDL1 status. This preliminary analysis will be the starting point for further evaluations in order to improve the personalized approach to aNSCLC. [Table: see text]

LIBELULE: A randomized phase III study to evaluate the clinical relevance of early liquid biopsy (LB) in patients with suspicious metastatic lung cancer.

9019 Background: Timeliness of molecular testing is essential to minimize the time to appropriate 1st line treatment initiation (TTI) in advanced NSCLC pts. We hypothesized that LB-based molecular testing performed at the patient's 1st visit could reduce this TTI. Methods: LIBELULE is a multicenter, randomized, comparative, open-label study, enrolling pts with radiological suspicion of stage IV lung cancer, no prior biopsy or cytology for advanced NSCLC diagnosis. In Arm A, a LB was performed at the 1st visit using the InVisionFirst-Lung assay, an amplicon-based NGS panel covering 37-NSCLC associated genes. When LB results were informative, treatment was initiated on the basis of: InVisionFirst-Lung alone for pts with EGFR, BRAF V600E mutation (mut), ALK or ROS1 rearrangement (category 1 alterations); InVisionFirst-Lung and pathological results for pts with ERBB2, MET exon 14, KRAS, BRAF non V600 and/or LKB1 mut, RET or NTRK rearrangement (category 2 alterations). In control Arm B, histological sampling was planned with genomic analysis when indicated (local LB allowed) and treatment was initiated according to ESMO guidelines. The primary endpoint was the time from randomization to initiation of appropriate treatment based on informative genomic and pathological results. 286 pts were needed to detect a 21% shortening in TTI (α = 0.05, 1-β = 90%). Results: 319 pts were randomized between Arm A (n = 161) and B (n = 158): median age 68 y (39-97), 56.1% male, 28.5% non-smokers, PS0-1 82%, PS≥2 18,1%; adenocarcinoma: 56.7%, squamous cell carcinoma: 11%, SCLC: 10%, other tumor types: 5%. 5.3% of pts had no cancer. In Arm A, 81% of pts had ctDNA findings; category 1 and category 2 alterations were identified on tissue and/or LB in 29.2% ( EGFR mut: 21.7%) and 24%, respectively, whereas in Arm B, 23.2% of pts had category 1 ( EGFR mut: 20.3%) and 20% had category 2 alterations detected. Systemic treatment was initiated in 74.5% and 65.8% of pts in Arm A and B, respectively. Main reasons for not initiating treatment were diagnosis other than cancer, local treatment and palliative care. The mean TTI was 29.0 days (d) (95%CI 25.9-32.1) in Arm A vs 33.9 d (95%CI 28.4-39.5) in Arm B in the intention-to-treat population (p = 0.28), but significantly shorter in Arm A 29.1 d vs 38.8 d in Arm B for pts receiving systemic treatment (p = 0.01). The mean TTI was significantly shorter (21 d vs 37.4 d) in pts with category 1 alterations (p = 0.004) as well as the time to contributive genomic analysis (17.9 d vs 25.6 d; p &lt; 0.001) in Arm A and B, respectively. In Arm A, 7.4% of pts vs 13.3% in Arm B initiated a treatment without genomic analysis available. Conclusions: Early LB significantly reduces the time to contributive molecular analysis and the time to initiation of appropriate 1st line therapy in pts eligible for systemic treatment, especially for pts with actionable alterations indicating targeted 1st line therapy. Clinical trial information: NCT03721120 .

The impact of single gene testing (SGT) on subsequent comprehensive genomic profiling (CGP) success in community oncology practice for advanced non-small cell lung cancer (NSCLC): Results from a prospective observational reference laboratory testing program.

6506 Background: CGP concurrently tests tumor tissue for guideline-recommended predictive biomarkers for optimal therapy selection and identification of clinical trials in NSCLC patients. A common practice in the community oncology setting is to first order SGT, then consider CGP when SGT results are negative or after treatment failure. Given the limited tissue available and the scope of alterations not tested by SGT, we sought to characterize the effects of prior SGT on subsequent CGP testing success, and therapeutic opportunities identified by CGP beyond SGT for NSCLC patients in the community setting. Methods: Reference laboratory information systems were used to prospectively contact clinicians across 80 community practices who ordered at least 1 SGT for guideline-recommended genomic variants in NSCLC. CGP was offered for their patients prior to SGT or upon receipt of negative SGT results. SGT included individual assays for BRAF, EGFR, KRAS, MET exon 14 skipping mutations; ALK, RET, and ROS1 rearrangements; and PD-L1 IHC . CGP included DNA-seq for mutations, copy number variants in 523 genes including guideline-recommended genomic variants, MSI, and tumor mutational burden; RNA-seq for rearrangements/fusions/splice variants; and PD-L1 IHC. Results: Among a total of 580 NSCLC patients with CGP ordered (May 2021-December 2022), 168 (29%) had ≥1 SGT ordered prior to CGP (median=5). No patients had all SGT performed, with untested cases ranging from 10% for ALK to 90% for MET exon 14. The same FFPE tissue block was used for CGP in 150/168 (89%) of cases with prior negative SGT. Compared to CGP-only cases, CGP for cases with prior negative SGT was canceled twice as often at tissue review (16% vs 7%; p=.001), had higher DNA extraction failures (13% vs 8%; p=.09), and lower DNA sequencing success (70% vs 83%, p&lt;.001). CGP detected guideline-recommended variants in 51% of all cases. Among prior negative SGT cases, frequencies were higher in genes where SGT was performed less often and is less sensitive than CGP RNA-seq ( RET fusions , MET exon 14 skipping). CGP also identified guideline-recommended variants in genes with no SGT offered during the study period ( ERBB2 mutations , NTRK2/3 fusions), as well as variants with emerging evidence for FDA expedited program-designated clinical trial therapies in 28% of patients, including NRG1 fusions and BRAF non-V600E , KEAP1, KRAS non-G12C, NFE2L2, STK11, and TP53 Y220C mutations. Conclusions: Prior negative SGT doubled subsequent CGP test cancellations for NSCLC due to tissue insufficiency and increased CGP DNA extraction failures. SGT practice in the community oncology setting does not meet practice guideline recommendations and negatively impacts the potential benefit of subsequent CGP for NSCLC patients.

Approach to risk stratification for papillary thyroid carcinoma based on molecular profiling: institutional analysis

Currently, treatment recommendations for papillary thyroid carcinoma are not based on the genetic background causing tumourigenesis. The aim of the present study was to correlate the mutational profile of papillary thyroid carcinoma with clinical parameters of tumour aggressiveness, to establish recommendations for risk-stratified surgical treatment. Papillary thyroid carcinoma tumour tissue of patients undergoing thyroid surgery at the University Medical Centre Mainz underwent analysis of BRAF, TERT promoter and RAS mutational status as well as potential RET and NTRK rearrangements. Mutation status was correlated with clinical course of disease. One hundred and seventy-one patients operated for papillary thyroid carcinoma were included. The median age was 48 years (range 8-85) and 69 per cent (118/171) of patients were females. One hundred and nine papillary thyroid carcinomas were BRAF-V600E mutant, 16 TERT promotor mutant and 12 RAS mutant; 12 papillary thyroid carcinomas harboured RET rearrangements and two papillary thyroid carcinomas showed NTRK rearrangements. TERT promoter mutant papillary thyroid carcinomas had a higher risk of distant metastasis (OR 51.3, 7.0 to 1048.2, P < 0.001) and radioiodine-refractory disease (OR 37.8, 9.9 to 169.5, P < 0.001). Concomitant BRAF and TERT promoter mutations increased the risk of radioiodine-refractory disease in papillary thyroid carcinoma (OR 21.7, 5.6 to 88.9, P < 0.001). RET rearrangements were associated with a higher count of tumour-affected lymph nodes (OR 7950.9, 233.7 to 270495.7, P < 0.001) but did not influence distant metastasis or radioiodine-refractory disease. Papillary thyroid carcinoma with concomitant BRAF-V600E and TERT promoter mutations demonstrated an aggressive course of disease, suggesting the need for a more extensive surgical strategy. RET rearrangement-positive papillary thyroid carcinoma did not affect the clinical outcome, potentially obviating the need for prophylactic lymphadenectomy.

RET rearrangements are relevant to histopathologic subtypes and clinicopathological features in Thai papillary thyroid carcinoma patients.

Background: Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. The RET gene rearrangements CCDC6::RET and NCOA4::RET are the most common RET gene rearrangements in PTC patients. Different RET::PTC rearrangements are associated with different PTC phenotypes. Methods: Eighty-three formalin-fixed paraffin-embedded (FFPE) PTC samples were examined. The prevalence and expression levels of CCDC6::RET and NCOA4::RET were determined using semi-quantitative polymerase chain reaction (qRT-PCR). The association of these rearrangements with clinicopathological data was investigated. Results: The presence of CCDC6::RET rearrangement was significantly associated with the classic subtype and absence of angio/lymphatic invasion (p < 0.05). While NCOA4::RET was associated with the tall-cell subtype, and presence of angio/lymphatic invasion and lymph node metastasis (p < 0.05). Multivariate analysis demonstrated that an absence of extrathyroidal extension and extranodal extension were independent predictive factors for CCDC6::RET, whereas the tall-cell subtype, large tumor size, angioinvasion, lymphatic invasion and perineural invasion were independent predictive factors for NCOA4::RET (p < 0.05). However, the mRNA expression level of CCDC6::RET and of NCOA4::RET were not significantly associated with clinicopathological data. Conclusion: CCDC6::RET was correlated with an innocent PTC subtype and characteristics, but NCOA4::RET correlated with an aggressive phenotype of PTC. Therefore, these RET rearrangements strongly associated with clinicopathological phenotypes and can be used as predictive markers in PTC patients.

Fluorescence in situ hybridization for ETV6 gene rearrangements on destained cytological smears: Role in diagnosis of secretory carcinoma of salivary gland.

Fine-needle aspiration cytology (FNAC) is the first-line diagnostic procedure for salivary gland masses. Secretory carcinoma (SC) is characterized by ETV6 and RET rearrangements detected by fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction optimized for paraffin-embedded and fresh-frozen tissue, respectively. The authors performed FISH on cytological material to assess its role in the diagnosis of SC. FNACs with SC as a diagnostic consideration and cases diagnosed as SC on histology with a corresponding FNAC with any diagnosis were evaluated for ETV6 rearrangement by FISH. If acinic cell carcinoma (ACC) was a differential diagnosis and ETV6 rearrangement was absent, NR4A3 FISH was performed. FISH results were compared with those on histological specimens, where available. Fifteen cases were included. FISH initially performed on three cell blocks did not yield good results, was then performed on direct smears, and was interpretable in 14 cases (93.3%). An ETV6 rearrangement was identified in seven cases (50%), and an NR4A3 rearrangement was identified in three cases (21.4%), providing a confirmatory diagnosis in 10 of 15 cases (66.7%). The Milan System for Reporting Salivary Gland Cytopathology category was altered in two cases (6.7%). Complete correlation (100%) was seen with FISH on corresponding histological specimens. With minor modifications, the FISH procedure can be optimized for FNAC smears with results comparable to those on histological specimens. ETV6 FISH testing on cytological smears in cases suspected as SC improves the diagnostic accuracy of FNAC and can help lower the proportion of the Milan categories salivary gland neoplasm of uncertain malignant potential and suspicious for malignancy, maximizing diagnostic information from less invasive samples and aiding in patient management.

Pathological characteristics and tumour immune microenvironment of lung malignancies with RET rearrangement

BackgroundFor patients with lung malignancies with RET rearrangement, the efficacy of immune checkpoint inhibitors is limited. The characteristics of the tumour immune microenvironment (TIME) and molecular pathological features of these patients have not been well elucidated. We aimed to investigate their clinical outcomes and explore characteristics of TIME, using multiplex immunohistochemistry technology (mIHC). Patients and MethodsThe pathology and TIME characteristics of 29 patients with lung malignancies with RET rearrangement were retrospectively analysed, and their relationships with clinical efficacy and prognosis were investigated. Gene detection relied on high-throughput sequencing, and TIME detection was based on mIHC. ResultsOf 29 patients, 25(86%) had adenocarcinoma, and the acinar type accounted for the greatest percentage of patients, followed by the solid type, regardless of whether the disease was early or locally advanced and metastatic. In addition, we report a novel KIF5B-RET(k24:R8) rearrangement in pulmonary sarcoma. The density of CD8+ T cells in tumour stroma in early-stage patients was significantly higher than that in locally advanced and metastatic patients (P = 0.014). The proportion of M2 macrophages in tumour stroma was significantly higher than that in tumour parenchyma (P = 0.046). Although the difference was not statistically significant (P = 0.098), patients positive for M2 macrophage infiltration into the tumour parenchyma (≥5%) may have a better prognosis. Seven patients received immunotherapy and disease control rate was 85.7%. ConclusionsA novel KIF5B-RET rearrangement variant in pulmonary sarcoma shows similar TIME characteristics to lung cancer. amongst patients with lung malignancies with RET rearrangement, patients with M2 macrophage infiltration into the tumour parenchyma may have a better prognosis, but further studies with larger cohorts are needed.

Oncogenic driver mutations in Vietnamese patients with lung adenocarcinoma

Mutation profiles of oncogenes play essential roles in cancer therapy, but data on the prevalence of lung cancer oncogenic driver mutations in Vietnamese patients are limited. This study aims to evaluate the mutation status of EGFR, ALK, ROS1, RET and MET and analyze the association of gene mutations with clinicopathological characteristics of lung adenocarcinoma. Of the 179 lung adenocarcinoma tissues, EGFR, ALK, ROS1, RET and MET mutation frequencies were 44.6; 7.9; 3.0; 3.0 and 2.0 percent, respectively. EGFR, ALK, ROS1 and RET alterations tended to be higher in females. Moreover, rearrangements of ALK, ROS and RET were more prevalent in younger lung adenocarcinoma, but skipping at exon 14 MET was more frequent in male and older patients. Stages III and IV seem to accumulate more ALK, RET and MET abnormalities. These findings identified the variation of frequencies of Vietnamese lung adenocarcinoma with different clinicopathological characteristics and established the pioneer data for oncogenic diver mutation of lung cancer in Vietnam.

Lung cancer presenting with central nervous system metastasis: Clinicopathological and molecular analysis of 171 cases

A subset of lung carcinoma presents initially with brain metastasis. Precise subtyping is mandatory for optimized treatment of these advanced aggressive carcinomas. We herein analyzed surgical biopsies from 171 Patients (99 males and 72 females aged 48–96; mean, 72), who presented with brain metastasis of lung cancer. In addition to conventional subtyping, we applied an extended immunohistochemistry (IHC) panel and performed several molecular tests looking for potential therapeutic targets other than EGFR mutations. Non-small cell carcinoma (NSCLC) comprised 157 (91.8 %) of cases: 109 (63.7 %) adenocarcinomas, 27 (15.8 %) squamous cell (SCC), 18 (10.5 %) large cell undifferentiated, 1 (0.6 %) adenosquamous and 2 (1.2 %) unclassified carcinomas. Of the adenocarcinomas, 81.7 % were TTF1+. Notably, 45 % of those TTF1-negative cases expressed HepPar1. SMARCA4 and SMARCA2 loss was observed in 13/171 (7.6 %) and 32/163 (19.6 %) cases, respectively; mainly TTF1- (40.0 %) and HepPar1+ (38.1 %) adenocarcinomas were affected by SMARCA2/4 loss. Loss of at least one mismatch repair (MMR) protein was observed in 3/156 (1.9 %) cases (2 adenocarcinomas and 1 large cell neuroendocrine carcinoma/LCNEC). Limited available data on mutation testing showed a frequency of EGFR mutations of 4.3% and of KRAS mutations of 57%. HER2 expression (2+/3+) was found in 45/166 (27.1 %) of cases with amplification verified by CISH in 18/38 (47.4 % of immunopositive cases and 10.5 % of the whole cohort); all but one were adenocarcinomas. Other genetic abnormalities detected included EML4::ALK rearrangements in 3 (1.8 %; 2 TTF1+ adenocarcinomas and 1 LCNEC) and RET rearrangements in one SCNEC. Variable subsets of tumors revealed amplifications of several potentially therapeutically targetable genes including MYC (30.0 %), MET (10.1 %), HER2 (10 %), FGFR1 (9.6 %), FGFR3 (4.6 %), and FGFR2 (3.4 %). This study highlights a highly heterogeneous molecular background in lung cancer presenting with CNS metastases. These findings highlight the need for individualized tumor testing strategies looking for potential therapeutic targets for this aggressive disease.

Ultrasound images-based deep learning radiomics nomogram for preoperative prediction of RET rearrangement in papillary thyroid carcinoma.

To create an ultrasound -based deep learning radiomics nomogram (DLRN) for preoperatively predicting the presence of RET rearrangement among patients with papillary thyroid carcinoma (PTC). We retrospectively enrolled 650 patients with PTC. Patients were divided into the RET/PTC rearrangement group (n = 103) and the non-RET/PTC rearrangement group (n = 547). Radiomics features were extracted based on hand-crafted features from the ultrasound images, and deep learning networks were used to extract deep transfer learning features. The least absolute shrinkage and selection operator regression was applied to select the features of nonzero coefficients from radiomics and deep transfer learning features; then, we established the deep learning radiomics signature. DLRN was constructed using a logistic regression algorithm by combining clinical and deep learning radiomics signatures. The prediction performance was evaluated using the receiver operating characteristic curve, calibration curve, and decision curve analysis. Comparing the effectiveness of the models by linking the area under the receiver operating characteristic curve of each model, we found that the area under the curve of DLRN could reach 0.9545 (95% confidence interval: 0.9133-0.9558) in the test cohort and 0.9396 (95% confidence interval: 0.9185-0.9607) in the training cohort, indicating that the model has an excellent performance in predicting RET rearrangement in PTC. The decision curve analysis demonstrated that the combined model was clinically useful. The novel ultrasonic-based DLRN has an important clinical value for predicting RET rearrangement in PTC. It can provide physicians with a preoperative non-invasive primary screening method for RET rearrangement diagnosis, thus facilitating targeted patients with purposeful molecular sequencing to avoid unnecessary medical investment and improve treatment outcomes.

Emerging Role of Targeted Therapy in Metastatic Pancreatic Adenocarcinoma.

The aggressive biology of pancreatic ductal adenocarcinoma (PDAC), along with its limited sensitivity to many systemic therapies, presents a major challenge in the management of patients with metastatic PDAC. Over the past decade, the incorporation of combinatorial cytotoxic chemotherapy regimens has improved patient outcomes. Despite these advances, resistance to cytotoxic chemotherapy inevitably occurs, and there is a great need for effective therapies. A major focus of research has been to identify molecularly defined subpopulations of patients with PDAC who may benefit from targeted therapies that are matched to their molecular profile. Recent successes include the demonstration of the efficacy of maintenance PARP inhibition in PDAC tumors harboring deleterious BRCA1, BRCA2, and PALB2 alterations. In addition, while therapeutic targeting of KRAS was long thought to be infeasible, emerging data on the efficacy of KRAS G12C inhibitors have increased optimism about next-generation KRAS-directed therapies in PDAC. Meanwhile, KRAS wild-type PDAC encompasses a unique molecular subpopulation of PDAC that is enriched for targetable genetic alterations, such as oncogenic BRAF alterations, mismatch repair deficiency, and FGFR2, ALK, NTRK, ROS1, NRG1, and RET rearrangements. As more molecularly targeted therapies are developed, precision medicine has the potential to revolutionize the treatment of patients with metastatic PDAC.

Clinicopathological significance of major fusion oncogenes in papillary thyroid carcinoma: An individual patient data meta-analysis.

Fusion oncogenes (e.g., NTRK, RET, ALK, BRAF) are rare genetic events in papillary thyroid carcinoma (PTC). It is still unclear regarding the similarities and differences in clinicopathological manifestations and prognostic outcomes of these genetic alterations. This individual patient data (IPD) meta-analysis analyzed the clinicopathological significance and prognoses of different types of oncogenic fusions in PTC patients. Categorical variables were compared by using Chi-square and Fisher's exact tests while Wilcoxon rank-sum and analysis of variance (ANOVA) tests were utilized for continuous covariates. Progression-free survival (PFS) and overall survival (OS) were computed using Kaplan-Meier analysis and log-rank test. We included 27 studies for meta-analyses. NTRK-, RET-, BRAF-, and ALK-rearranged PTCs had a unique demographic/clinicopathological profile but similar PFS and OS. NTRK1-positive PTCs demonstrated more aggressive clinical behaviors and shorter PFS in comparison to NTRK3-positive PTCs whereas RET rearrangement variants shared comparable clinicopathological backgrounds. This study provides new insights and facilitates our current understanding of clinicopathological features and survival outcomes of different fusion oncogenes in PTCs. It may help clinicians better counsel the patients and tailor appropriate treatment decisions.

Genomic landscape of lung adenocarcinomas in different races.

BackgroundLung adenocarcinoma is a molecularly heterogeneous disease. Several studies, including The Cancer Genome Atlas Research Network (TCGA) and Lung Cancer Mutation Consortium (LCMC), explored the genetic alterations among different ethnic groups. However, minority groups are often under-represented in these relevant studies and the genomic alterations among racial groups are not fully understood.MethodsWe analyze genomic characteristics among racial groups to understand the diversities and their impact on clinical outcomes.ResultsNative Americans had significantly higher rates of insertions and deletions than other races (P<0.001). Among patients with lung adenocarcinomas, EGFR and KRAS were the highest discrepancy genes in the different racial groups (P<0.001). The EGFR exon 21 L858R point mutation was three times higher in Asians than in all other races (P<0.001). Asians, Whites, and Blacks had 4.7%, 3.1%, and 1.8% ALK rearrangement, respectively (P<0.001). White patients had the highest rates of reported KRAS G12C (15.51%) than other races (P<0.001). Whites (17.2%), Blacks (15.1%), and Other (15.7%) had higher rates of STK11 mutation than Asians (3.94%) (P<0.001). RET rearrangement and ERBB2 amplification were more common in Asian patients than in Other racial groups. Apart from point mutations, structural variations, and fusion genes, we identified a significant amount of copy number alterations in each race.ConclusionsThe tumor genomic landscape is significantly distinct in different races. This data would shed light on the understanding of molecular alterations and their impacts on clinical management in different lung cancer patients.