BRAF V600E-mutated large cell neuroendocrine carcinoma responding to targeted therapy: a case report and review of the literature.

Large cell neuroendocrine carcinoma (LCNEC) is a rare and aggressive high-grade neuroendocrine tumor, commonly arising in the lung or in the gastrointestinal tract, with a frequent proportion of unknown primary origin (20%). In the metastatic setting, platinum-based or fluoropyrimidine-based chemotherapeutic regimens are as considered the first-line treatment, despite the limited duration of response. To date, the prognosis of advanced high-grade neuroendocrine carcinoma remains poor, suggesting the need to explore new treatment strategies in this orphan tumor. The evolving molecular landscape of LCNEC, not yet been completely defined, could explain the heterogeneous response to different chemotherapeutic regimens and suggest that treatment strategy could be driven by molecular features. v-Raf murine sarcoma viral oncogene homolog B (BRAF) mutations, well described in melanoma, thyroid cancer, colon cancer and lung adenocarcinoma, account for approximately 2% of cases in lung LCNEC. Here, we describe the case of a patient with a BRAF V600E-mutated LCNEC of unknown primary origin who partially responded to BRAF/mitogen-activated protein kinase kinase inhibitors after standard treatment. Additionally, BRAF V600E circulating tumor DNA was used to monitor disease response. Thereafter, we reviewed the available literature about the role of targeted therapy in high-grade neuroendocrine neoplasms to provide insight for future research to identify patients with driver oncogenic mutations, who can potentially benefit from target therapy.

Expanding the Spectrum of BRAF Non-V600E Mutations in Thyroid Nodules: Evidence-Based Data from a Tertiary Referral Centre.

The BRAF p.V600E mutation represents the most specific marker for papillary thyroid carcinoma and is potentially related to aggressive behavior and persistent disease. BRAF alterations other than the p.V600E are less common in thyroid carcinoma and represent an alternative mechanism of BRAF activation with unclear clinical significance. The study aims to describe the frequency and clinicopathologic characteristics of BRAF non-V600E mutations in a large cohort (1654 samples) of thyroid lesions characterized by next-generation sequencing. BRAF mutations have been found in 20.3% (337/1654) of thyroid nodules, including classic (p.V600E) mutation in 19.2% (317/1654) of samples and non-V600E variants in 1.1% of cases (19/1654). BRAF non-V600E alterations include 5 cases harboring p.K601E, 2 harboring p.V600K substitutions, 2 with a p.K601G variant, and 10 cases with other BRAF non-V600E alterations. BRAF non-V600E mutations have been reported in one case of follicular adenoma, three cases of conventional papillary thyroid carcinoma, eight cases of follicular variant of papillary carcinomas, one case of columnar cell variant papillary thyroid carcinoma, one case of oncocytic follicular carcinoma, and two bone metastasis of follicular thyroid carcinoma. We confirm that BRAF non-V600E mutations are uncommon and typically found in indolent follicular-patterned tumors. Indeed, we show that BRAF non-V600E mutations can be found in tumors with metastatic potential. However, in both aggressive cases, the BRAF mutations were concomitant with other molecular alterations, such as TERT promoter mutation.

Multi-Gene Next-Generation Sequencing Panel for Analysis of BRCA1/BRCA2 and Homologous Recombination Repair Genes Alterations Metastatic Castration-Resistant Prostate Cancer.

Despite significant therapeutic advances, metastatic CRPC (mCRPC) remains a lethal disease. Mutations in homologous recombination repair (HRR) genes are frequent in mCRPC, and tumors harboring these mutations are known to be sensitive to PARP inhibitors. The aim of this study was to verify the technical effectiveness of this panel in the analysis of mCRPC, the frequency and type of mutations in the BRCA1/BRCA2 genes, as well as in the homologous recombination repair (HRR) genes. A total of 50 mCRPC cases were analyzed using a multi-gene next-generation sequencing panel evaluating a total of 1360 amplicons in 24 HRR genes. Of the 50 cases, 23 specimens (46.0%) had an mCRPC harboring a pathogenic variant or a variant of uncertain significance (VUS), whereas in 27 mCRPCs (54.0%), no mutations were detected (wild-type tumors). BRCA2 was the most commonly mutated gene (14.0% of samples), followed by ATM (12.0%), and BRCA1 (6.0%). In conclusion, we have set up an NGS multi-gene panel that is capable of analyzing BRCA1/BRCA2 and HRR alterations in mCRPC. Moreover, our clinical algorithm is currently being used in clinical practice for the management of patients with mCRPC.

Next-generation sequencing revealing TP53 mutation as potential genetic driver in dermal deep-seated melanoma arising in giant congenital nevus in adult patients: A unique case report and review of the literature.

Melanoma in giant congenital nevus (M-GCN) is a rare and potentially lethal neoplasm. In children, M-GCN appears as a dermal/deep-seated melanoma (DDM-GCN) with histopathologic features difficult to distinguish from proliferative nodules (PNs-GCN). DDM-GCN in adults is an anecdotal entity and only 8 cases have been described and genetically characterized. We report the first case of DDM-GCN in a 34-year-old man characterized with a large-panel next-generation sequence (NGS) highlighting a TP53 mutation with a UV-signature (C>T substitution) in DDM but not in PNs-GCN and GCN. Curiously, DDM showed an aberrant p16 overexpression without detection of CDKN2A mutation at NGS. In line with previous studies, it supports a different pathway in children and adults: UV-induced mutations may be involved in the latter not only by CDKN2A but also by TP53 mutations, with a potentially confusing overexpression of p16 protein. While these data need to be confirmed in larger cases series, our results show that NGS could be an additional genetic diagnostic tool in DDM-GCN.

Interferon-γ is increased in the gut of patients with irritable bowel syndrome and modulates serotonin metabolism.

Mucosal immune activation and altered serotonin metabolism participate in the pathophysiology of irritable bowel syndrome (IBS). However, the reciprocal interplay between these two systems remains unknown. We evaluated the expression and release of interferon (IFN)-γ from the colonic mucosa of patients with IBS and its impact on serotonin reuptake transporter (SERT) gene expression in Caco-2 cells. qPCR was used to evaluate IFN-γ gene expression in colonic mucosal biopsies, whereas IFN-γ protein amount was assessed by ELISA. Colonic T box expressed in T cells (T-bet) and phosphorylated signal transducer and activator of transcription 4 protein amount were evaluated by Western blot. The impact of colonic mucosal mediators on SERT gene expression was evaluated in Caco-2 cells using qPCR. IFN-γ receptor was silenced in Caco-2 cells to determine the effect of IFN-γ released by mucosal biopsies. Compared with asymptomatic controls (ACs), the expression of IFN-γ gene and its transcription factor T-bet were markedly increased in the colonic mucosa of patients with IBS. Compared with ACs, IFN-γ protein tissue levels and its release by mucosal biopsies were significantly increased in IBS. The exposure of Caco-2 cells to IBS supernatants induced a significant decrease in SERT gene expression, independently of IBS subtypes, compared with AC mucosal supernatants. In Caco-2 cells, IFN-γ receptor silencing reversed the reduction of SERT expression evoked by IBS supernatants vs. nonsilenced cell lines. IFN-γ gene, its transcription factor T-bet, IFN-γ protein expression, and its release are increased in the colonic mucosa of patients with IBS and downregulate SERT gene expression in vitro. These results suggest that IFN-γ downregulates SERT expression, hence likely playing a role in altered serotonin metabolism of patients with IBS.

Successful treatment with personalized dosage of imatinib in elderly patients with gastrointestinal stromal tumors.

Imatinib is the standard first-line therapy for metastatic gastrointestinal stromal tumors. It has markedly improved the prognosis and outcome of patients affected by gastrointestinal stromal tumors, especially in the case of exon 11 KIT mutations. Imatinib-associated adverse events are generally mild to moderate; however, in clinical practice, intolerance caused by chronic toxicities frequently leads to breaks in treatment. This is particularly true in elderly patients in whom age, decline in drug metabolism, and polypharmacy, with a possible drug-drug interaction, may influence the tolerability of imatinib. In the present article, we report our extensive experience with the management of imatinib therapy in a 'real' population, in particular in very elderly patients, discussing whether the use of personalized imatinib dosage could be a safe and advantageous option, enabling continuous administration, thus ensuring effective treatment. Only a few case reports in the literature provide data on outcome with low tailored dosage of imatinib and none of them has been carried out on a Western population. Here, we report four cases treated with low imatinib dosage as a safe and useful option enabling continued treatment with imatinib, improving tolerance, and maintaining good and lasting disease control.

Plasma citrulline after small bowel transplantation: effect of time from transplantation, acute cellular rejection, and renal failure.

BACKGROUND: Plasma citrulline concentration (CIT) depends on its synthesis by enterocytes and its catabolism by renal tubules. To evaluate CIT applicability as a marker of acute cellular rejection (ACR) after intestinal transplantation (ITx), CIT was investigated according to time from ITx, episodes of ACR, and creatinine clearance (CrCl). METHODS: Twenty-four adult ITx recipients were prospectively studied. The results were compared with those of 19 healthy controls (HCs) and of 29 patients with chronic renal failure (CRF). RESULTS: In ITx recipients, CIT was lower than in HCs during the first two postoperative weeks; it then progressively increased and reached the range observed in HCs, approximately between the 31st and the 45th postoperative day. A positive association with postoperative days (R = 0.63; p < 0.0001) and a negative association with CrCl (R = -0.57; p < 0.0001) were observed. CIT was higher in patients with CRF than in HCs (p < 0.0001). CIT sensitivity and specificity in detecting ACR after the 45th postoperative day were 38% and 83%, using CIT threshold observed in HCs, and 69% and 77%, respectively, using CIT threshold adjusted for CRF degree. CONCLUSIONS: Adjusting CIT threshold for CRF degree almost doubled the sensitivity of CIT as a non-invasive marker of ACR in ITx recipients.

Virologic and genetic evaluation of vemurafenib-induced skin cancers.

We describe the occurrence of a giant squamous cell carcinoma in a patient receiving vemurafenib for the treatment of late melanoma mestastases. Although the development of keratoacanthomas and squamous cell carcinomas (SCC) has been described during vemurafenib therapy, most of the reported cases are treated with surgical excision. In the present case, SCC regressed after drug withdrawal.

Do not Judge a book by its Cover: A Secretory Carcinoma Arising from a Salivary Gland Heterotopia in Laterocervical lymph-node.

Heterotopia, the occurrence of specific tissues in ectopic sites during embryogenesis, includes the presence of salivary gland tissue in unusual locations. Salivary gland neoplasms arising from heterotopic sites are rare. Secretory Carcinoma (SC) is a rare salivary gland carcinoma characterized by ETV6-NTRK3 fusion gene, very rarely described in salivary gland heterotopia. Here a case of SC originating from salivary gland heterotopia in a neck lymph node is reported, together with a literature review.A 66-year-old male presented with a left neck mass. Imaging and fine needle aspiration cytology indicated a preliminary diagnosis of a benign/low-grade malignancy neoplasm.Following surgery (superficial parotidectomy and mass excision), histological examination revealed SC within an intranodal salivary heterotopia, confirmed by molecular analysis.Heterotopic salivary gland tissue (HSGT) is rare, and its association with neoplasms is even rarer. Tumours arising on HSGT, share histological similarities with those affecting orthotopic salivary glands. This unique case expands the understanding of SC occurrences on HSGT.

Challenges and future perspectives for the use of temozolomide in the treatment of SCLC.

Small-cell lung cancer (SCLC), accounting for 10-20 % of all lung tumors, represents the most aggressive high-grade neuroendocrine carcinoma. Most patients are diagnosed with extensive-stage SCLC (ES-SCLC), with brian metastases identified in âˆ¼ 80 % of cases during the disease cours, and the prognosis is dismal, with a 5-year survival rate of less than 5 %. Current available treatments in the second-line setting are limited, and topotecan has long been the only FDA-approved drug in relapsed or refractory ES-SCLC, until the recent approval of lurbinectedin, a selective inhibitor of RNA polymerase II. Temozolomide (TMZ) is an oral alkylating agent, which showed single-agent activity in SCLC, particularly among patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation. Several studies have revealed the synergistic activity of temozolomide with poly-ADP-ribose polymerase (PARP) inhibitors, that prevent repair of TMZ-induced DNA damage. This review focuses on the rationale for the use of TMZ in ES-SCLC and provides an overview of the main trials that have evaluated and are currently investigating its role, both as a single-agent and in combinations, in relapse or refractory disease.

Molecular Characterization of Advanced-Stage Melanomas in Clinical Practice Using a Laboratory-Developed Next-Generation Sequencing Panel.

Cutaneous melanoma is one of the most lethal tumors among skin cancers, characterized by complex genetic and molecular alterations that result in uncontrolled cell proliferation and metastatic spread. Next-generation sequencing (NGS) enables the simultaneous examination of numerous genes, making this molecular technique essential for melanoma diagnosis, prognostic stratification, and therapy planning. Herein, we present the experience with our laboratory-designed NGS panel for the routine assessment of advanced-stage melanoma. A total of 260 specimens of advanced-stage melanomas were evaluated utilizing a laboratory-developed multi-gene NGS panel, which allowed the investigation of 229 amplicons in 25 oncogene/oncosuppressor genes. The NGS panel proved to be a reliable tool, failing to produce results in only 1.2% of the samples tested. BRAF and TERT were the two more commonly altered genes in 44.0% and 59.9% of samples, respectively. In 59.3% of the mutated cases, at least two concomitant variants were detected. In eight cases, both primary lesion and metastatic disease were analyzed by NGS. In all specimens (8/8, 100%), a perfect concordance in variants harbored by the primary and recurrence lesions was observed. Finally, this study described the validity of a laboratory-developed multi-gene NGS panel built specifically for advanced-stage melanomas in ordinary clinical practice.

Non-Small Cell Lung Cancer Testing on Reference Specimens: An Italian Multicenter Experience.

INTRODUCTION: Biomarker testing is mandatory for the clinical management of patients with advanced non-small cell lung cancer (NSCLC). Myriads of technical platforms are now available for biomarker analysis with differences in terms of multiplexing capability, analytical sensitivity, and turnaround time (TAT). We evaluated the technical performance of the diagnostic workflows of 24 representative Italian institutions performing molecular tests on a series of artificial reference specimens built to mimic routine diagnostic samples. METHODS: Sample sets of eight slides from cell blocks of artificial reference specimens harboring exon 19 EGFR (epidermal growth factor receptor) p.E746_AT50del, exon 2 KRAS (Kirsten rat sarcoma viral oncogene homologue) p.G12C, ROS1 (c-ros oncogene 1)-unknown gene fusion, and MET (MET proto-oncogene, receptor tyrosine kinase) Δ exon 14 skipping were distributed to each participating institution. Two independent cell block specimens were validated by the University of Naples Federico II before shipment. Methodological and molecular data from reference specimens were annotated. RESULTS: Overall, a median DNA concentration of 3.3 ng/µL (range 0.1-10.0 ng/µL) and 13.4 ng/µL (range 2.0-45.8 ng/µL) were obtained with automated and manual technical procedures, respectively. RNA concentrations of 5.7 ng/µL (range 0.2-11.9 ng/µL) and 9.3 ng/µL (range 0.5-18.0 ng/µL) were also detected. KRAS exon 2 p.G12C, EGFR exon 19 p.E736_A750del hotspot mutations, and ROS1 aberrant transcripts were identified in all tested cases, whereas 15 out of 16 (93.7%) centers detected MET exon 14 skipping mutation. CONCLUSIONS: Optimized technical workflows are crucial in the decision-making strategy of patients with NSCLC. Artificial reference specimens enable optimization of diagnostic workflows for predictive molecular analysis in routine clinical practice.

Integrated clinicopathologic and molecular analysis of endometrial carcinoma: Prognostic impact of the new ESGO-ESTRO-ESP endometrial cancer risk classification and proposal of histopathologic algorithm for its implementation in clinical practice.

Introduction: The European Society of Gynecologic Oncology/European Society of Radiation Therapy and Oncology/European Society of Pathology (ESGO/ESTRO/ESP) committee recently proposed a new risk stratification system for endometrial carcinoma (EC) patients that incorporates clinicopathologic and molecular features. The aim of the study is to compare the new ESGO/ESTRO/ESP risk classification system with the previous 2016 recommendations, evaluating the impact of molecular classification and defining a new algorithm for selecting cases for molecular analysis to assign the appropriate risk class. Methods: The cohort included 211 consecutive EC patients. Immunohistochemistry and next-generation sequencing were used to assign molecular subgroups of EC: POLE mutant (POLE), mismatch repair deficient (MMRd), p53 mutant (p53abn), and no specific molecular profile (NSMP). Results: Immuno-molecular analysis was successful in all cases, identifying the four molecular subgroups: 7.6% POLE, 32.2% MMRd, 20.9% p53abn, and 39.3% NSMP. The recent 2020 guidelines showed a 32.7% risk group change compared with the previous 2016 classification system: the reassignment is due to POLE mutations, abnormal p53 expression, and a better definition of lymphovascular space invasion. The 2020 system assigns more patients to lower-risk groups (42.2%) than the 2016 recommendation (25.6%). Considering the 2020 risk classification system that includes the difference between "unknown molecular classification" and "known," the integration of molecular subgroups allowed 6.6% of patients to be recategorized into a different risk class. In addition, the use of the proposed algorithm based on histopathologic parameters would have resulted in a 62.6% reduction in molecular analysis, compared to applying molecular classification to all patients. Conclusion: Application of the new 2020 risk classification integrating clinicopathologic and molecular parameters provided more accurate identification of low-and high-risk patients, potentially allowing a more specific selection of patients for post-operative adjuvant therapy. The proposed histopathologic algorithm significantly decreases the number of tests needed and could be a promising tool for cost reduction without compromising prognostic stratification.

Reference standards for gene fusion molecular assays on cytological samples: an international validation study.

AIMS: Gene fusions assays are key for personalised treatments of advanced human cancers. Their implementation on cytological material requires a preliminary validation that may make use of cell line slides mimicking cytological samples. In this international multi-institutional study, gene fusion reference standards were developed and validated. METHODS: Cell lines harbouring EML4(13)-ALK(20) and SLC34A2(4)-ROS1(32) gene fusions were adopted to prepare reference standards. Eight laboratories (five adopting amplicon-based and three hybridisation-based platforms) received, at different dilution points two sets of slides (slide A 50.0%, slide B 25.0%, slide C 12.5% and slide D wild type) stained by Papanicolaou (Pap) and May Grunwald Giemsa (MGG). Analysis was carried out on a total of 64 slides. RESULTS: Four (50.0%) out of eight laboratories reported results on all slides and dilution points. While 12 (37.5%) out of 32 MGG slides were inadequate, 27 (84.4%) out of 32 Pap slides produced libraries adequate for variant calling. The laboratories using hybridisation-based platforms showed the highest rate of inadequate results (13/24 slides, 54.2%). Conversely, only 10.0% (4/40 slides) of inadequate results were reported by laboratories adopting amplicon-based platforms. CONCLUSIONS: Reference standards in cytological format yield better results when Pap staining and processed by amplicon-based assays. Further investigation is required to optimise these standards for MGG stained cells and for hybridisation-based approaches.

Prognostic Impact of Pathologic Features in Molecular Subgroups of Endometrial Carcinoma.

The molecular characterization of endometrial carcinoma (EC) has recently been included in the ESGO/ESTRO/ESP guidelines. The study aims to evaluate the impact of integrated molecular and pathologic risk stratification in the clinical practice and the relevance of pathologic parameters in predicting prognosis in each EC molecular subgroup. ECs were classified using immunohistochemistry and next-generation sequencing into the four molecular classes: POLE mutant (POLE), mismatch repair deficient (MMRd), p53 mutant (p53abn), and no specific molecular profile (NSMP). According to the WHO algorithm, 219 ECs were subdivided into the following molecular subgroups: 7.8% POLE, 31% MMRd, 21% p53abn, 40.2% NSMP. Molecular classes as well as ESGO/ESTRO/ESP 2020 risk groups were statistically correlated with disease-free survival. Considering the impact of histopathologic features in each molecular class, stage was found to be the strongest prognostic factor in MMRd ECs, whereas in the p53abn subgroup, only lymph node status was associated with recurrent disease. Interestingly, in the NSMP tumor, several histopathologic features were correlated with recurrence: histotype, grade, stage, tumor necrosis, and substantial lymphovascular space invasion. Considering early-stage NSMP ECs, substantial lymphovascular space invasion was the only independent prognostic factor. Our study supports the prognostic importance of EC molecular classification and demonstrated the essential role of histopathologic assessment in patients' management.

Sarcomatoid change in adenocarcinoma arising in adulthood congenital pulmonary airway malformation.

Congenital pulmonary airway malformations (CPAM) are rare conditions generally diagnosed in childhood and possibly harboring malignant tumor growths. We describe a unique case of pleomorphic carcinoma in a longstanding type 1 CPAM diagnosed by wedge resection. The patient underwent completion left lower lobectomy and lymphadenectomy, but cancer recurred in nodal station #7 six months later. Clinicians should keep in mind that CPAM may hide radiologically undetectable malignancy in a relevant rate of cases, then requiring surgery in all patients. While MIA is the most common histology in type 1 CPAM, sarcomatoid change has herein been demonstrated.

Clinical characteristics and treatment outcomes of non-V600 E/K BRAF mutant melanoma patients: a single-institution experience.

The widespread use of more sensitive detection tools, such as next-generation sequencing, has increased the identification of a variety of BRAF mutations other than V600E/K in melanoma patients. However, there is a lack of established data regarding the efficacy of BRAF/MEK inhibitors and immune-checkpoint immune inhibitors (ICI) for these patients. We performed a retrospective study, including all the patients diagnosed with stage III or IV melanoma that were referred to the University Hospital of Bologna from 2011 to 2021, carrying a non-V600E or V600K mutation of BRAF and who were started on systemic treatment. We found 14 patients with stage III or IV melanoma harboring the following BRAF mutations: V600R, V600_K601delinsE, K601E, p.T599_V600insT, L597V, G466R, S467L, and A598T. Of note, G466R and A598T BRAF mutations have never been previously reported in melanoma. Four patients received combined BRAF/MEK inhibitors, two patients BRAF inhibitor monotherapy, and six patients were treated with ICI for advanced melanoma; four patients received adjuvant treatment with nivolumab. Given the few cases and the absence of randomized clinical trials, it is important to report clinical experiences, which can guide physicians in the treatment of melanomas harboring rare BRAF mutations.

Correlation of molecular alterations with pathological features in hepatocellular carcinoma: Literature review and experience of an Italian center.

Hepatocellular carcinoma (HCC) represents the primary carcinoma of the liver and the fourth leading cause of cancer-related deaths. The World Health Organization estimates an increase in cases in the coming years. The risk factors of HCC are multiple, and the incidence in different countries is closely related to the different risk factors to which the population is exposed. The molecular mechanisms that drive HCC tumorigenesis are extremely complex, but understanding this multistep process is essential for the identification of diagnostic, prognostic, and therapeutic markers. The development of multigenic next-generation sequencing panels through the parallel analysis of multiple markers can provide a landscape of the genomic status of the tumor. Considering the literature and our preliminary data based on 36 HCCs, the most frequently altered genes in HCCs are TERT, CTNNB1, and TP53. Over the years, many groups have attempted to classify HCCs on a molecular basis, but a univocal classification has never been achieved. Nevertheless, statistically significant correlations have been found in HCCs between the molecular signature and morphologic features, and this leads us to think that it would be desirable to integrate the approach between anatomic pathology and molecular laboratories.

ADK-VR2, a cell line derived from a treatment-naïve patient with SDC4-ROS1 fusion-positive primarily crizotinib-resistant NSCLC: a novel preclinical model for new drug development of ROS1-rearranged NSCLC.

Background: ROS1 fusions are driver molecular alterations in 1-2% of non-small cell lung cancers (NSCLCs). Several tyrosine kinase inhibitors (TKIs) have shown high efficacy in patients whose tumors harbour a ROS1 fusion. However, the limited availability of preclinical models of ROS1-positive NSCLC hinders the discovery of new drugs and the understanding of the mechanisms underlying drug resistance and strategies to overcome it. Methods: The ADK-VR2 cell line was derived from the pleural effusion of a treatment-naïve NSCLC patient bearing SDC4-ROS1 gene fusion. The sensitivity of ADK-VR2 and its crizotinib-resistant clone ADK-VR2 AG143 (selected in 3D culture in the presence of crizotinib) to different TKIs was tested in vitro, in both 2D and 3D conditions. Tumorigenic and metastatic ability was assessed in highly immunodeficient mice. In addition, crizotinib efficacy on ADK-VR2 was evaluated in vivo. Results: 2D-growth of ADK-VR2 cells was partially inhibited by crizotinib. On the contrary, the treatment with other TKIs, such as lorlatinib, entrectinib and DS-6051b, did not result in cell growth inhibition. TKIs showed dramatically different efficacy on ADK-VR2 cells, depending on the cell culture conditions. In 3D culture, ADK-VR2 growth was indeed almost totally inhibited by lorlatinib and DS-6051b. The clone ADK-VR2 AG143 showed higher resistance to crizotinib treatment in vitro, compared to its parental cell line, in both 2D and 3D cultures. Similarly to ADK-VR2, ADK-VR2 AG143 growth was strongly inhibited by lorlatinib in 3D conditions. Nevertheless, ADK-VR2 AG143 sphere formation was less affected by TKIs treatment, compared to the parental cell line. In vivo experiments highlighted the high tumorigenic and metastatic ability of ADK-VR2 cell line, which, once injected in immunodeficient mice, gave rise to both spontaneous and experimental lung metastases while the crizotinib-resistant clone ADK-VR2 AG143 showed a slower growth in vivo. In addition, ADK-VR2 tumor growth was significantly reduced but not eradicated by crizotinib treatment. Conclusions: The ADK-VR2 cell line is a promising NSCLC preclinical model for the study of novel targeted therapies against ROS1 fusions and the mechanisms of resistance to TKI therapies.