Resistance to ALK Inhibitors in Routine Practice

more frequently after second- or third-generation TKI treatment (54%-71%) than after crizotinib (20% of patients).¹¹ Meanwhile, 2 second-generation TKIs, ceritinib and alectinib, have shown efficacy as first-line treatment,⁴,²⁵ and ongoing clinical trials are currently evaluating next-generation TKIs (NCT03052608, NCT02737501, NCT02767804) in this context. Although no report so far has provided rates of ALK mutations after disease progression during treatment with second-generation TKIs given as first-line therapy, it is reasonable to speculate that they will exceed the 20% observed after failure of crizotinib treatment. Data further suggest that the ALK mutation rate also increases when several ALK TKIs are given sequentially. Solomon et al reported ALK mutation rates of 16.6% (14/84) after first-line ALK TKI treatment and 29.2% (31/106) after treatment with at least 2 ALK TKIs.²⁶ We have likewise observed a 15% ALK mutation rate after treatment failure of a first ALK TKI and a 33% ALK mutation rate after failure of 2 ALK TKI treatments. Whether this is because of the number of ALK TKIs administered or to the use of next-generation TKIs remains to be determined.

All of these findings, and because ALK-positive patients have been shown to benefit from receiving multiple TKIs,⁸,²⁷,²⁸ suggest that the future will see an increase in the proportion of patients exposed to next-generation ALK TKIs, along with an increase in the rate of ALK mutations.

Detection of ALK mutations in patients with disease progression after ALK TKI treatment is becoming crucial to determining the optimal therapeutic strategy. In vitro, different ALK mutations have been reported to predict different sensitivities to a single ALK TKI, and ALK TKIs do not all share the same spectrum of activity against ALK mutants.¹¹,²⁹ Similar findings have been reported in patients, although extrapolating from in vitro data to patients requires caution. In 2 patients, for example, the I1171N mutation was reported to drive resistance to alectinib and sensitivity to ceritinib.³⁰,³¹ In another patient, the L1152R mutation has on the contrary been reported to confer resistance to ceritinib and to predict a response to alectinib.³² Consistently, we have found that all 6 patients who were treated with an ALK TKI presumed to act against the detected mutation achieved disease control, whereas the only patient who was treated with an ALK TKI not presumed to act effectively against the detected mutation showed progressive disease at first assessment. Interestingly, 2 patients harboring a G1202R mutation achieved a partial response during treatment with brigatinib, which is reported to exert intermediate activity on G1202R mutants in vitro.

To date, no clinical trial has assessed the clinical relevance of identifying ALK mutations at the time of disease progression during treatment with an ALK TKI. Besides the previously mentioned case reports, data from prospective trials suggest that patients harboring an ALK mutation are more likely to respond to subsequent treatments with ALK TKIs. In patients who receive the third-generation TKI lorlatinib after failure of treatment with 2 or 3 previous ALK TKIs, Shaw et al reported objective response rates of 26% in patients with no ALK mutation and 61% in patients with at least 1 ALK mutation.³³ Interestingly, we have found a similar trend in our set of patients treated in routine practice: we observed a 71% ORR with subsequent TKI treatment in patients known to have an ALK mutation and a 33% ORR in patients with no ALK mutation, after treatment with a third TKI; the presence of an ALK mutation might reveal sustained dependence on the ALK pathway and thus predict sensitivity to ALK

inhibition,²⁶ and the type of ALK mutation should guide the choice of the ALK TKI to be used. Inversely, the absence of any ALK mutation suggests that resistance might be because of an ALK pathway-independent mechanism that is not sensitive to ALK inhibition. Various ALK-independent resistance mechanisms have been described, including activation of EGFR, insulin growth factor 1 receptor, and viral oncogene homolog mutation of KRAS or phosphatidylinositol 3-kinase, amplification of stem cell growth factor receptor (SCFRI/cKIT) and gene fusions.¹²,¹³,¹⁸,³⁴,³⁷ In in vitro studies, cells having acquired resistance to an ALK TKI via an ALK-independent mechanism showed cross-resistance to other ALK TKIs, but sensitivity to ALK TKIs could be restored by combined treatment with another TKI.³⁴,³⁷,³⁸ Efficacy of ALK TKI in patients having acquired resistance to a given ALK TKI via an ALK-independent mechanism is still poorly known.³⁷ Patients having progressed during crizotinib treatment with no evidence of an ALK mutation have been shown to benefit from second-generation ALK TKIs,³⁹ but whether their resistance was because of an ALK-independent mechanism, an ALK-dependent mechanism not involving an ALK mutation, or an ALK mutation that escaped detection because of sensitivity issues remains unknown.

Sequential treatment with several ALK TKIs might select resistant clones harboring multiple (ie, compound) ALK mutations. In a recent study, Yoda et al reported a 35% compound mutation rate in patients treated with lorlatinib⁴⁰; in our set, 2 patients had disease progression with lorlatinib treatment and a compound mutation was detected at the time of disease progression in a patient known to harbor a G1202R mutation. In vitro, compound mutations have been reported to confer resistance to most ALK TKIs, including lorlatinib.⁴⁰ However, a patient having acquired resistance to crizotinib, ceritinib, and lorlatinib successively and who was found to harbor a C1156Y/ L1196M compound mutation achieved an objective response with crizotinib treatment. It is necessary to further determine the sensitivity of compound mutants to ALK TKIs.¹⁶

Finally, other molecular alterations might affect patient outcomes. Recent findings have shown that TP53 mutations or ALK variants might be associated with shorter progression-free survival and OS in ALK-positive NSCLC patients.⁴¹,⁴² In our study, no difference in OS was found between patients with or without TP53 mutations, but the number of patients in each subgroup was too low to draw any conclusion from this result. ALK variants were not assessed in our study.

Conclusion

Our findings show that mechanisms of resistance to ALK TKIs can be detected in routine practice in a third of ALK-positive NSCLC patients. Identifying ALK resistance mutations might help in selecting the best subsequent treatment.

Clinical Practice Points

* Anaplastic lymphome kinase (ALK) mutations confer resistance to ALK TKIs in ALK-rearranged NSCLC. The frequency and type of ALK mutations that occur at progression depends on the type of ALK TKI received. These mutations are still rarely investigated in routine practice.

* Using targeted NGS, we identified 8 patients harboring an ALK mutation among 23 ALK-rearranged NSCLC patients whose
disease progressed during ALK TKI treatment. Our results suggest that targeted NGS is suitable for detecting ALK resistance mutations in ALK-rearranged NSCLC patients in routine practice.

* The ALK mutation rate was 15% after failure of the first ALK TKI and 33% after failure of treatment with 2 ALK TKIs. Furthermore, all of the patients who received a TKI presumed to act on the detected ALK mutant achieved disease control, suggesting that the identification of ALK mutation could help to select the best treatment at progression.

## Disclosure

A.B.C. participated in advisory boards or received honoraria from Astra-Zeneca, Bristol-Myers Squibb, Merck & Co, Pfizer, Roche, Novartis, and Takeda, and received grants paid to A.B.C.'s institution from Novartis and Merck. P.J. reports nonfinancial support from Chugai Pharma, and personal fees from Boehringer Ingelheim, outside the submitted work. E.D. reports personal fees from Bristol-Myers Squibb, Merck & Co, Astra-Zeneca, Lilly oncology, and grants from Roche, outside the submitted work. S.B. reports personal fees and nonfinancial support from Lilly, GSK, Roche, and Pfizer, and a grant from the Intergroup Francophone de Cancérológica Thoracique outside the submitted work. M.W. received honorarium or personal fees from Astra-Zeneca, Bristol-Myers Squibb, Merck & Co, Roche, Boehringer Ingelheim, and Amgen. R.G. participated in advisory boards or received honorarium from Astra-Zeneca, BMS, MSD, Pfizer, Roche, Novartis, and Boehringer, and received grants paid to R.G.'s institution from Roche. N.Ri. reports personal fees from AstraZeneca, and personal fees from Boehringer Ingelheim, outside the submitted work. M.C.C. participates in advisory boards for Pfizer and Roche. C.D. reports personal fees and nonfinancial support from AstraZeneca, Novartis Pharma SAS, Roche SAS, Boehringer Ingelheim France, and Pfizer, outside the submitted work. The remaining authors have stated that they have no conflicts of interest.

## Supplemental Data

Supplemental tables accompanying this article can be found in the online version at https://doi.org/10.1016/j.cllc.2019.02.013.

## References

1. Solomon B, Varella-Garcia M, Camidge DR. ALK gene rearrangements: a new therapeutic target in a molecularly defined subset of non-small-cell lung cancer. *J Thorac Oncol* 2009; 4:1450-4.

2. Chiarle R, Voena C, Ambrogio C, Piva R, Inghiram G. The anaplastic lymphoma kinase in the pathogenesis of cancer. *Nat Rev Cancer* 2008; 8:11-23.

3. Lin JJ, Riely GJ, Shaw AT. Targeting ALK: precision medicine takes on drug resistance. *Cancer Discov* 2017; 7:137-55.

4. Soria JC, Tan DS, Chiari R, et al. First-line ceritinib versus platinum-based chemotherapy in advanced ALK-rearranged non-small-cell lung cancer (ASCEND-4): a randomised, open-label, phase 3 study. *Lancet* 2017; 389:917-29.

5. Shaw AT, Kim DW, Nakagawa K, et al. Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. *N Engl J Med* 2013; 368:2385-94.

6. Hida T, Nokihara H, Kondo M, et al. Alectinib versus crizotinib in patients with ALK-positive non-small-cell lung cancer (J-ALEX): an open-label, randomised phase 3 trial. *Lancet* 2017; 390:29-39.

7. Friboulet L, Li N, Katayama R, et al. The ALK inhibitor ceritinib overcomes crizotinib resistance in non-small cell lung cancer. *Cancer Discov* 2014; 4: 662-73.

Philippe Jamme et al

8. Shaw AT, Gandhi L, Gadged S, et al. Alectinib in ALK-positive, crizotinib-resistant, non-small-cell lung cancer: a single-group, multicentre, phase 2 trial. *Lancet Oncol* 2016; 17:234-42.

9. Mezquita L, Planchard D. The role of brigatinib in crizotinib-resistant non-small-cell lung cancer. *Cancer Manag Res* 2018; 10:123-30.

10. Shaw AT, Felip E, Bauer TM, et al. Lorlatinib in non-small-cell lung cancer with ALK or ROS1 rearrangement: an international, multicentre, open-label, single-arm first-in-man phase 1 trial. *Lancet Oncol* 2017; 18:1590-9.

11. Gainor JF, Dardaei L, Yoda S, et al. Molecular mechanisms of resistance to first- and second-generation ALK inhibitors in ALK-rearranged lung cancer. *Cancer Discov* 2016; 6:1118-33.

12. Katayama R, Shaw AT, Khan TM, et al. Mechanisms of acquired crizotinib resistance in ALK-rearranged lung cancers. *Sci Transl Med* 2012; 4:120ra17.

13. Sasaki T, Koivunen J, Ogino A, et al. A novel ALK secondary mutation and EGFR signaling cause resistance to ALK kinase inhibitors. *Cancer Res* 2011; 71:6051-60.

14. Choi YL, Soda M, Yamashita Y, et al. EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. *N Engl J Med* 2010; 363:1734-9.

15. Sasaki T, Okuda K, Zheng W, et al. The neuroblastoma-associated F1174L ALK mutation causes resistance to an ALK kinase inhibitor in ALK-translocated cancers. *Cancer Res* 2010; 70:10038-43.

16. Shaw AT, Friboulet L, Leshchiner I, et al. Resensitization to crizotinib by the lorlatinib ALK resistance mutation L1198F. *N Engl J Med* 2016; 374:54-61.

17. Kim S, Kim TM, Kim DW, et al. Heterogeneity of genetic changes associated with acquired crizotinib resistance in ALK-rearranged lung cancer. *J Thorac Oncol* 2013; 8:415-22.

18. Dobele RC, Pilling AB, Aisner DL, et al. Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer. *Clin Cancer Res* 2012; 18:1472-82.

19. Toyokawa G, Inamatsu E, Shimamatsu S, et al. Identification of a novel ALK G1235S mutation in a patient with ALK-rearranged non-small-cell lung cancer exhibiting resistance to ceritinib. *J Thorac Oncol* 2015; 10:e55-7.

20. Qiao H, Lovly CM. Cracking the code of resistance across multiple lines of ALK inhibitor therapy in lung cancer. *Cancer Discov* 2016; 6:1084-6.

21. Ou SH, Azada M, Hsiang DJ, et al. Next-generation sequencing reveals a novel NSCLC ALK F1174V mutation and confirms ALK G1202R mutation confers high-level resistance to alectinib (CH5424802/RO5424802) in ALK-rearranged NSCLC patients who progressed on crizotinib. *J Thorac Oncol* 2014; 9:549-53.

22. Zou HY, Friboulet L, Kodack DP, et al. PF-06463922, an ALK/ROS1 inhibitor, overcomes resistance to first and second generation ALK inhibitors in predinical models. *Cancer Cell* 2015; 28:70-81.

23. Infarinato NR, Park JH, Krytska K, et al. The ALK/ROS1 inhibitor PF-06463922 overcomes primary resistance to crizotinib in ALK-driven neuroblastoma. *Cancer Discov* 2016; 6:96-107.

24. Passaro A, Prelaj A, Pochesi A, et al. Brigatinib for the treatment of ALK-positive advanced non-small-cell lung cancer patients. *Drug Today (Barc)* 2017; 53:435-46.

25. Peters S, Camidge DR, Shaw AT, et al. Alectinib versus crizotinib in untreated ALK-positive non-small-cell lung cancer. *N Engl J Med* 2017; 377:829-38.

26. Solomon BJ, Bessé B, Bauer TM, et al. Lorlatinib in patients with ALK-positive non-small-cell lung cancer: results from a global phase 2 study. *Lancet Oncol* 2018; 19:1654-67.

27. Duruisseaux M, Bessé B, Cadranel J, et al. Overall survival with crizotinib and next-generation ALK inhibitors in ALK-positive non-small-cell lung cancer (IFCT-1302 CLINALK): a French nationwide cohort retrospective study. *Oncotarget* 2017; 8:21903-17.

28. Mok T, Spigel D, Felip E, et al. ASCEND-2: a single-arm, open-label, multicenter phase II study of ceritinib in adult patients (pts) with ALK-rearranged (ALK+) non-small-cell lung cancer (NSCLC) previously treated with chemotherapy and crizotinib (CRZ). *J Clin Oncol* 2015; 33:8059.

29. Zhang S, Anjum R, Squillace R, et al. The potent ALK inhibitor brigatinib (AP26113) overcomes mechanisms of resistance to first- and second-generation ALK inhibitors in predinical models. *Clin Cancer Res* 2016; 22:5527-38.

30. Johnson AC, D6 P, Richard N, et al. Identification of I1171N resistance mutation in ALK-positive non-small-cell lung cancer tumor sample and circulating tumor DNA. *Lung Cancer* 2016; 99:38-40.

31. Ou SH, Greenbowe J, Khan ZU, et al. I1171 missense mutation (particularly I1171N) is a common resistance mutation in ALK-positive NSCLC patients who have progressive disease while on alectinib and is sensitive to ceritinib. *Lung Cancer* 2015; 88:231-4.

32. Tchekmetyian N, Ali SM, Miller VA, Haura EB. Acquired ALK L1152R mutation confers resistance to ceritinib and predicts response to alectinib. *J Thorac Oncol* 2016; 11:e87-8.

33. Shaw AT, Martini J-F, Bessé B, et al. Abstract CT044: Efficacy of lorlatinib in patients (pts) with advanced ALK-positive non-small cell lung cancer (NSCLC) and ALK kinase domain mutations. *Cancer Res* 2018; 78(suppl 13), CT044.

34. Miyawaki M, Yasuda H, Tani T, et al. Overcoming EGFR bypass signal-induced acquired resistance to ALK tyrosine kinase inhibitors in ALK-translocated lung cancer. *Mol Cancer Res* 2017; 15:106-14.

35. Crystal AS, Shaw AT, Sequir LV, et al. Patient-derived models of acquired resistance can identify effective drug combinations for cancer. *Science* 2014; 346: 1480-6.
Resistance to ALK Inhibitors in Routine Practice

36. Lovly CM, McDonald NT, Chen H, et al. Rationale for co-targeting IGF-1R and ALK in ALK fusion-positive lung cancer. *Nat Med* 2014; 20:1027-34.

37. McCoach CE, Le AT, Gowan K, et al. Resistance mechanisms to targeted therapies in ROS1 + and ALK+ non-small cell lung cancer. *Clin Cancer Res* 2018; 24:3334-47.

38. Tani T, Yanuda H, Hamamoto J, et al. Activation of EGFR bypass signaling by TGFβ overexpression induces acquired resistance to alclerib in ALK-translocated lung cancer cells. *Mol Cancer Ther* 2016; 15:162-71.

39. Shaw AT, Kim DW, Mehra D, et al. Ceritinib in ALK-rearranged non-small-cell lung cancer. *N Engl J Med* 2014; 370:1189-97.

40. Yoda S, Lin JJ, Lawrence MS, et al. Sequential ALK inhibitors can select forlordarinib-resistant compound ALK mutations in ALK-positive lung cancer. *Cancer Discov* 2018; 8:14-29.

41. Kron A, Alldouty C, Scheffler M, et al. Impact of TP53 mutation status on systemic treatment outcome in ALK-rearranged non-small-cell lung cancer. *Ann Oncol* 2018; 29:2068-75.

42. Lin JJ, Zhu VW, Yoda S, et al. Impact of EMIA-ALK variant on resistance mechanisms and clinical outcomes in ALK-positive lung cancer. *J Clin Oncol* 2018; 36:1199-206.
Supplemental Data

<table><thead><tr><th colspan="2">Supplemental Table 1 List of Genes and Exons Tested in the Colon & Lung V2 (Thermofisher Scientific, Waltham, MA) Panel</th></tr><tr><th>Gene</th><th>Exon</th></tr></thead><tbody><tr><td>AKT1</td><td>3</td></tr><tr><td>ALK</td><td>22, 23</td></tr><tr><td>BRAF</td><td>11</td></tr><tr><td>CTNNB1</td><td>3</td></tr><tr><td>DDR2</td><td>6, 9, 13-16, 18</td></tr><tr><td>EGFR</td><td>12, 18-21</td></tr><tr><td>ERBB2</td><td>19-21</td></tr><tr><td>ERBB4</td><td>3-4, 6-9, 15, 23</td></tr><tr><td>FBXW7</td><td>5, 8-11</td></tr><tr><td>FGFR1</td><td>4, 7</td></tr><tr><td>FGFR2</td><td>7, 9, 12</td></tr><tr><td>FGFR3</td><td>7, 9, 14, 16, 18</td></tr><tr><td>KRAS</td><td>2-4</td></tr><tr><td>MAP2K1</td><td>2</td></tr><tr><td>MET</td><td>2, 16</td></tr><tr><td>NOTCH1</td><td>1, 3, 6, 7, 8</td></tr><tr><td>SMAD4</td><td>3, 5-6, 8-12</td></tr><tr><td>STK11</td><td>1, 4-6, 8</td></tr><tr><td>TP53</td><td>2, 4-8, 10</td></tr></tbody></table>

<table><caption>Supplemental Table 2 Sites of Tissue Biopsy (n = 26)</caption><thead><tr><th>Biopsy Site</th><th>n (%)</th></tr></thead><tbody><tr><td>Lung</td><td>8 (30.8)</td></tr><tr><td>Pleural Effusion</td><td>5 (19.2)</td></tr><tr><td>Liver</td><td>4 (15.4)</td></tr><tr><td>Breast</td><td>2 (7.7)</td></tr><tr><td>Lymph Node</td><td>2 (7.7)</td></tr><tr><td>Brain</td><td>1 (3.8)</td></tr><tr><td>Cerebrospinal Fluid</td><td>1 (3.8)</td></tr><tr><td>Bone</td><td>1 (3.8)</td></tr><tr><td>Peritoneal</td><td>1 (3.8)</td></tr><tr><td>Pericardial Effusion</td><td>1 (3.8)</td></tr></tbody></table>
# 7 DISCUSSION

La médecine de précision a pour but de proposer un traitement adapté aux caractéristiques moléculaires des tumeurs. La détection d'altérations moléculaires susceptibles de répondre à des thérapies ciblées ainsi que l'identification des mécanismes de résistance à ces thérapies représentent deux aspects majeurs de la médecine de précision qui ont été étudiés dans mes travaux de thèse.

Nous nous sommes d'abord intéressés aux mécanismes de résistance primaire aux ITK MET dans les CBNPC avec mutations METex14 (représentant entre 2 et 3 % des CBNPC) (article n°1). Dans les CBNPC METex14, l'efficacité des ITK MET est plus faible que dans les autres modèles d'addiction oncogénique, ce qui suggère la présence de mécanismes de résistance primaires. Compte tenu des interactions entre la voie PI3K et le récepteur MET (Hervieu et Kermorgant 2018), nous nous sommes focalisés sur l'étude des altérations de la voie PI3K-AKT comme potentiel mécanisme de résistance aux ITK MET. En constituant une cohorte de 65 patients, nous avons étudié les anomalies de la voie PI3K et mis en évidence deux patients porteurs d'une mutation PIK3CA et six porteurs d'une perte totale d'expression de PTEN (Phosphatase and TENsin homolog). En corrélant les données cliniques avec les données histologiques et moléculaires, nous avons observé que le taux de réponse à un ITK MET des patients porteurs d'une co-altération MET/PI3K était sensiblement inférieur (0 %) à celui de ceux ne présentant pas d'altération de la voie PI3K (63 %). L'étude in vitro de trois lignées cellulaires porteuses de co-altération MET/PI3K a permis de confirmer l'implication de ces altérations dans la résistance aux ITK MET. L'utilisation d'un inhibiteur PI3K en combinaison avec un ITK MET a permis de bloquer la prolifération de ces lignées. Ces résultats peuvent permettre d'envisager, à terme, un essai thérapeutique pour les patients porteurs d'un CBNPC METex14 avec altération de la voie PI3K.

Par la suite, nous nous sommes intéressés aux mutations rares susceptibles de répondre aux ITK MET (article n°2). Nous avons identifié un patient porteur d'un cholangiocarcinome avec fusion et amplification de MET. Ce patient, initialement réfractaire à la chimiothérapie, a reçu un ITK MET permettant l'obtention d'une réponse objective. Après plusieurs mois de traitement, une progression tumorale nous a conduit à rechercher un mécanisme de
résistance acquis. Nous avons mis en évidence l'émergence d'une mutation du domaine
kinase de MET connue pour induire une résistance aux ITK MET de type 1.

Enfin, nous nous sommes intéressés à la faisabilité de la recherche des mutations de résistance ALK par NGS ciblé chez les patients porteurs d'un CBNPC ALK+, traités par un ITK ALK (article n°3). Les mécanismes de résistance aux ITK ALK sont majoritairement des secondes mutations ALK affectant le domaine kinase. Les ITK ALK de deuxième et troisième génération ont été développés afin de surmonter ces mécanismes. Dans ce travail, nous avons établi une cohorte rétrospective de 23 patients ayant reçu un ITK ALK et pour lesquels une biopsie à progression était disponible. Une analyse par NGS des tumeurs à progression a été réalisée. Nous avons identifié neuf patients porteurs d'une altération ALK (neuf mutations du domaine kinase ALK et une amplification ALK) conférant une résistance aux ITK ALK. Parmi ces patients, sept ont reçu un ITK ALK de nouvelle génération à progression. Parmi les six patients ayant reçu un ITK ALK présumé actif contre la mutation ALK de résistance, cinq ont présenté une réponse partielle et un une stabilité prolongée. Le patient ayant reçu un ITK présumé non actif sur la mutation de résistance a progressé à la première évaluation. Ces données semblent confirmer l'utilité de rechercher les mécanismes de résistance afin d'orienter les choix thérapeutiques.

Dans cette discussion, nous aborderons, tout d'abord, l'étude des mécanismes de résistance aux ITK, en proposant une revue des mécanismes identifiés aux ITK MET. Ensuite, nous nous intéresserons à la voie PI3K-AKT et aux enjeux soulevés par la découverte de ces co-altérations. Enfin, nous évoquerons le challenge de la détection des anomalies moléculaires.

## 7.1 Médecine de précision et mécanisme de résistance

### 7.1.1 Définition d'un mécanisme de résistance

Nous avons vu, précédemment, que l'identification des mécanismes conduisant à la résistance aux thérapies ciblées a permis le développement de stratégies thérapeutiques innovantes afin de surmonter cette résistance. Dès lors, la recherche des mécanismes de résistance est essentielle si l'on souhaite améliorer la prise en charge des patients. Il existe deux notions
importantes afin de définir un mécanisme de résistance : sa temporalité par rapport à la thérapie reçue et sa position par rapport à la cible thérapeutique.

Concernant la temporalité, les altérations responsables d'une résistance peuvent être présentes initialement dans la tumeur (mécanisme primaire) ou émerger après un traitement (mécanisme acquis) (Lin et Shaw, n. d.). Les mécanismes de résistance acquis sont définis comme l'apparition d'une progression sous traitement après une réponse initiale (Westover et al. 2018). Plusieurs critères doivent être respectés : la thérapie reçue par le patient doit être délivrée en monothérapie et la progression doit survenir après une première réponse documentée partielle ou complète ou après une période de six mois de stabilité. Toutefois, il est intéressant de noter que l'amélioration des techniques de détection a permis d'observer que certains mécanismes de résistance acquis, émergeant sous la pression sélective des thérapies, peuvent être présents initialement dans la tumeur. Dans le cadre de nos travaux de thèse, nous avons identifié les altérations de la voie PI3K comme un mécanisme primaire de résistance conduisant à une progression sous ITK MET (article n°1). Nous avons également identifié les mutations du domaine kinase de MET et ALK comme des mécanismes acquis ayant émergé sous ITK MET et ALK (articles n°2 et n°3).

S'agissant de la position par rapport à la cible thérapeutique, les mécanismes de résistance peuvent survenir sur la cible (mécanisme on-target) ou survenir en dehors de la cible (mécanisme off-target). Les mécanismes on-target correspondent généralement à des mutations du domaine kinase pouvant augmenter l'affinité du récepteur à l'ATP et induisant un blocage stérique du site de fixation. En prenant pour exemple les CBNPC EGFR mutés et ALK+, la mutation EGFR T790M représente 50 à 60 % des résistances aux ITK EGFR de première et deuxième génération tandis que la mutation du domaine kinase ALK représente entre 20 et 40 % des résistances aux ITK ALK (Lin et Shaw, n. d). D'autres mécanismes on target ont par ailleurs été identifiés comme l'amplification du gène cible retrouvé dans 8 à 10 % des CBNPC EGFR mutés traités par ITK EGFR et 7 à 18 % des CBNPC ALK+ traités par ITK ALK (Sequist et al. 2011 ; Katayama et al. 2012 ; Doebele et al. 2012). Les mécanismes de résistance off target sont plus hétérogènes, regroupant l'activation de voies de signalisation parallèles et les transformations phénotypiques. Les voies de signalisation parallèles peuvent être activées via la présence d'altérations sur d'autres RTK, comme, par exemple, les amplifications de MET dans les CBNPC EGFR-mutés et les CBNPC ALK+ (Baldacci et al. 2018 ; Gouji et al. 2014), mais
également par la présence de mutations en aval, comme les mutations de MEK et de PIK3CA (Crystal et al. 2014). Les transformations phénotypiques, quant à elles, correspondent le plus souvent dans les CBNPC EGFR mutés et ALK+ à de modifications histologiques en carcinome à petites cellules (CPC) (Lin et Shaw n. d. ; Miyamoto et al. 2016 ; Sequist et al. 2011). Les mécanismes conduisant à la transformation des CBNPC en CPC sont inconnus. La présence au sein de la tumeur initiale d'un contingent cellulaire de CPC qui aurait été sélectionné sous ITK EGFR est une hypothèse proposée (Cortot et Janne 2014b).

Dans le cadre de mes travaux de thèse, les altérations de la voie PI3K sont des mécanismes de résistance off target car activant une voie de signalisation parallèle (article n°1) alors que les mutations du domaine kinase de MET et ALK ainsi que l'amplification du gène ALK sont des mécanismes on target (articles n°2 et 3).

## 7.1.2 Revue des mécanismes de résistance et des biomarqueurs de réponse aux ITK MET

À ce jour, différents mécanismes de résistance (acquis et primaires) aux ITK MET ont été observés en clinique. Dans mes travaux de thèse, nous avons identifié les altérations de la voie PI3K comme mécanismes de résistance primaires (article n°1) et les mutations du domaine kinase de MET comme mécanisme de résistance acquis (article n°2). Dans la pratique clinique, les ITK MET sont uniquement recommandés chez les patients porteurs d'un CBNPC METex14. Dès lors, l'essentiel des mécanismes de résistance ayant été identifiés concerne les patients porteurs d'un CBNPC METex14.

### 7.1.2.1 Les mécanismes de résistance primaires aux ITK MET

À ce jour, aucun mécanisme de résistance on target responsable d'une résistance primaire aux ITK MET n'a été observé dans les CBNPC METex14.

Concernant les mécanismes off target, deux principaux mécanismes de résistance primaires ont été décrits : les mutations de KRAS et les altérations de la voie de signalisation PI3K. Des mutations du gène KRAS ont été identifiées de novo chez respectivement 4,4 % et 5,2 % des patients atteints de CBNPC METex14 (Rotow et al. 2018 ; Suzawa et al. 2019). Il s'agit
majoritairement de substitutions impliquant la guanine en position 12 (G12 C/D/S/V) et, plus rarement, des mutations KRAS G13C, Q22K et Q61H. Les données précliniques suggèrent que la présence d'une mutation de *KRAS* entraîne une résistance aux ITK MET dans le CBNPC METex14 et que la combinaison des ITK MET et d'un inhibiteur MEK pourrait surmonter cette résistance (Rotow *et al.* 2018). En pratique clinique, la sensibilité aux ITK MET reste mal connue. Suzawa *et al.* ont décrit cinq patients présentant un CBNPC avec une co-altération METex14 et *KRAS*. Dans quatre cas, la mutation a été identifiée dans la tumeur initiale. Le traitement par un ITK MET a pu être initié dans deux cas et a entraîné une stabilisation transitoire de la maladie. Dans l'étude METROS, l'utilisation du crizotinib chez deux patients atteints d'un CBNPC METex14 avec une mutation de *KRAS* a conduit à une absence de réponse objective (Landi *et al.* 2019). L'amplification du gène *KRAS* a également été décrite *de novo* chez 2,6 % des patients, mais l'impact sur l'efficacité d'un TKI MET n'est pas connu (Suzawa *et al.* 2019).

Concernant les altérations de la voie PI3K, nous avons observé que les mutations du gène *PI3K* et les pertes d'expression de la protéine PTEN étaient fréquentes dans les CBNPC METex14. Nous avons également rapporté, après une analyse d'une cohorte de 65 patients porteurs d'un CBNPC METex14, un taux de réponse aux ITK MET plus faible chez les patients porteurs d'une altération de la voie PI3K. De récentes données semblent confirmer nos observations. L'analyse d'une cohorte de 75 patients ayant reçu un ITK MET pour un CBNPC METex14 a mis en évidence un taux de réponse objective ainsi qu'une survie plus faible pour les patients porteurs d'une altération de la voie PI3K par rapport aux patients non porteurs de ces altérations (13 % versus 41 %) (Guo *et al.* 2020). Toutefois, nous notons des différences par rapport à nos travaux. Concernant la fréquence des altérations, Guo *et al.* ont retrouvé une altération de la voie PI3K dans 11 % des cas. Bien que nous ayons identifié une altération de la voie PI3K dans 12 % des cas, cette fréquence est probablement sous-estimée compte tenu du fait que l'analyse de l'expression de PTEN (principale altération identifiée) n'a pu être réalisée que sur 40 % des tumeurs. Concernant les altérations de PTEN, Guo *et al.* se sont intéressés aux mutations survenant sur le gène *PTEN*, alors que nous nous sommes focalisés sur l'expression de la protéine PTEN. Il existe de nombreuses altérations post-transcriptionnelles pouvant moduler l'expression de la protéine PTEN, ce qui peut expliquer la différence de fréquence observée dans nos deux études (Lee, Chen et Pandolfi 2018).
### 7.1.2.2 Les mécanismes de résistance acquis aux ITK MET

À ce jour, nous disposons des résultats d'analyse de deux cohortes de patients porteurs d'un CBNPC METex14, traités par ITK MET et pour lesquels une biopsie à progression a été réalisée. Une première cohorte de 20 patients a mis en évidence un mécanisme *on target* dans 35 % des tumeurs et un mécanisme *off target* dans 45 % d'entre elles (Recondo et al. 2020). Une seconde cohorte de quinze patients a permis de retrouver un mécanisme *on target* dans 20 % des cas et un mécanisme *off target* dans 33 % des cas (Guo et al. 2020).

#### 7.1.2.2.1 Les mécanismes de résistance acquis et *on target*

L'un des mécanismes de résistance acquis et *on target* aux ITK MET le plus décrit chez les patients atteints d'un CBNPC METex14 est la survenue d'une mutation secondaire du gène *MET* (le plus souvent dans le domaine kinase). Nous avons également observé un cas similaire d'émergence d'une mutation du domaine kinase de MET après un traitement par capmatinib (article n°2). Les mutations de *MET* sont responsables d'une modification de la conformation du domaine kinase de la protéine MET interférant avec la liaison à l'ITK MET (Fujino et al. 2019). À ce jour, plus de dix mutations différentes ont été rapportées. Les plus fréquentes se situent sur la leucine 1195, la lysine 1228, 1246 et la tyrosine 1230 (Heist et al. 2016 ; Guo et al. 2019a ; Li et al. 2017 ; Jin et al. 2019 ; Dong et al. 2016 ; Rotow et al. 2018 ; Zhang, Yin et Peng 2017 ; Ou et al. 2017 ; Awad et al. 2018 ; 2020). Par ailleurs, la présence de mutations kinases composites a également été observée. En effet, Awad et al. ont rapporté trois cas de mutations composites : un après un traitement au crizotinib (MET Y1230H ; D1228N ; D1228S) et deux (MET D1228N ; G1163R ; L1195V et H1049Y ; L1195V ; L1195F) après un traitement au glesatinib.

Parmi les autres mécanismes *on target* décrits, on retrouve une amplification du gène *MET* (17 copies), survenue chez un patient traité par glesatinib ainsi qu'un cas d'amplification du gène *HGF* après traitement par crizotinib (Guo et al. 2019b ; Recondo et al. 2020).

Il est intéressant de noter que la sécrétion autocrine d'HGF et l'amplification *EGFR* ont été décrits comme des mécanismes de résistance potentiels aux ITK EGFR (Yano et al. 2008).
### 7.1.2.2.2 Les mécanismes de résistance acquise et off target

L'amplification du gène *KRAS* est le mécanisme de résistance acquis et *off target* le plus fréquemment mis en évidence. En 2018, Bahcall *et al*. ont identifié trois patients atteints d'un CBNPC METex14 ayant acquis, après une réponse initiale au crizotinib, une amplification du gène sauvage de *KRAS* ((Bahcall *et al*. 2018). En 2019, Rotow *et al*. ont décrit un cas d'amplification du gène *KRAS* (22 copies du gène) après une réponse initiale au crizotinib (Rotow *et al*. 2018). La même année, Han *et al*. ont rapporté un cas d'amplification de *KRAS* après neuf mois de traitement par savolitinib chez un patient atteint d'un CBNPC METex14 (Han *et al*. 2019). Plus récemment Awad *et al*. ont observé l'émergence d'une amplification de *KRAS* chez deux patients préalablement traités par crizotinib. Il est intéressant de noter que chez ces deux patients une amplification *EGFR* était également retrouvée (Recondo *et al*. 2020). Parmi les autres mécanismes identifiés, la mutation KRASG12D a été retrouvée chez un patient traité par crizotinib et une co-amplification de *ERBB3* et *EGFR* a été identifiée chez deux patients traités par capmatinib (Recondo *et al*. 2020).

### 7.1.2.3 Les biomarqueurs prédictifs de réponse aux ITK MET

Il existe des données significatives concernant des marqueurs prédictifs de réponse. En 2020, Guo *et al*. se sont intéressés à l'impact de l'expression du récepteur MET, analysé par spectrométrie de masse (SM) et par immunohistochimie (IHC), sur la réponse aux ITK MET dans les CBNPC METex14. Les auteurs ont ainsi observé que les tumeurs n'exprimant pas le récepteur MET étaient résistantes aux ITK MET (0 % versus 60 %). De plus, le taux de réponse était dépendant du H-score (proportion de cellules positives) avec un taux de 62 % si le H score était > 200 versus 28 % pour un H-score compris entre 1 et 199. La survie était également impactée, avec une survie sans progression à 10,4 mois pour les H-scores > 200 contre 5,5 mois pour les H-score < 200 (Guo *et al*. 2020).

Concernant la recherche de biomarqueurs de réponse, au sein du laboratoire nous travaillons actuellement sur l'évaluation du rôle de l'HGF dans les CBNPC METex14 (cf. annexe article 1). Dans un premier temps, nous avons évalué le rôle de l'HGF dans le potentiel oncogénique des mutations METex14. Pour cela, nous avons inséré (via une technique de CRISPR-Cas9) une
mutation ponctuelle de *MET* responsable d'un saut de l'exon 14 de *MET* dans une lignée épithéliale bronchique immortalisée mais non transformée (16HBE14o-). Ainsi, nous avons obtenu la lignée 16HBE-Ex14. Nous avons également établi une lignée 16HBE14o- exprimant le récepteur MET sauvage, 16HBE-WT. Plusieurs expérimentations ont été menées dont des expériences de tumorigenèse *in vivo* pour lesquelles des xénogreffes de cellules 16HBE-Ex14 et16HBE-WT ont été réalisées dans deux modèles de souris SCID. Nous avons utilisé un modèle de souris SCID exprimant l'HGF humain (hHGFki-SCID) et un modèle de souris SCID contrôle (NSG). Nous avons, dans un premier temps, observé que les cellules 16HBE-Ex14 injectées dans des souris hHGFki-SCID, formaient plus de tumeurs comparées aux cellules 16HBE-WT (100% versus 14%) (Annexe n°1 A). Par la suite, nous avons pu observer que le pourcentage de souris présentant des tumeurs était nettement supérieur lorsque les cellules 16HBE-ex14 étaient injectées dans les souris hHGFki-SCID que dans les souris NSG (86 % versus 35 %) (Annexe n°1 B). Nous avons également démontré, que l'ajout d'un inhibiteur de MET, permettait de bloquer la prolifération des cellules 16HBE-ex14 dans les souris hHGFki-SCID (Annexe n°1 C). Ces résultats témoignent de l'impact de l'HGF dans le potentiel oncogénique des altérations METex14. Par la suite, nous nous sommes intéressés au rôle de l'HGF comme potentiel biomarqueur de réponse prédictif aux ITK MET dans les CBNPC METex14. Nous avons constaté que 33 % des tumeurs expriment l'HGF, ce qui confirme la faisabilité de cette recherche. Toutefois, aucune donnée clinique d'efficacité des ITK MET n'est disponible pour ces patients, ce qui ne permet pas, à ce jour, d'évaluer l'HGF comme un éventuel biomarqueur de réponse.

### 7.1.2.4 Stratégies proposées afin de surmonter les résistances acquises on target aux ITK MET

Nos travaux de recherche sur les CBNPC ALK+ (article n°3) nous ont permis de montrer que l'utilisation d'un ITK ALK, choisi en fonction du mécanisme de résistance identifié, était associée à un taux de réponse important. Concernant les mécanismes de résistance aux ITK MET, à l'instar d'autres modèles d'addiction oncogénique, la survenue de mutations dans les exons codant pour le domaine kinase du récepteur MET est le mécanisme de résistance acquis et on target le plus fréquemment observé. S'agissant de la prise en charge des patients présentant ces mutations de résistance, des données précliniques, obtenues dans le modèle
cellulaire Ba/F3 exprimant la mutation METex14, ont permis de classer les différentes
sensibilités aux ITK MET selon le type de mutation kinase de MET (Fujino et al. 2019). Ainsi, il
a été observé que les mutations impliquant la lysine 1228 et la tyrosine 1230 sont toutes
résistantes aux ITK de type Ia et Ib et pourraient être surmontées, pour la majorité d'entre
elles, par un ITK de type II, à l'exception des mutations D1228A et D1228Y. En revanche, les
mutations impliquant la leucine 1195 et la phénylalanine 1120 sont principalement sensibles
aux ITK MET de type 1 et résistantes aux ITK de type 2. Des données cliniques semblent
confirmer ces observations.

Le cas d'un patient suivi pour un CBNPC METex14 et ayant présenté l'émergence d'une mutation de résistance du domaine kinase de MET (Y1230H) après un traitement par crizotinib a été rapporté. Un traitement par glesatinib, un ITK MET de type II, a pu être initié, ce qui a permis l'obtention d'une réponse partielle (Engstrom et al. 2017). Recondo et al. ont également rapporté plusieurs cas de patients porteurs d'un CBNPC METex14 et ayant développé un ou plusieurs mécanismes de résistance on target. Un premier cas concernait un patient ayant développé une mutation MET Y1230H après un traitement de quinze mois de crizotinib, un traitement par mérestinib, un inhibiteur de type II, a été introduit et a permis l'obtention d'une réponse. Un deuxième cas rapportait l'émergence d'une amplification de MET (17 copies) sous glesatinib, un traitement par crizotinib a été initié conduisant à une réponse partielle pendant dix mois. Enfin, un troisième cas était celui d'un patient traité par crizotinib et pour lequel six mutations ont été identifiées (les mutations MET D1228N/H, Y1230S/H et G1163R, connues pour induire une résistance aux ITK MET de type 1, et la mutation MET L1195V, responsable d'une résistance aux ITK MET de type II), le glesatinib a été introduit et a conduit à une progression après un mois de traitement (Recondo et al. 2020).

Ces données offrent des perspectives intéressantes pour les patients porteurs d'un cancer
avec une addiction oncogénique à *MET* et développant une mutation du domaine kinase de
MET sous ITK. En effet, nous avons observé chez les patients porteurs d'un CBNPC ALK+ avec
une mutation du domaine kinase ALK que l'utilisation d'une stratégie thérapeutique visant à
surmonter la résistance est associée à un taux de réponse (86 %) (article n°3).

Il est également intéressant d'explorer d'autres stratégies en cas de mécanismes on target,
comme l'inhibition de voies de signalisation parallèles pouvant être suractivées, et ce, en
l'absence d'altération spécifique de ces voies. Par exemple, des données précliniques suggèrent que la voie NOTCH est suractivée en présence de la mutation de résistance aux ITK EGFR de première génération (EGFR T790M) et également en présence de la mutation de résistance à l'osimertinib (EGFR C797S). Il a aussi été observé que l'utilisation d'un inhibiteur de la voie NOTCH permet de restaurer la sensibilité aux ITK EGFR (Mur et al. 2020).

## 7.2 Voie PI3K et résistance aux thérapies anticancéreuses

Durant mes travaux de thèses, nous nous sommes spécifiquement focalisés sur les altérations de la voie PI3K comme mécanismes de résistance aux ITK. Ce choix tient à l'existence d'une forte interaction entre MET et la voie PI3K.

L'impact réel des altérations de PI3K dans la survenue de résistances aux thérapies anticancéreuses est peu connu. Toutefois, les altérations de PI3K ont été décrites comme un mécanisme de résistance aux thérapies ciblées, à la chimiothérapie, à l'hormonothérapie et l'immunothérapie témoignant, ainsi, d'un rôle prépondérant des acteurs de cette voie dans l'échec thérapeutique (Huang et Fu 2015 ; Mills, Rutkovsky et Giordano 2018 ; Wang et al. 2016 ; Hwu et Davies n. d. ; Barrueto et al. 2020).

Dans les CBNPC avec addiction oncogénique, les altérations de PI3K (tout particulièrement les mutations *PIK3CA*) ont été décrites comme des mécanismes de résistances aux ITK. Engelman et al. ont démontré qu'insérer une mutation *PIK3CA* (E545K) dans une lignée cellulaire de CBNPC (HCC827) sensible aux ITK EGFR conduit à une résistance à ces derniers (Engelman et al. 2006). Dans les adénocarcinomes pulmonaires, la présence d'une co-altération EGFR-PI3K est détectée dans 13 % des cas. Ce taux est ailleurs enrichi après exposition à un ITK EGFR (Yu et al. 2018). La présence d'une co-altération EGFR-PI3K est associée à un plus mauvais pronostic (survie globale de 18 mois *versus* 33, p : 0,006) sans pour autant modifier le taux de réponse aux ITK EGFR (83 % *versus* 62 %, p = 0,80) (Engelman et al. 2015).

Des données issues de 324 patients ayant progressé sous ITK EGFR de première génération ont montré que 14,9 % des tumeurs étaient porteuses d'une altération de PI3K (mutation de *PIK3CA*, mutation de *AKT* ou mutation de *PTEN*), suggérant que cette dernière est un potentiel mécanisme de résistance acquis (Fang et al. 2020).

Les résultats récents obtenus avec les inhibiteurs de la voie PI3K dans les cancers du sein offrent l'espoir d'utiliser ces thérapeutiques dans les années à venir dans différents cancers
(Verret et al. 2019). Toutefois, de nombreux essais ayant évalué les inhibiteurs PI3K en combinaison avec la chimiothérapie ou des thérapies ciblées ont échoué à démontrer la supériorité de l'ajout de ce traitement (Rodon et al. 2018).

La dérégulation conjointe de MET et de PI3K a été identifiée dans de nombreux cancers (Hervieu et Kermorgant 2018). Il s'agit principalement d'études précliniques ayant relevé la présence d'une activation conjointe de MET et de PI3K dans des lignées cellulaires résistantes à la chimiothérapie et à la radiothérapie (Jung, Choi et Kwak 2015 ; Ettl et al. 2015).

Les stratégies visant à combiner des ITK de PI3K et des inhibiteurs de MET ont été évaluées à plusieurs reprises. Nous avons pu montrer que l'utilisation d'un inhibiteur de la voie PI3K permettait de restaurer la sensibilité aux ITK MET en cas de co-altération MET/PI3K (article n°1). Une observation similaire a également été effectuée en cas d'amplification de *KRAS*. En effet, dans ce modèle, la voie PI3K-AKT (médiée par la sécrétion autocrine du ligand EGF) était activée. L'ajout d'un inhibiteur de la voie PI3K couplé à une inhibition de MET a permis de surmonter la résistance. Sur le plan clinique, à ce jour, nous disposons d'une seule étude ayant évalué la possibilité d'une co-inhibition de MET et de PI3K. Cette étude s'est intéressée aux patients porteurs d'un glioblastome avec altération de PTEN. Il s'agit d'un essai de phase Ib/II, ayant évalué le capmatinib en combinaison avec le buparlisib. En raison d'un manque d'efficacité clinique notable, caractérisé par l'absence de réponse objective et un taux de stabilité de 30 %, cet essai a été interrompu. Toutefois, il est intéressant de noter un profil de tolérance acceptable avec un taux d'évènement indésirable de grade III de 36,4 %, suggérant, à terme, la possibilité d'évaluer cette combinaison chez des patients porteurs d'un CBNPC METex14 avec une altération de la voie PI3K.

## 7.3 Détection des biomarqueurs : enjeux et problématiques

Durant mes travaux de thèses, la détection des biomarqueurs de réponse aux thérapies ciblées était centrale. Dans un premier temps, nous avons cherché à optimiser nos outils de détection afin de mieux identifier les patients porteurs d'un CBNPC METex14 (Descarpentries et al. 2018). Afin de mieux identifier les altérations de PTEN, nous avons proposé une analyse par IHC de l'expression de PTEN (article n°1). Nous avons travaillé à la détection d'un nouveau driver oncogénique, la fusion de *MET* (article n°2). Nous avons essayé d'étudier la faisabilité de la recherche des mécanismes de résistance en routine clinique par NGS ciblé réalisé sur
biopsie solide et liquide (article n°3). Enfin, nous travaillons actuellement sur le rôle de l'HGF en tant que biomarqueur de réponse aux ITK MET et proposons une analyse par IHC de l'HGF sur tumeur (Fernandes et al.).

### 7.3.1 Place des différents outils de biologie cellulaire et moléculaire dans la détection des altérations moléculaires en oncologie

Nos travaux sur les mutations METex14 et les fusions de MET soulignent la difficulté d'identifier certaines cibles thérapeutiques. La première étape indispensable précédant la mise en place d'une thérapie ciblée consiste à identifier les patients porteurs d'une altération moléculaire actionnable. Historiquement, la détection de ces altérations était effectuée selon une stratégie de test d'un gène unique. Plusieurs outils sont disponibles (dont certains encore largement utilisés) tels que la technique de Sanger, la RT-PCR (réaction de polymérisation en chaîne par transcription inverse) ou la FISH (Sabari et al. 2017). La technique de Sanger a été l'une des premières méthodes de détection des mutations dans le cancer du poumon, dont, notamment, les mutations des gènes EGFR et KRAS. La RT-PCR et la FISH sont utilisées afin de détecter des fusions de gènes.

L'arrivée des ITK en oncologie coïncide avec une révolution en biologie moléculaire : le séquençage à haut débit ou de nouvelle génération (NGS). Contrairement à la technique de Sanger, le NGS permet la lecture en parallèle de milliers de brins d'ADN. Ce progrès technologique a, notamment, réduit la vitesse de séquençage du génome humain initialement de quinze ans à une journée (Sabari et al. 2017). Il existe trois techniques différentes : le séquençage ciblé, le séquençage des régions codantes (Whole exome sequencing) et le séquençage du génome entier (Whole genome sequencing). À ce jour, le séquençage d'un panel de gènes est la technique la plus couramment employée en routine clinique pour des raisons de cout et de rapidité d'interprétation.

En cancérologie pulmonaire, le NGS a révolutionné la détection des mutations. En 2014, le TCGA (the cancer genome atlas) a publié les profils moléculaires de 230 adénocarcinomes pulmonaires obtenus par Whole exome sequencing. Le NGS a permis de détecter des mutations qui n'étaient pas retrouvées par les techniques anciennes. En 2015, une étude rétrospective effectuée sur des patients porteurs d'un adénocarcinome, peu ou non fumeurs
et ne présentant pas d'altération actionnable après analyse par les techniques antérieures au
NGS, a permis d'observer que le séquençage à haut débit d'un panel conduisait à identifier
65 % des patients avec des altérations moléculaires actionnables (Drilon et al. 2015).

Comme toute technique, le NGS présente des limites. Le WES et le WGS sont des techniques
couteuses et difficiles d'interprétation compte tenu des données produites et du nombre de
variants identifiés (Kamps et al. 2017). Le séquençage d'un panel de gènes, quant à lui, est
limité par rapport aux cibles que l'on souhaite analyser. Dans le cas de nos travaux de
recherche, nous avons été confronté à la problématique que pose la détection des mutations
METex14 car, contrairement aux mutations *EGFR*, ces dernières sont nombreuses (plus d'une
centaine ont été décrites) et hétérogènes (délétions, insertions et substitutions). De plus, elles
surviennent dans les introns, qui sont des régions habituellement non couvertes par les panels
de NGS classiques. L'utilisation du panel *Colon and Lung* (largement utilisé en routine clinique),
ne permettait d'identifier que 0,2 % des cas nécessitant d'optimiser la technique. En
développant un panel spécifique, couplé à une analyse des fragments longs, nous avons pu
obtenir un taux de détection des mutations METex14 dans les CBNPC de 2,2 % (Annexe 2)
(Descarpentries et al. 2018).

Une autre problématique repose sur les fusions de gènes. Au cours des dernières décennies,
le nombre de fusions impliquant des oncogènes identifiés n'a cessé de croître. L'arrivée de
thérapies efficaces contre ces altérations a rendu indispensable leur recherche en routine
clinique. Au-delà des fusions bien connues comme celles impliquant ALK ou ROS1, on peut par
exemple citer les fusions NTRK, NRG1, RET. Concernant le récepteur MET, le nombre de
fusions de *MET* détectées augmente dans de nombreux cancers et l'utilisation d'un ITK MET
semble efficace en présence de cette altération (article n°2). Néanmoins, les techniques
actuelles de détection des fusions utilisées en routine clinique reposent essentiellement sur
la FISH. La recherche de l'ensemble des fusions par FISH est chronophage, car cette technique
permet l'analyse d'un seul gène. Afin de pallier cette problématique, de nouvelles techniques
de NGS basées sur la détection d'ARN ont été développés et sont disponibles
commercialement (Schram et al. 2017). Ces techniques, sensibles et permettant de tester
plusieurs échantillons en même temps, se heurtent néanmoins aux problèmes liés à la qualité
des échantillons et au coût.
### 7.3.2 Enjeu et défi de la détection des mécanismes de résistances en routine clinique

La détection des mécanismes de résistance est un enjeu majeur de la médecine de précision. Toutefois, cette détection est délicate car elle impose de disposer de matériel tumoral suffisant et dépend du mécanisme recherché.

L'accessibilité du matériel tumoral concerne essentiellement la recherche de mécanismes acquis. Elle suppose que le site de progression soit accessible à une biopsie et que le patient présente un état général compatible avec la réalisation de ce geste invasif. En effet, une étude menée par le groupe français de pneumo-cancérologie (GFPC) a montré que sur 100 patients pour lesquels une biopsie était proposée, 18 % n'étaient pas éligibles à sa réalisation. De plus, sur l'ensemble des biopsies effectuées, 34 % n'étaient pas contributives (Chouaid et al. 2014). Une alternative, que nous présentons dans les articles n°2 et n°3, consiste à utiliser de la biopsie liquide. Cette dernière regroupe les analyses des cellules tumorales circulantes (CTC), de l'ADN tumoral circulant (ADNc), des miARN circulants et des exosomes (petites vésicules extracellulaires). La biopsie liquide offre plusieurs avantages : peu invasive, ce qui implique une notion de répétabilité, elle peut être utilisée pour suivre une réponse sous traitement (Lianidou et Pantel 2019) et permet, enfin, de mieux refléter l'hétérogénéité tumorale et de mieux identifier le mécanisme de résistance. Par ailleurs, cette technique est recommandée dans la détection des mutations *EGFR*. Néanmoins, l'utilisation de la biopsie liquide comporte une limite importante : une sensibilité plus faible que celle de la biopsie tissulaire dans la détection des mutations, généralement entre 40 et 70 % selon les techniques. En outre, les biopsies liquides ne permettent pas de détecter l'ensemble des mécanismes de résistance, comme les amplifications et les transformations histologiques (Chang et al. 2020).

L'autre point important concerne le type de mécanisme recherché. Dans l'article n°1, nous présentons la perte d'expression de PTEN comme mécanisme de résistance. Nous avons défini un seuil de négativité lorsque 0 % des cellules exprime la protéine. La mesure de l'expression de PTEN est bien codifiée et de nombreux anticorps sont disponibles pour l'analyse. Toutefois, l'analyse de l'expression de PTEN n'est pas réalisée en routine dans tous les centres, ce qui limite la détection de ce mécanisme. De surcroît, le seuil de négativité peut varier, certaines équipes incluant une expression faible de la protéine < 10 % comme un résultat négatif (Lotan
et al. 2011). Il convient alors de bien standardiser les techniques afin d'offrir une meilleure reproductibilité.

### 7.3.3 Impact de la détection de co-altérations sur le choix thérapeutique

Comme nous l'avons décrit précédemment, l'utilisation de traitements capables de surmonter les mécanismes de résistance a considérablement modifié la prise en charge des patients porteurs de tumeurs avec une addiction oncogénique. De plus, des stratégies visant à surmonter les résistances avant que ces dernières n'émergent semblent offrir des résultats supérieurs aux stratégies développées initialement.

La plupart des travaux ayant étudié des co-altérations ont établi des stratégies thérapeutiques dont le plus grand nombre repose sur l'utilisation de combinaisons de traitement afin de bloquer les différentes altérations. Bien que les résultats obtenus soient prometteurs, en pratique clinique aucune combinaison n'est actuellement recommandée.

L'une des raisons principales tient au contraste observé entre les données obtenues sur des modèles précliniques et les données de vie réelle. En prenant pour exemple la co-altération de la voie RAS identifiée chez des patients porteurs de CBNPC METex14, des données publiées par Guo et al. n'ont pas mis en évidence de différence de survie et de taux de réponse selon le statut RAS de la tumeur chez des patients traités par ITK MET. Ce contraste entre les données cliniques et fondamentales repose essentiellement sur l'hétérogénéité intratumorale définie par la présence de plusieurs populations cellulaires dont les anomalies moléculaires diffèrent au sein d'une même tumeur (Hinohara et Polyak 2019). En effet, il n'est pas rare d'observer la présence d'une co-altération sur une fraction de la tumeur analysée, suggérant qu'une partie de la tumeur ne porte pas de co-altération susceptible de conférer une résistance.

## 7.4 Conclusion

En conclusion, ce travail de thèse a permis d'identifier une nouvelle voie de résistance aux ITK MET dans les CBNPC METex14 et de proposer une stratégie thérapeutique afin de surmonter ce mécanisme. Ce travail de thèse a également offert l'opportunité de confirmer la fusion de
MET comme une cible potentielle et d'identifier la mutation kinase de MET comme mécanisme de résistance acquise, dans un autre modèle d'addiction à MET. Enfin, il a donné la possibilité d'évaluer la faisabilité et la pertinence de la recherche de mutations de résistance dans les CBNPC ALK+. Ces résultats illustrent la nécessité de poursuivre dans la recherche des mécanismes de résistance afin de proposer des stratégies thérapeutiques innovantes.
Abella, Jasmine V, Pascal Peschard, Monica A Naujokas, Tong Lin, Caroline Saucier, Sylvie Urbé, and Morag Park. 2005.
"Met/Hepatocyte Growth Factor Receptor Ubiquitination Suppresses Transformation and Is Required for Hrs
Phosphorylation." *Molecular and Cellular Biology* 25 (21): 9632-45. https://doi.org/10.1128/MCB.25.21.9632-9645.2005.

Aisner, Dara L., Lynette M. Sholl, Lynne D. Berry, Michael R. Rossi, Heidi Chen, Junya Fujimoto, Andre L. Moreira, et al. 2018.
"The Impact of Smoking and TP53 Mutations in Lung Adenocarcinoma Patients with Targetable Mutations—The Lung
Cancer Mutation Consortium (LCMC2)." *Clinical Cancer Research* 24 (5): 1038–47. https://doi.org/10.1158/1078-0432.CCR-17-2289.

Ancot, Frédéric, Catherine Leroy, Ghaffar Muharram, Jonathan Lefebvre, Jérôme Vicogne, Arnaud Lemiere, Zoulika Kherrouche, et al. 2012. "Shedding-Generated Met Receptor Fragments Can Be Routed to Either the Proteasomal or the Lysosomal Degradation Pathway." *Traffic* 13 (9): 1261–72. https://doi.org/10.1111/j.1600-0854.2012.01384.x.

Andermarcher, E., M. A. Surani, and E. Gherardi. 1996. "Co-Expression of the HGF/SF and c-Met Genes during Early Mouse Embryogenesis Precedes Reciprocal Expression in Adjacent Tissues during Organogenesis." *Developmental Genetics* 18 (3): 254–66. https://doi.org/10.1002/(SICI)1520-6408(1996)18:3<254::AID-DVG6>3.0.CO;2-8.

Asses, Yasmine, Vincent Leroux, Safia Tairi-Kellou, Rosanna Dono, Flavio Maina, and Bernard Maigret. 2009. "Analysis of C-Met Kinase Domain Complexes: A New Specific Catalytic Site Receptor Model for Defining Binding Modes of Atp-Competitive Ligands." *Chemical Biology and Drug Design* 74 (6): 560–70. https://doi.org/10.1111/j.1747-0285.2009.00895.x.

Awad, Mark M., Magda Bahcall, Lynette M. Sholl, Frederick Hugh Wilson, Cloud Paweletz, Marzia Capelletti, Giulia Costanza Leonardi, et al. 2018. "Mechanisms of Acquired Resistance to MET Tyrosine Kinase Inhibitors (TKIs) in MET Exon 14 (METex14) Mutant Non-Small Cell Lung Cancer (NSCLC)." *Journal of Clinical Oncology* 36 (15_suppl): 9069–9069. https://doi.org/10.1200/JCO.2018.36.15_suppl.9069.

Awad, Mark M., Jessica Kim Lee, Russell Madison, Anthony Classon, Jamie Kmak, Garrett M. Frampton, Brian Michael Alexander, Jeffrey Venstrom, and Alexa Betzig Schrock. 2020. "Characterization of 1,387 NSCLCs with MET Exon 14 (METex14) Skipping Alterations (SA) and Potential Acquired Resistance (AR) Mechanisms." *Journal of Clinical Oncology* 38 (15_suppl): 9511–9511. https://doi.org/10.1200/jco.2020.38.15_suppl.9511.

Awad, Mark M, Geoffrey R Oxnard, David M Jackman, Daniel O Savukoski, Dimity Hall, Priyanka Shivdasani, Jennifer C Heng, et al. 2016. "MET Exon 14 Mutations in Non-Small-Cell Lung Cancer Are Associated With Advanced Age and Stage-Dependent MET Genomic Amplification and c-Met Overexpression." *Journal of Clinical Oncology* 34 (7): JCO.2015.63.4600-. https://doi.org/10.1200/JCO.2015.63.4600.

Ayati, Adileh, Setareh Moghimi, Somayeh Salarinejad, Maliheh Safavi, Behjat Pouramiri, and Alireza Foroumadi. 2020. "A Review on Progression of Epidermal Growth Factor Receptor (EGFR) Inhibitors as an Efficient Approach in Cancer Targeted Therapy." *Bioorganic Chemistry*. Academic Press Inc. https://doi.org/10.1016/j.bioorg.2020.103811.

Bahcall, Magda, Mark M. Awad, Lynette M. Sholl, Frederick H Wilson, Man Xu, Stephen Wang, Sangeetha Palakurthi, et al. 2018. "Amplification of Wild Type KRAS Imparts Resistance to Crizotinib in MET Exon 14 Mutant Non-Small Cell Lung Cancer." *Clinical Cancer Research*, August, clincanres.0876.2018. https://doi.org/10.1158/1078-0432.CCR-18-0876.

Baldacci, Simon, Zoulika Kherrouche, Vincent Cockenpot, Luc Stoven, Marie Christine Copin, Elisabeth Werkmeister, Nathalie Marchand, Maéva Kyheng, David Tulasne, and Alexis B. Cortot. 2018. "MET Amplification Increases the Metastatic Spread of EGFR-Mutated NSCLC." *Lung Cancer* 125 (November): 57–67.
https://doi.org/10.1016/j.lungcan.2018.09.008.

Baltschukat, Sabrina, Barbara Schacher Engstler, Alan Huang, Huai-Xiang Hao, Angela Tam, Hui Qin Wang, Jinsheng Liang, et al. 2019. "Capmatinib (INC280) Is Active Against Models of Non-Small Cell Lung Cancer and Other Cancer Types with Defined Mechanisms of MET Activation." *Clinical Cancer Research*, January, clincanres.2814.2018.
https://doi.org/10.1158/1078-0432.CCR-18-2814.

Bao, Zhao Shi, Hui Min Chen, Ming Yu Yang, Chuan Bao Zhang, Kai Yu, Wan Lu Ye, Bo Qiang Hu, et al. 2014. "RNA-Seq of 272 Gliomas Revealed a Novel, Recurrent PTPRZ1-MET Fusion Transcript in Secondary Glioblastomas." *Genome Research* 24 (11): 1765-73. https://doi.org/10.1101/gr.165126.113.

Barrueto, Luisa, Francheska Caminero, Lindsay Cash, Courtney Makris, Purushottam Lamichhane, and Rahul R. Deshmukh. 2020. "Resistance to Checkpoint Inhibition in Cancer Immunotherapy." *Translational Oncology*. Neoplasia Press, Inc. https://doi.org/10.1016/j.tranon.2019.12.010.

Bent, Martin van den, Analia Azaro, Filip Vos, Juan Sepulveda, W K Alfred Yung, Patrick Wen, Andrew Lassman, et al. 2017. "ACTR-74. A PHASE IB/II, OPEN-LABEL, MULTICENTER STUDY OF CAPMATINIB (INC280) ALONE AND IN COMBINATION WITH BUPARLISIB (BKM120) IN ADULT PATIENTS WITH RECURRENT GLIOBLASTOMA." *Neuro-Oncology* 19 (suppl_6): vi16-17. https://doi.org/10.1093/neuonc/nox168.061.

Birchmeier, Carmen, Walter Birchmeier, Ermanno Gherardi, and George F Vande Woude. 2003. "Met, Metastasis, Motility and More." *Nature Reviews. Molecular Cell Biology* 4 (12): 915-25. https://doi.org/10.1038/nrm1261.

Blanc-Durand, Félix, Raafat Alameddine, Anthony J Iafrate, Danh Tran-Thanh, Ying-Chun Lo, Normand Blais, Bertrand Routy, et al. 2020. "Tepotinib Efficacy in an NSCLC Patient with Brain Metastasis Harboring an HLA-DRB1-MET Gene Fusion." *The Oncologist*, July. https://doi.org/10.1634/theoncologist.2020-0502.

Bleeker, F E, L Felicioni, F Buttitta, S Lamba, L Cardone, M Rodolfo, A Scarpa, et al. 2008. "AKT1E17K in Human Solid Tumours." *Oncogene* 27 (42): 5648-50. https://doi.org/10.1038/onc.2008.170.

Boccaccio, Carla, Margherita Andò, Luca Tamagnone, Alberto Bardelli, Paolo Michieli, Carlo Battistini, and Paolo M. Comoglio. 1998. "Induction of Epithelial Tubules by Growth Factor HGF Depends on the STAT Pathway." *Nature* 391 (6664): 285-88. https://doi.org/10.1038/34657.

Bononi, Angela, and Paolo Pinton. 2015. "Study of PTEN Subcellular Localization." *Methods (San Diego, Calif.)* 77-78 (May): 92-103. https://doi.org/10.1016/j.ymeth.2014.10.002.

Bos, Johannes L., Holger Rehmann, and Alfred Wittinghofer. 2007. "GEFs and GAPs: Critical Elements in the Control of Small G Proteins." *Cell* 129 (5): 865-77. https://doi.org/10.1016/J CELL.2007.05.018.

Boumber, Yanis. 2018. "Tumor Mutational Burden (TMB) as a Biomarker of Response to Immunotherapy in Small Cell Lung Cancer." *Journal of Thoracic Disease*. AME Publishing Company. https://doi.org/10.21037/jtd.2018.07.120.

Bussolino, F., M. F. Di Renzo, M. Ziche, E. Bocchietto, M. Olivero, L. Naldini, G. Gaudino, L. Tamagnone, A. Coffer, and P. M. Comoglio. 1992. "Hepatocyte Growth Factor Is a Potent Angiogenic Factor Which Stimulates Endothelial Cell Motility and Growth." *Journal of Cell Biology* 119 (3): 629-41. https://doi.org/10.1083/jcb.119.3.629.

Calvi, Carla, Megan Podowski, Armando Lopez-Mercado, Shana Metzger, Kaori Misono, Alla Malinina, Dustin Dikeman, et al. 2013. "Hepatocyte Growth Factor, a Determinant of Airspace Homeostasis in the Murine Lung." Edited by Steven D. Shapiro. *PLoS Genetics* 9 (2): e1003228. https://doi.org/10.1371/journal.pgen.1003228.

Camidge, D. Ross, Fabrice Barlesi, Jonathan Wade Goldman, Daniel Morgensztern, Rebecca Suk Heist, Everett E. Vokes, Alexander I. Spira, et al. 2019. "Results of the Phase 1b Study of ABBV-399 (Telisotuzumab Vedotin; Teliso-v) in Combination with Erlotinib in Patients with c-Met+ Non-Small Cell Lung Cancer by EGFR Mutation Status." *Journal of Clinical Oncology* 37 (15_suppl): 3011-3011. https://doi.org/10.1200/jco.2019.37.15_suppl.3011.

Camidge, D. Ross, Hye Ryun Kim, Myung-Ju Ahn, James Chih-Hsin Yang, Ji-Youn Han, Jong-Seok Lee, Maximilian J. Hochmair,
et al. 2018. " Brigatinib versus Crizotinib in ALK-Positive Non-Small-Cell Lung Cancer. " *New England Journal of Medicine* 379 (21): 2027-39. https://doi.org/10.1056/nejmoa1810171.

Camidge, D. Ross, Sai-Hong Ignatius Ou, Geoffrey Shapiro, Gregory Alan Otterson, Liza Cosca Villaruz, Miguel Angel Villalona-Calero, A. John lafrate, et al. 2014. " Efficacy and Safety of Crizotinib in Patients with Advanced C-MET - Amplified Non-Small Cell Lung Cancer (NSCLC). " *Journal of Clinical Oncology* 32 (15_suppl): 8001-8001.
https://doi.org/10.1200/jco.2014.32.15_suppl.8001.

Camidge, D Ross, Yung-Jue Bang, Eunice L Kwak, A John lafrate, Marileila Varella-Garcia, Stephen B Fox, Gregory J Riely, et al. 2012. "Activity and Safety of Crizotinib in Patients with ALK-Positive Non-Small-Cell Lung Cancer: Updated Results from a Phase 1 Study." *The Lancet Oncology* 13 (10): 1011-19. https://doi.org/10.1016/S1470-2045(12)70344-3.

Castellano, Esther, and Julian Downward. 2011. "RAS Interaction with PI3K: More Than Just Another Effector Pathway." *Genes & Cancer* 2 (3): 261-74. https://doi.org/10.1177/1947601911408079.

Chang, Sunhee, Jae Young Hur, Yoon La Choi, Chang Hun Lee, and Wan Seop Kim. 2020. "Current Status and Future Perspectives of Liquid Biopsy in Non-Small Cell Lung Cancer." *Journal of Pathology and Translational Medicine* 54 (3): 204-12. https://doi.org/10.4132/jptm.2020.02.27.

"Chemotherapy and Supportive Care versus Supportive Care Alone for Advanced Non-Small Cell Lung Cancer." 2010.
*Cochrane Database of Systematic Reviews*, May. https://doi.org/10.1002/14651858.cd007309.pub2.

Chen, Hui Min, Kai Yu, Xiao Yan Tang, Zhao Shi Bao, Tao Jiang, Xiao Long Fan, Xiao Wei Chen, and Xiao Dong Su. 2015.
"Enhanced Expression and Phosphorylation of the MET Oncoprotein by Glioma-Specific PTPRZ1-MET Fusions." *FEBS Letters* 589 (13): 1437-43. https://doi.org/10.1016/j.febslet.2015.04.032.

Chen, Ya Wen, Zhenghong Xu, Adrienne N.H. Baksh, Jehng Kang Wang, Chiu Yuan Chen, Richard Swanson, Steve T. Olson, Hiroaki Kataoka, Michael D. Johnson, and Chen Yong Lin. 2013. "Antithrombin Regulates Matriptase Activity Involved in Plasmin Generation, Syndecan Shedding, and HGF Activation in Keratinocytes." *PLoS ONE* 8 (5).
https://doi.org/10.1371/journal.pone.0062826.

Chiarle, Roberto, Claudia Voena, Chiara Ambrogio, Roberto Piva, and Giorgio Inghirami. 2008. "The Anaplastic Lymphoma Kinase in the Pathogenesis of Cancer." *Nature Reviews Cancer* 8 (1): 11-23. https://doi.org/10.1038/nrc2291.

Cho, Byoung Chul, Alexander E. Drilon, Robert Charles Doebele, Dong-Wan Kim, Jessica Jiyeong Lin, Jeeyun Lee, Myung-Ju Ahn, et al. 2019. " Safety and Preliminary Clinical Activity of Repotrectinib in Patients with Advanced ROS1 Fusion-Positive Non-Small Cell Lung Cancer (TRIDENT-1 Study). ." *Journal of Clinical Oncology* 37 (15_suppl): 9011-9011.
https://doi.org/10.1200/jco.2019.37.15_suppl.9011.

Cho, Byoung Chul, Sun Min Lim, Hye Ryun Kim, Jong Seok Lee, Ki Hyeong Lee, Yun Gyoo Lee, Young Joo Min, et al. 2017.
"Open-Label, Multicenter, Phase II Study of Ceritinib in Patients with Non-Small-Cell Lung Cancer Harboring ROS1 Rearrangement." *Journal of Clinical Oncology* 35 (23): 2613-18. https://doi.org/10.1200/JCO.2016.71.3701.

Choi, Young Lim, Manabu Soda, Yoshihiro Yamashita, Toshihide Ueno, Junpei Takashima, Takahiro Nakajima, Yasushi Yatabe, et al. 2010. "EML4-ALK Mutations in Lung Cancer That Confer Resistance to ALK Inhibitors." *New England Journal of Medicine* 363 (18): 1734-39. https://doi.org/10.1056/NEJMoa1007478.

Chouaid, Christos, Cecile Dujon, Pascal Do, Isabelle Monnet, Anne Madroszyk, Herve Le Caer, Jean Bernard Auliac, et al. 2014. "Feasibility and Clinical Impact of Re-Biopsy in Advanced Non-Small-Cell Lung Cancer: A Prospective Multicenter Study in a Real-World Setting (GFPC Study 12-01)." *Lung Cancer* 86 (2): 170-73.
https://doi.org/10.1016/j.lungcan.2014.08.016.

Chowdhury, Tamrin, Yeajina Lee, Sojin Kim, Hyeon Jong Yu, So Young Ji, Jeong Mo Bae, Jae Kyung Won, et al. 2020. "A Glioneuronal Tumor with CLIP2-MET Fusion." *Npj Genomic Medicine* 5 (1): 1-7. https://doi.org/10.1038/s41525-020-0131-6.
Cocco, Emiliano, Maurizio Scaltriti, and Alexander Drilon. 2018. "NTRK Fusion-Positive Cancers and TRK Inhibitor Therapy." *Nature Reviews Clinical Oncology*. Nature Publishing Group. https://doi.org/10.1038/s41571-018-0113-0.

Cooper, Colin S., Morag Park, Donald G. Blair, Michael A. Tainsky, Kay Huebner, Carlo M. Croce, and George F. Vande Woude. 1984. "Molecular Cloning of a New Transforming Gene from a Chemically Transformed Human Cell Line." *Nature* 311 (5981): 29-33. https://doi.org/10.1038/311029a0.

Cortot, A. B., and P. A. Janne. 2014a. "Molecular Mechanisms of Resistance in Epidermal Growth Factor Receptor-Mutant Lung Adenocarcinomas." *European Respiratory Review* 23 (133): 356-66. https://doi.org/10.1183/09059180.00004614.

— —. 2014b. "Molecular Mechanisms of Resistance in Epidermal Growth Factor Receptor-Mutant Lung Adenocarcinomas." *European Respiratory Review* 23 (133): 356-66. https://doi.org/10.1183/09059180.00004614.

Cortot, Alexis B., Zoulika Kherrouche, Clotilde Descarp entries, Marie Wislez, Simon Baldacci, Alessandro Furlan, and David Tulasne. 2017. "Exon 14 Deleted MET Receptor as a New Biomarker and Target in Cancers." *JNCI: Journal of the National Cancer Institute* 109 (5). https://doi.org/10.1093/jnci/djw262.

Cramer, Alexander, Sandra Kleiner, Martin Westermann, Anja Meissner, Anika Lange, and Karlheinz Friedrich. 2005. "Activation of the C-Met Receptor Complex in Fibroblasts Drives Invasive Cell Behavior by Signaling through Transcription Factor STAT3." *Journal of Cellular Biochemistry* 95 (4): 805-16. https://doi.org/10.1002/jcb.20459.

Crystal, Adam S., Alice T. Shaw, Lecia V. Sequist, Luc Friboulet, Matthew J. Niederst, Elizabeth L. Lockerman, Rosa L. Frias, et al. 2014. "Patient-Derived Models of Acquired Resistance Can Identify Effective Drug Combinations for Cancer." *Science* 346 (6216): 1480-86. https://doi.org/10.1126/science.1254721.

Cutsem, Eric Van, Boguslawa Karaszewska, Yoon Koo Kang, Hyun Cheol Chung, Veena Shankaran, Salvatore Siena, Ning F. Go, Hui Yang, Marco Schupp, and David Cunningham. 2019. "A Multicenter Phase II Study of AMG 337 in Patients with MET-Amplified Gastric/Gastroesophageal Junction/Esophageal Adenocarcinoma and Other MET-Amplified Solid Tumors." *Clinical Cancer Research* 25 (8): 2414-23. https://doi.org/10.1158/1078-0432.CCR-18-1337.

Dagogo-Jack, I, and A T Shaw. 2016. "Crizotinib Resistance: Implications for Therapeutic Strategies." *Annals of Oncology*: *Official Journal of the European Society for Medical Oncology* 27 Suppl 3 (Suppl 3): iii42-50. https://doi.org/10.1093/annonc/mdw305.

Dagogo-Jack, Ibiyai, Satoshi Yoda, Jochen K. Lennerz, Adam Langenbucher, Jessica J. Lin, Marguerite M. Rooney, Kylie Prutisto-Chang, et al. 2020. "MET Alterations Are a Recurring and Actionable Resistance Mechanism in ALK-Positive Lung Cancer." *Clinical Cancer Research* 26 (11): 2535-45. https://doi.org/10.1158/1078-0432.CCR-19-3906.

Davies, Kurtis D., and Robert C. Doebele. 2013. "Molecular Pathways: ROS1 Fusion Proteins in Cancer." *Clinical Cancer Research* 19 (15): 4040-45. https://doi.org/10.1158/1078-0432.CCR-12-2851.

Davies, Kurtis D., Terry L. Ng, Adriana Estrada-Bernal, Anh T. Le, Peter R. Ennever, D. Ross Camidge, Robert C. Doebele, and Dara L. Aisner. 2017. " Dramatic Response to Crizotinib in a Patient With Lung Cancer Positive for an HLA-DRB1-MET Gene Fusion ." *JCO Precision Oncology* 2017 (1): 1-6. https://doi.org/10.1200/po.17.00117.

Descarp entries, C., F. Leprêtre, F. Escande, Z. Kherrouche, M. Figeac, S. Sebda, S. Baldacci, et al. 2018. "Optimization of Routine Testing for MET Exon 14 Splice Site Mutations in NSCLC Patients." *Journal of Thoracic Oncology* 13 (12). https://doi.org/10.1016/j.jtho.2018.08.2023.

Descarp entries, Clotilde, Frédéric Leprêtre, Fabienne Escande, Zoulika Kherrouche, Martin Figeac, Shéhérazade Sebda, Simon Baldacci, et al. 2018. "Optimization of Routine Testing for MET Exon 14 Splice Site Mutations in Non-Small Cell Lung Cancer Patients." *Journal of Thoracic Oncology*, September. https://doi.org/10.1016/j.jtho.2018.08.2023.

Do, Hongdo, Benjamin Solomon, Paul L Mitchell, Stephen B Fox, and Alexander Dobrovic. 2008. "Detection of the Transforming AKT1 Mutation E17K in Non-Small Cell Lung Cancer by High Resolution Melting." *BMC Research Notes* 1
(1): 14. https://doi.org/10.1186/1756-0500-1-14.

Doebele, Robert C., Amanda B. Pilling, Dara L. Aisner, Tatiana G. Kutateladze, Anh T. Le, Andrew J. Weickhardt, Kimi L. Kondo, et al. 2012. "Mechanisms of Resistance to Crizotinib in Patients with ALK Gene Rearranged Non-Small Cell Lung Cancer." *Clinical Cancer Research* 18 (5). http://clincancerres.aacrjournals.org/content/18/5/1472.long.

Dong, Hua-Jie, Peng Li, Chang-Ling Wu, Xiao-Yue Zhou, Hong-Jun Lu, and Tong Zhou. 2016. "Response and Acquired Resistance to Crizotinib in Chinese Patients with Lung Adenocarcinomas Harboring MET Exon 14 Splicing Alternations." *Lung Cancer* 102 (December): 118-21. https://doi.org/10.1016/j.lungcan.2016.11.006.

Drilon, A., J. Clark, J. Weiss, S. Ou, D.R. Camidge, B. Solomon, G. Otterson, et al. 2018a. "OA12.02 Updated Antitumor Activity of Crizotinib in Patients with MET Exon 14-Altered Advanced Non-Small Cell Lung Cancer." *Journal of Thoracic Oncology* 13 (10): S348. https://doi.org/10.1016/j.jtho.2018.08.300.

— —. 2018b. "OA12.02 Updated Antitumor Activity of Crizotinib in Patients with MET Exon 14-Altered Advanced Non-Small Cell Lung Cancer." *Journal of Thoracic Oncology* 13 (10): S348. https://doi.org/10.1016/j.jtho.2018.08.300.

Drilon, Alexander, Federico Cappuzzo, Sai Hong Ignatius Ou, and D. Ross Camidge. 2017. "Targeting MET in Lung Cancer: Will Expectations Finally Be MET?" *Journal of Thoracic Oncology*. Elsevier Inc. https://doi.org/10.1016/j.jtho.2016.10.014.

Drilon, Alexander, Theodore W. Laetsch, Shivaani Kummar, Steven G. DuBois, Ulrik N. Lassen, George D. Demetri, Michael Nathenson, et al. 2018. "Efficacy of Larotrectinib in TRK Fusion-Positive Cancers in Adults and Children." *New England Journal of Medicine* 378 (8): 731-39. https://doi.org/10.1056/nejmoa1714448.

Drilon, Alexander, Geoffrey R. Oxnard, Daniel S.W. Tan, Herbert H.F. Loong, Melissa Johnson, Justin Gainor, Caroline E. McCoach, et al. 2020. "Efficacy of Selpercatinib in RET Fusion-Positive Non-Small-Cell Lung Cancer." *New England Journal of Medicine* 383 (9): 813-24. https://doi.org/10.1056/nejmoa2005653.

Drilon, Alexander, Natasha Rekhtman, Maria Arcila, Lu Wang, Andy Ni, Melanie Albano, Martine Van Voorthuysen, et al. 2016. "Cabozantinib in Patients with Advanced RET-Rearranged Non-Small-Cell Lung Cancer: An Open-Label, Single-Centre, Phase 2, Single-Arm Trial." *The Lancet Oncology* 17 (12): 1653-60. https://doi.org/10.1016/S1470-2045(16)30562-9.

Drilon, Alexander, Salvatore Siena, Sai Hong Ignatius Ou, Manish Patel, Myung Ju Ahn, Jeeyun Lee, Todd M. Bauer, et al. 2017. "Safety and Antitumor Activity of the Multitargeted Pan-TRK, ROS1, and ALK Inhibitor Entrectinib: Combined Results from Two Phase I Trials (ALKA-372-001 and STARTRK-1)." *Cancer Discovery* 7 (4): 400-409. https://doi.org/10.1158/2159-8290.CD-16-1237.

Drilon, Alexander, Lu Wang, Maria E. Arcila, Sohail Balasubramanian, Joel R. Greenbowe, Jeffrey S. Ross, Phil Stephens, et al. 2015. "Broad, Hybrid Capture-Based next-Generation Sequencing Identifies Actionable Genomic Alterations in Lung Adenocarcinomas Otherwise Negative for Such Alterations by Other Genomic Testing Approaches." *Clinical Cancer Research* 21 (16): 3631-39. https://doi.org/10.1158/1078-0432.CCR-14-2683.

Druker, Brian J., Moshe Talpaz, Debra J. Resta, Bin Peng, Elisabeth Buchdunger, John M. Ford, Nicholas B. Lydon, et al. 2001. "Efficacy and Safety of a Specific Inhibitor of the BCR-ABL Tyrosine Kinase in Chronic Myeloid Leukemia." *New England Journal of Medicine* 344 (14): 1031-37. https://doi.org/10.1056/nejm200104053441401.

Du, Xue, Yun Shao, Hai Feng Qin, Yan Hong Tai, and Hong Jun Gao. 2018. "ALK-Rearrangement in Non-Small-Cell Lung Cancer (NSCLC)." *Thoracic Cancer* 9 (4): 423-30. https://doi.org/10.1111/1759-7714.12613.

Duh, Fuh Mei, Stephen W. Scherer, Lap Chee Tsui, Michael I. Lerman, Berton Zbar, and Laura Schmidt. 1997. "Gene Structure of the Human MET Proto-Oncogene." *Oncogene* 15 (13): 1583-86. https://doi.org/10.1038/sj.onc.1201338.

Duplaquet, Leslie, Catherine Leroy, Audrey Vinchent, Sonia Paget, Jonathan Lefebvre, Fabien Vanden Abeele, Steve Lancel, et al. 2020. "Control of Cell Death/Survival Balance by the Met Dependence Receptor." *ELife* 9 (February).
https://doi.org/10.7554/eLife.50041.

Duruisseaux, Michaël, Benjamin Besse, Jacques Cadranel, Maurice Pérol, Bertrand Mennecier, Laurence Bigay-Game, Renaud Descourt, et al. 2017. "Overall Survival with Crizotinib and Next-Generation ALK Inhibitors in ALK-Positive Non-Small-Cell Lung Cancer (IFCT-1302 CLINALK): A French Nationwide Cohort Retrospective Study." *Oncotarget* 8 (13): 21903-17. https://doi.org/10.18632/oncotarget.15746.

Eifert, Cheryl, and R. Scott Powers. 2012. "From Cancer Genomes to Oncogenic Drivers, Tumour Dependencies and Therapeutic Targets." *Nature Reviews Cancer*. Nat Rev Cancer. https://doi.org/10.1038/nrc3299.

Endoh, Hideki, Yasushi Yatabe, Takayuki Kosaka, Hiroyuki Kuwano, and Tetsuya Mitsudomi. 2006. "PTEN and PIK3CA Expression Is Associated with Prolonged Survival after Gefitinib Treatment in EGFR-Mutated Lung Cancer Patients." *Journal of Thoracic Oncology: Official Publication of the International Association for the Study of Lung Cancer* 1 (7): 629-34. https://doi.org/10.1016/S1556-0864(15)30374-9.

Eng, Juliana, Kaitlin M. Woo, Camelia S. Sima, Andrew Plodkowski, Matthew D. Hellmann, Jamie E. Chaft, Mark G. Kris, Maria E. Arcila, Marc Ladanyi, and Alexander Drilon. 2015. "Impact of Concurrent PIK3CA Mutations on Response to EGFR Tyrosine Kinase Inhibition in EGFR-Mutant Lung Cancers and on Prognosis in Oncogene-Driven Lung Adenocarcinomas." *Journal of Thoracic Oncology* 10 (12): 1713-19. https://doi.org/10.1097/JTO.0000000000000671.

Engelman, J. A., T. Mukohara, K. Zejnullahu, E. Lifshits, A. M. Borras, C.-M. Gale, G. N. Naumov, et al. 2006. "Allelic Dilution Obscures Detection of a Biologically Significant Resistance Mutation in EGFR-Amplified Lung Cancer." *Journal of Clinical Investigation* 116 (10): 2695-2706. https://doi.org/10.1172/JCI28656.

Engelman, Jeffrey A., Ji Luo, and Lewis C. Cantley. 2006. "The Evolution of Phosphatidylinositol 3-Kinases as Regulators of Growth and Metabolism." *Nature Reviews Genetics* 7 (8): 606-19. https://doi.org/10.1038/nrg1879.

Engstrom, Lars D., Ruth Aranda, Matthew Lee, Elizabeth A. Tovar, Curt J. Essenburg, Zachary Madaj, Harrah Chiang, et al. 2017. "Glesatinib Exhibits Antitumor Activity in Lung Cancer Models and Patients Harboring MET Exon 14 Mutations and Overcomes Mutation-Mediated Resistance to Type I MET Inhibitors in Nonclinical Models." *Clinical Cancer Research* 23 (21): 6661-72. https://doi.org/10.1158/1078-0432.CCR-17-1192.

Ettl, Tobias, Sandra Viale-Bouroncle, Matthias G. Hautmann, Martin Gosau, Oliver Kölbl, Torsten E. Reichert, and Christian Morsczeck. 2015. "AKT and MET Signalling Mediates Antiapoptotic Radioresistance in Head Neck Cancer Cell Lines." *Oral Oncology* 51 (2): 158-63. https://doi.org/10.1016/j.oraloncology.2014.11.005.

Fan, Saijun, Yong Xian Ma, Min Gao, Ren-Qi Yuan, Qinghui Meng, Itzhak D. Goldberg, and Eliot M. Rosen. 2001. "The Multisubstrate Adapter Gab1 Regulates Hepatocyte Growth Factor (Scatter Factor)-c-Met Signaling for Cell Survival and DNA Repair." *Molecular and Cellular Biology* 21 (15): 4968-84. https://doi.org/10.1128/mcb.21.15.4968-4984.2001.

Fang, Wenfeng, Yihua Huang, Weiguang Gu, Jiadi Gan, Wenjing Wang, Shiyue Zhang, Kai Wang, et al. 2020. "PI3K-AKT-MTOR Pathway Alterations in Advanced NSCLC Patients after Progression on EGFR-TKI and Clinical Response to EGFR-TKI plus Everolimus Combination Therapy." *Translational Lung Cancer Research* 9 (4): 1258-67. https://doi.org/10.21037/tlcr-20-141.

Felip, Enriqueta, Leora Horn, Jyoti D. Patel, Hiroshi Sakai, Jürgen Scheele, Rolf Bruns, and Paul K. Paik. 2018. "Tepotinib in Patients with Advanced Non-Small Cell Lung Cancer (NSCLC) Harboring *MET* Exon 14-Skipping Mutations: Phase II Trial." *Journal of Clinical Oncology* 36 (15_suppl): 9016-9016. https://doi.org/10.1200/JCO.2018.36.15_suppl.9016.

Felip Font, E., A.T. Shaw, B.J. Solomon, T.M. Bauer, S.-H.I. Ou, S. Gadgeel, R.A. Soo, et al. 2017. "Efficacy and Safety of Lorlatinib in Patients (Pts) with ALK+ Non-Small Cell Lung Cancer (NSCLC) Previously Treated with 2nd-Generation ALK TKIs." *Annals of Oncology* 28 (September): v478-79. https://doi.org/10.1093/annonc/mdx380.045.

Finocchiaro, Giovanna, Luca Toschi, Letizia Gianoncelli, Marina Baretti, and Armando Santoro. 2015. "Prognostic and
Predictive Value of MET Deregulation in Non-Small Cell Lung Cancer." *Annals of Translational Medicine*. AME Publishing Company. https://doi.org/10.3978/j.issn.2305-5839.2015.03.43.

Fixman, Elizabeth D., Tanya M. Fournier, Darren M. Kamikura, Monica A. Naujokas, and Morag Park. 1996. "Pathways Downstream of Shc and Grb2 Are Required for Cell Transformation by the Tpr-Met Oncoprotein." *Journal of Biological Chemistry* 271 (22): 13116-22. https://doi.org/10.1074/jbc.271.22.13116.

Fontana, Diletta, Monica Ceccon, Carlo Gambacorti-Passerini, and Luca Mologni. 2015. "Activity of Second-Generation ALK Inhibitors against Crizotinib-Resistant Mutants in an NPM-ALK Model Compared to EML4-ALK." *Cancer Medicine* 4 (7): 953-65. https://doi.org/10.1002/cam4.413.

Foveau, Bénédicte, Frédéric Ancot, Catherine Leroy, Annalisa Petrelli, Karina Reiss, Valérie Vingtdeux, Silvia Giordano, Véronique Fafeur, and David Tulasne. 2009. "Down-Regulation of the Met Receptor Tyrosine Kinase by Presenilin-Dependent Regulated Intramembrane Proteolysis." *Molecular Biology of the Cell* 20 (9): 2495-2507. https://doi.org/10.1091/mbc.E08-09-0969.

Frampton, Garrett M, Siraj M Ali, Mark Rosenzweig, Juliann Chmielecki, Xinyuan Lu, Todd M Bauer, Mikhail Akimov, et al. 2015. "Activation of MET via Diverse Exon 14 Splicing Alterations Occurs in Multiple Tumor Types and Confers Clinical Sensitivity to MET Inhibitors." *Cancer Discovery* 5 (8): 850-59. https://doi.org/10.1158/2159-8290.CD-15-0285.

Friboulet, Luc, Nanxin Li, Ryohei Katayama, Christian C. Lee, Justin F. Gainor, Adam S. Crystal, Pierre-Yves Michellys, et al. 2014. "The ALK Inhibitor Ceritinib Overcomes Crizotinib Resistance in Non-Small Cell Lung Cancer." *Cancer Discovery* 4 (6). http://cancerdiscovery.aacrjournals.org/content/4/6/662.long.

Fruman, David A., Honyin Chiu, Benjamin D. Hopkins, Shubha Bagrodia, Lewis C. Cantley, and Robert T. Abraham. 2017. "The PI3K Pathway in Human Disease." *Cell* 170 (4): 605-35. https://doi.org/10.1016/j.cell.2017.07.029.

Fujino, Toshio, Yoshihisa Kobayashi, Kenichi Suda, Takamasa Koga, Masaya Nishino, Shuta Ohara, Masato Chiba, et al. 2019. "Sensitivity and Resistance of MET Exon 14 Mutations in Lung Cancer to Eight MET Tyrosine Kinase Inhibitors In Vitro." *Journal of Thoracic Oncology* 14 (10): 1753-65. https://doi.org/10.1016/j.jtho.2019.06.023.

Fukuyama, Ryuichi, Yuuka Ichijoh, Shinsei Minoshima, Naomi Kitamura, and Nobuyoshi Shimizu. 1991. "Regional Localization of the Hepatocyte Growth Factor (HGF) Gene to Human Chromosome 7 Band Q21.1." *Genomics* 11 (2): 410-15. https://doi.org/10.1016/0888-7543(91)90149-9.

Gainor, Justin F., Dae Ho Lee, Giuseppe Curigliano, Robert Charles Doebele, Dong-Wan Kim, Christina S Baik, Daniel Shao-Weng Tan, et al. 2019. "Clinical Activity and Tolerability of BLU-667, a Highly Potent and Selective RET Inhibitor, in Patients (Pts) with Advanced RET-Fusion+ Non-Small Cell Lung Cancer (NSCLC)." *Journal of Clinical Oncology* 37 (15_suppl): 9008-9008. https://doi.org/10.1200/jco.2019.37.15_suppl.9008.

Gainor, Justin F, Leila Dardaei, Satoshi Yoda, Luc Friboulet, Ignaty Leshchiner, Ryohei Katayama, Ibiayi Dagogo-Jack, et al. 2016. "Molecular Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in ALK-Rearranged Lung Cancer." *Cancer Discovery* 6 (10): 1118-33. https://doi.org/10.1158/2159-8290.CD-16-0596.

Gandino, Lucia, Paola Longatis, Enzo Medico, Maria Prat, and Paolo M Comoglioo. 1994. "Phosphorylation of Serine 985 Negatively Regulates the Hepatocyte Growth Factor Receptor Kinase*." *THE JOURNAL OF BIOLAXICAL CHEMISTRY*. Vol. 269.

Garcia-Guzman, Miguel, Fabrizio Dolfi, Karin Zeh, and Kristiina Vuori. 1999. "Met-Induced JNK Activation Is Mediated by the Adapter Protein Crk and Correlates with the Gab1-Crk Signaling Complex Formation." *Oncogene* 18 (54): 7775-86. https://doi.org/10.1038/sj.onc.1203198.

Gettinger, Scott N, Lyudmila A Bazhenova, Corey J Langer, Ravi Salgia, Kathryn A Gold, Rafael Rosell, Alice T Shaw, et al. 2016. "Activity and Safety of Brigatinib in ALK-Rearranged Non-Small-Cell Lung Cancer and Other Malignancies: A Single-Arm, Open-Label, Phase 1/2 Trial." *The Lancet. Oncology* 17 (12): 1683-96. https://doi.org/10.1016/S1470-
2045(16)30392-8.

Ghadjar, Pirus, Wieslawa Blank-Liss, Mathew Simcock, Ivan Hegyi, Karl T. Beer, Holger Moch, Daniel M. Aebersold, and Yitzhak Zimmer. 2009. "MET Y1253D-Activating Point Mutation and Development of Distant Metastasis in Advanced Head and Neck Cancers." *Clinical and Experimental Metastasis* 26 (7): 809–15. https://doi.org/10.1007/s10585-009-9280-9.

Giordano, S., C. Ponzetto, M. F. Di Renzo, C. S. Cooper, and P. M. Comoglio. 1989. "Tyrosine Kinase Receptor Indistinguishable from the C-Met Protein." *Nature* 339 (6220): 155–56. https://doi.org/10.1038/339155a0.

Girard, Nicolas. 2015. "Is Chemotherapy Still an Option in Oncogene-Addicted Non-Small Cell Lung Cancer? No." *Bulletin Du Cancer* 102 (6): S96–99. https://doi.org/10.1016/s0007-4551(15)31225-x.

Girard, Nicolas, Denis Moro-Sibilot, Stéphane Bouée, Corinne Emery, Elodie Torreton, Katell Le Lay, Laura Luciani, Christophe Maritaz, and Christos Chouaid. 2020. "Treatment Sequence of First and Second Generation Tyrosine Kinase Inhibitor Followed by Osimertinib in EGFR-Mutated Non-Small-Cell Lung Cancer: A Real Life Study." *Future Oncology* 16 (16): 1115–24. https://doi.org/10.2217/fon-2020-0084.

Goncalves, Marcus D., Benjamin D. Hopkins, and Lewis C. Cantley. 2018. "Phosphatidylinositol 3-Kinase, Growth Disorders, and Cancer." *New England Journal of Medicine* 379 (21): 2052–62. https://doi.org/10.1056/NEJMra1704560.

Gouji, Toyokawa, Seto Takashi, Takenoyama Mitsuhiro, and Ichinose Yukito. 2014. "Crizotinib Can Overcome Acquired Resistance to CH5424802: Is Amplification of the MET Gene a Key Factor?" *Journal of Thoracic Oncology*. J Thorac Oncol. https://doi.org/10.1097/JTO.0000000000000113.

Gow, Chien Hung, Yi Nan Liu, Huei Ying Li, Min Shu Hsieh, Shih Han Chang, Sheng Ching Luo, Tzu Hsiu Tsai, Pei Lung Chen, Meng Feng Tsai, and Jin Yuan Shih. 2018. "Oncogenic Function of a KIF5B-MET Fusion Variant in Non-Small Cell Lung Cancer." *Neoplasia (United States)* 20 (8): 838–47. https://doi.org/10.1016/j.neo.2018.06.007.

Graveel, Carrie, Yanli Su, Julie Koeman, Ling Mei Wang, Lino Tessarollo, Michele Fiscella, Carmen Birchmeier, Pamela Swiatek, Roderick Bronson, and George Vande Woude. 2004. "Activating Met Mutations Produce Unique Tumor Profiles in Mice with Selective Duplication of the Mutant Allele." *Proceedings of the National Academy of Sciences of the United States of America* 101 (49): 17198–203. https://doi.org/10.1073/pnas.0407651101.

Graziani, Andrea, Danjela Gramaglia, Paolo M.Comoglio. 1991. "The Tyrosine-Phosphorylated Hepatocyte Growth Factor/Scatter Factor Receptor Associates with Phosphatidylinositol 3-Kinase." *THE JOURNAL OF BIOLOGICAL CHEMISTR.*

Graziani, A., D. Gramaglia, P. Dalla Zonca, and P. M. Comoglio. 1993. "Hepatocyte Growth Factor/Scatter Factor Stimulates the Ras-Guanine Nucleotide Exchanger." *Journal of Biological Chemistry* 268 (13): 9165–68. https://europepmc.org/article/MED/8387483.

Gregg, Jeffrey P., Tianhong Li, and Ken Y. Yoneda. 2019. "Molecular Testing Strategies in Non-Small Cell Lung Cancer: Optimizing the Diagnostic Journey." *Translational Lung Cancer Research*. AME Publishing Company. https://doi.org/10.21037/tlcr.2019.04.14.

Guo, Robin, Michael Offin, A. Rose Brannon, Jason C. Chang, Andrew Chow, Lukas Delasos, Jeffrey Girshman, et al. 2020. "MET Exon 14-Altered Lung Cancers and MET Inhibitor Resistance." *Clinical Cancer Research*, November, clincanres.2861.2020. https://doi.org/10.1158/1078-0432.CCR-20-2861.

Guo, Robin, Michael Offin, A. Rose Brannon, Andrew Chow, Lukas Delasos, Romel Somwar, Olivia Wilkins, et al. 2019a. "MET Inhibitor Resistance in Patients with MET Exon 14-Altered Lung Cancers." *Journal of Clinical Oncology* 37 (15_suppl): 9006–9006. https://doi.org/10.1200/jco.2019.37.15_suppl.9006.

Guo, Robin, Michael Offin, A Rose Brannon, Andrew Chow, Lukas Delasos, Romel Somwar, Olivia Wilkins, et al. 2019b. "MET Inhibitor Resistance in Patients with MET Exon 14-Altered Lung Cancers." *Journal of Clinical Oncology* 37 (15_suppl):
9006. https://doi.org/10.1200/JCO.2019.37.15_suppl.9006.

Hamanoue, Masahiro, Kouichi Kawaida, Sonshin Takao, Hisaaki Shimazu, Sumihare Noji, Kunio Matsumoto, and Toshikazu Nakamura. 1992. "Rapid and Marked Induction of Hepatocyte Growth Factor during Liver Regeneration after Ischemic or Crush Injury." *Hepatology* 16 (6): 1485-92. https://doi.org/10.1002/hep.1840160626.

Han, Sen, Jian Fang, Shun Lu, Linfang Wang, Jing Li, Min Cheng, Yongxin Ren, and Weiguo Su. 2019. "Response and Acquired Resistance to Savolitinib in a Patient with Pulmonary Sarcomatoid Carcinoma Harboring <em>MET</em> Exon 14 Skipping Mutation: A Case Report." *OncoTargets and Therapy* Volume 12 (September): 7323-28.
https://doi.org/10.2147/OTT.S210365.

Hashigasako, A., M. Machide, T. Nakamura, K. Matsumoto, and T. Nakamura. 2004. "Bi-Directional Regulation of Ser-985 Phosphorylation of c-Met via Protein Kinase C and Protein Phosphatase 2A Involves c-Met Activation and Cellular Responsiveness to Hepatocyte Growth Factor." *Journal of Biological Chemistry* 279 (25): 26445-52.
https://doi.org/10.1074/jbc.M314254200.

Heist, Rebecca S., Lecia V. Sequist, Darrell Borger, Justin F. Gainor, Ronald S. Arellano, Long P. Le, Dora Dias-Santagata, et al. 2016. "Acquired Resistance to Crizotinib in NSCLC with MET Exon 14 Skipping." *Journal of Thoracic Oncology* 11 (8): 1242-45. https://doi.org/10.1016/j.jtho.2016.06.013.

Hervieu, Alexia, and Stéphanie Kermorgant. 2018. "The Role of PI3K in Met Driven Cancer: A Recap." *Frontiers in Molecular Biosciences* 5: 86. https://doi.org/10.3389/fmolb.2018.00086.

Hida, Toyoaki, Hiroshi Nokihara, Masashi Kondo, Young Hak Kim, Koichi Azuma, Takashi Seto, Yuichi Takiguchi, et al. 2017. "Alectinib versus Crizotinib in Patients with ALK-Positive Non-Small-Cell Lung Cancer (J-ALEX): An Open-Label, Randomised Phase 3 Trial." *Lancet (London, England)* 390 (10089): 29-39. https://doi.org/10.1016/S0140-6736(17)30565-2.

Hida, Toyoaki, Vamsidhar Velcheti, Karen L. Reckamp, Hiroshi Nokihara, Pallavi Sachdev, Tomoki Kubota, Takuya Nakada, Corina E. Dutcus, Min Ren, and Tomohide Tamura. 2019. "A Phase 2 Study of Lenvatinib in Patients with RET Fusion-Positive Lung Adenocarcinoma." *Lung Cancer* 138 (December): 124-30.
https://doi.org/10.1016/j.lungcan.2019.09.011.

Hinohara, Kunihiko, and Kornelia Polyak. 2019. "Intratumoral Heterogeneity: More Than Just Mutations." *Trends in Cell Biology*. Elsevier Ltd. https://doi.org/10.1016/j.tcb.2019.03.003.

Hofman, Paul. 2017. "ALK in Non-Small Cell Lung Cancer (NSCLC) Pathobiology, Epidemiology, Detection from Tumor Tissue and Algorithm Diagnosis in a Daily Practice." *Cancers*. MDPI AG. https://doi.org/10.3390/cancers9080107.

Hong, David S., Marwan G. Fakih, John H. Strickler, Jayesh Desai, Gregory A. Durm, Geoffrey I. Shapiro, Gerald S. Falchook, et al. 2020. "KRAS G12C Inhibition with Sotorasib in Advanced Solid Tumors." *New England Journal of Medicine* 383 (13): 1207-17. https://doi.org/10.1056/nejmoa1917239.

Horiike, Atsushi, Kengo Takeuchi, Takeshi Uenami, Yuko Kawano, Azusa Tanimoto, Kyohei Kaburaki, Yuichi Tambo, et al. 2016. "Sorafenib Treatment for Patients with RET Fusion-Positive Non-Small Cell Lung Cancer." *Lung Cancer* 93 (March): 43-46. https://doi.org/10.1016/j.lungcan.2015.12.011.

Huang, Lihua, and Liwu Fu. 2015. "Mechanisms of Resistance to EGFR Tyrosine Kinase Inhibitors." *Acta Pharmaceutica Sinica B*. Chinese Academy of Medical Sciences. https://doi.org/10.1016/j.apsb.2015.07.001.

Hubbard, Stevan R. 2002. "Protein Tyrosine Kinases: Autoregulation and Small-Molecule Inhibition." *Current Opinion in Structural Biology*. Elsevier Ltd. https://doi.org/10.1016/S0959-440X(02)00383-4.

Hwu, Patrick, and Michael Davies. n.d. "The Role of the PI3K Pathway in Immune Resistance." Accessed November 25, 2020.
https://grantome.com/grant/NIH/R01-CA187076-01.

Igawa, T., K. Matsumoto, S. Kanda, Y. Saito, and T. Nakamura. 1993. "Hepatocyte Growth Factor May Function as a
Renotropic Factor for Regeneration in Rats with Acute Renal Injury." *American Journal of Physiology - Renal Fluid and Electrolyte Physiology* 265 (1 34-1). https://doi.org/10.1152/ajprenal.1993.265.1.f61.

"IMpower132: Efficacy of Atezolizumab (Atezo) + Carboplatin (Carbo)/Cisplatin (Cis) + Pemetrexed (Pem) as 1L Treatment in Key Subgroups with Stage I... | OncologyPRO." n.d. Accessed December 10, 2020.

https://oncologypro.esmo.org/meeting-resources/esmo-2018-congress/IMpower132-efficacy-of-atezolizumab-atezo-carboplatin-carbo-cisplatin-cis-pemetrexed-pem-as-1L-treatment-in-key-subgroups-with-stage-IV-non-squamous-non-small-cell-lung-cancer-NSCLC.

Infarinato, N. R., J. H. Park, K. Krytska, H. T. Ryles, R. Sano, K. M. Szigety, Y. Li, et al. 2016. "The ALK/ROS1 Inhibitor PF-06463922 Overcomes Primary Resistance to Crizotinib in ALK-Driven Neuroblastoma." *Cancer Discovery* 6 (1): 96-107. https://doi.org/10.1158/2159-8290.CD-15-1056.

Ishibe, Shuta, Dominique Joly, Zhen Xiang Liu, and Lloyd G. Cantley. 2004a. "Paxillin Serves as an ERK-Regulated Scaffold for Coordinating FAK and Rac Activation in Epithelial Morphogenesis." *Molecular Cell* 16 (2): 257-67.

https://doi.org/10.1016/j.molcel.2004.10.006.

— —. 2004b. "Paxillin Serves as an ERK-Regulated Scaffold for Coordinating FAK and Rac Activation in Epithelial Morphogenesis." *Molecular Cell* 16 (2): 257-67. https://doi.org/10.1016/j.molcel.2004.10.006.

Ishibe, Shuta, Anil Karihaloo, Hong Ma, Junhui Zhang, Arnaud Marlier, Mitchihiro Mitobe, Akashi Togawa, et al. 2009. "Met and the Epidermal Growth Factor Receptor Act Cooperatively to Regulate Final Nephron Number and Maintain Collecting Duct Morphology." *Development* 136 (2): 337-45. https://doi.org/10.1242/dev.024463.

Jardim, Denis L Fontes, Debora De Melo Gagliato, Gerald S. Falchook, Filip Janku, Ralph Zinner, Jennifer J. Wheler, Vivek Subbiah, et al. 2014. "MET Aberrations and C-MET Inhibitors in Patients with Gastric and Esophageal Cancers in a Phase I Unit." *Oncotarget* 5 (7): 1837-45. https://doi.org/10.18632/oncotarget.1828.

Jean, S., and A. A. Kiger. 2014. "Classes of Phosphoinositide 3-Kinases at a Glance." *Journal of Cell Science* 127 (5): 923-28.

https://doi.org/10.1242/jcs.093773.

Jeffers, M, L Schmidt, N Nakaigawa, C P Webb, G Weirich, T Kishida, B Zbar, and G F Vande Woude. 1997. "Activating Mutations for the Met Tyrosine Kinase Receptor in Human Cancer." *Proceedings of the National Academy of Sciences of the United States of America* 94 (21): 11445-50. http://www.ncbi.nlm.nih.gov/pubmed/9326629.

Jin, Wei, Benjie Shan, Hu Liu, Shoubing Zhou, Wenjuan Li, Jing Pan, Lin Lin, Dandan Hu, and Yueyin Pan. 2019. "Acquired Mechanism of Crizotinib Resistance in NSCLC with MET Exon 14 Skipping." *Journal of Thoracic Oncology: Official Publication of the International Association for the Study of Lung Cancer* 14 (7): e137-39.

https://doi.org/10.1016/j.jtho.2019.04.021.

Jing, Changwen, Xuhua Mao, Zhuo Wang, Kejing Sun, Rong Ma, Jianzhong Wu, and Haixia Cao. 2018a. "Next-generation Sequencing-based Detection of EGFR, KRAS, BRAF, NRAS, PIK3CA, Her-2 and TP53 Mutations in Patients with Non-small Cell Lung Cancer." *Molecular Medicine Reports*, June. https://doi.org/10.3892/mmr.2018.9210.

— —. 2018b. "Next-generation Sequencing-based Detection of EGFR, KRAS, BRAF, NRAS, PIK3CA, Her-2 and TP53 Mutations in Patients with Non-small Cell Lung Cancer." *Molecular Medicine Reports* 18 (2): 2191-97.

https://doi.org/10.3892/mmr.2018.9210.

Joffre, Carine, Rachel Barrow, Ludovic Ménard, Véronique Calleja, Ian R. Hart, and Stéphanie Kermorgant. 2011. "A Direct Role for Met Endocytosis in Tumorigenesis." *Nature Cell Biology* 13 (7): 827-37. https://doi.org/10.1038/ncb2257.

Johnson, Alison C., Pascal Dô, Nicolas Richard, Catherine Dubos, Jean Jacques Michels, Jessica Bonneau, and Radj Gervais. 2016. "Identification of I1171N Resistance Mutation in ALK-Positive Non-Small-Cell Lung Cancer Tumor Sample and Circulating Tumor DNA." *Lung Cancer* 99 (September): 38-40. https://doi.org/10.1016/j.lungcan.2016.06.010.

Jung, Kyeong Ah, Bo Hyun Choi, and Mi Kyoung Kwak. 2015. "The C-MET/PI3K Signaling Is Associated with Cancer
Resistance to Doxorubicin and Photodynamic Therapy by Elevating BCRP/ABCG2 Expression." *Molecular Pharmacology* 87 (3): 465-76. https://doi.org/10.1124/mol.114.096065.

Juric, Dejan, Jordi Rodon, Josep Tabernero, Filip Janku, Howard A Burris, Jan H M Schellens, Mark R Middleton, et al. 2018. "Phosphatidylinositol 3-Kinase α-Selective Inhibition With Alpelisib (BYL719) in PIK3CA-Altered Solid Tumors: Results From the First-in-Human Study." *Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology* 36 (13): 1291-99. https://doi.org/10.1200/JCO.2017.72.7107.

Kamps, Rick, Rita Brandão, Bianca Bosch, Aimee Paulussen, Sofia Xanthoulea, Marinus Blok, and Andrea Romano. 2017. "Next-Generation Sequencing in Oncology: Genetic Diagnosis, Risk Prediction and Cancer Classification." *International Journal of Molecular Sciences* 18 (2): 308. https://doi.org/10.3390/ijms18020308.

Kang, Hyo Jae, Hyo Jeong Lim, Jong Sun Park, Young Jae Cho, Ho Il Yoon, Jin Haeng Chung, Jae Ho Lee, and Choon Taek Lee. 2014. "Comparison of Clinical Characteristics between Patients with ALK-Positive and EGFR-Positive Lung Adenocarcinoma." *Respiratory Medicine* 108 (2): 388-94. https://doi.org/10.1016/j.rmed.2013.11.020.

Kannaiyan, Radhamani, and Daruka Mahadevan. 2018. "A Comprehensive Review of Protein Kinase Inhibitors for Cancer Therapy." *Expert Review of Anticancer Therapy*. Taylor and Francis Ltd. https://doi.org/10.1080/14737140.2018.1527688.

Katayama, R., A. T. Shaw, T. M. Khan, M. Mino-Kenudson, B. J. Solomon, B. Halmos, N. A. Jessop, et al. 2012. "Mechanisms of Acquired Crizotinib Resistance in ALK-Rearranged Lung Cancers." *Science Translational Medicine* 4 (120): 120ra17-120ra17. https://doi.org/10.1126/scitranslmed.3003316.

Kawakami, Hisato, Isamu Okamoto, Wataru Okamoto, Junko Tanizaki, Kazuhiko Nakagawa, and Kazuto Nishio. 2014. "Targeting MET Amplification as a New Oncogenic Driver." *Cancers* 6 (3): 1540-52. https://doi.org/10.3390/cancers6031540.

Kermorgant, Stéphanie, and Peter J. Parker. 2008. "Receptor Trafficking Controls Weak Signal Delivery: A Strategy Used by c-Met for STAT3 Nuclear Accumulation." *Journal of Cell Biology* 182 (5): 855-63. https://doi.org/10.1083/jcb.200806076.

Kherrouche, Zoulika, Didier Monte, Elisabeth Werkmeister, Luc Stoven, Yvan De Launit, Alexis B. Cortot, David Tulasne, and Anne Chotteau-Lelievre. 2015. "PEA3 Transcription Factors Are Downstream Effectors of Met Signaling Involved in Migration and Invasiveness of Met-Addicted Tumor Cells." *Molecular Oncology* 9 (9): 1852-67. https://doi.org/10.1016/j.molonc.2015.07.001.

Kim, Dong-Wan, Myung-Ju Ahn, Yuankai Shi, Tommaso Martino De Pas, Pan-Chyr Yang, Gregory J. Riely, Lucio Crinò, et al. 2012. "Results of a Global Phase II Study with Crizotinib in Advanced ALK-Positive Non-Small Cell Lung Cancer (NSCLC)." *Clinical Advances in Hematology and Oncology*. American Society of Clinical Oncology. https://doi.org/10.1200/jco.2012.30.15_suppl.7533.

Kim, Dong-Wan, Marcello Tiseo, Myung-Ju Ahn, Karen L. Reckamp, Karin Holmskov Hansen, Sang-We Kim, Rudolf M. Huber, et al. 2017. "Brigatinib in Patients With Crizotinib-Refractory Anaplastic Lymphoma Kinase-Positive Non-Small-Cell Lung Cancer: A Randomized, Multicenter Phase II Trial." *Journal of Clinical Oncology* 35 (22): 2490-98. https://doi.org/10.1200/JCO.2016.71.5904.

Kim, Soyeon, Tae Min Kim, Dong-Wan Kim, Heounjeong Go, Bhumsuk Keam, Se-Hoon Lee, Ja-Lok Ku, Doo Hyun Chung, and Dae Seog Heo. 2013. "Heterogeneity of Genetic Changes Associated with Acquired Crizotinib Resistance in ALK-Rearranged Lung Cancer." *Journal of Thoracic Oncology* 8 (4): 415-22. https://doi.org/10.1097/JTO.0b013e318283dcc0.

Kirchhofer, Daniel, Mark Peek, Michael T. Lipari, Karen Billeci, Bin Fan, and Paul Moran. 2005. "Hepsin Activates Pro-Hepatocyte Growth Factor and Is Inhibited by Hepatocyte Growth Factor Activator Inhibitor-1B (HAI-1B) and HAI-2."
FEBS Letters 579 (9): 1945-50. https://doi.org/10.1016/j.febslet.2005.01.085.

Klempner, Samuel J., Ali Borghei, Behrooz Hakimian, Siraj M. Ali, and Sai-Hong Ignatius Ou. 2017. "Intracranial Activity of Cabozantinib in MET Exon 14-Positive NSCLC with Brain Metastases." *Journal of Thoracic Oncology* 12 (1): 152-56.
https://doi.org/10.1016/j.jtho.2016.09.127.

Komada, Masayuki, Kiyotaka Hatsuzawa, Sayumi Shibamoto, Fumiaki Ito, Kazuhisa Nakayama, and Naomi Kitamura. 1993. "Proteolytic Processing of the Hepatocyte Growth Factor/Scatter Factor Receptor by Furin." *FEBS Letters* 328 (1-2): 25-29. https://doi.org/10.1016/0014-5793(93)80958-W.

Kong-Beltran, M. 2006. "Somatic Mutations Lead to an Oncogenic Deletion of Met in Lung Cancer." *Cancer Research* 66 (1): 283-89. https://doi.org/10.1158/0008-5472.CAN-05-2749.

Kong-Beltran, Monica, Jennifer Stamos, and Dineli Wickramasinghe. 2004. "The Sema Domain of Met Is Necessary for Receptor Dimerization and Activation." *Cancer Cell* 6 (1): 75-84. https://doi.org/10.1016/j.ccr.2004.06.013.

Kong, Li Ren, Nur Afiqah Binte Mohamed Salleh, Tuan Zea Tan, Dennis Kappei, and Boon Cher Goh. 2017. "P1.02-041 Characterization of MET-N375S as an Activating Mutation in Squamous Cell Carcinoma of the Lung." *Journal of Thoracic Oncology* 12 (1): S512. https://doi.org/10.1016/j.jtho.2016.11.624.

Kosai, Ken Ichiro, Kunio Matsumoto, Shigekazu Nagata, Yoshihide Tsujimoto, and Toshikazu Nakamura. 1998. "Abrogation of Fas-Induced Fulminant Hepatic Failure in Mice by Hepatocyte Growth Factor." *Biochemical and Biophysical Research Communications* 244 (3): 683-90. https://doi.org/10.1006/bbrc.1998.8293.

Kozlov, Guennadi, Audrey Perreault, Joseph D. Schrag, Morag Park, Miroslaw Cygler, Kalle Gehring, and Irena Ekiel. 2004. "Insights into Function of PSI Domains from Structure of the Met Receptor PSI Domain." *Biochemical and Biophysical Research Communications* 321 (1): 234-40. https://doi.org/10.1016/j.bbrc.2004.06.132.

Kris, Mark G., D. R. Camidge, G. Giaccone, T. Hida, B. T. Li, J. O'Connell, I. Taylor, et al. 2015. "Targeting HER2 Aberrations as Actionable Drivers in Lung Cancers: Phase II Trial of the Pan-HER Tyrosine Kinase Inhibitor Dacomitinib in Patients with HER2-Mutant or Amplified Tumors." *Annals of Oncology* 26 (7): 1421-27.
https://doi.org/10.1093/annonc/mdv186.

Kron, A, C Alidousty, M Scheffler, S Merkelbach-Bruse, D Seidel, R Riedel, M A Ihle, et al. 2018. "Impact of TP53 Mutation Status on Systemic Treatment Outcome in ALK-Rearranged Non-Small-Cell Lung Cancer." *Annals of Oncology : Official Journal of the European Society for Medical Oncology* 29 (10): 2068-75. https://doi.org/10.1093/annonc/mdy333.

Lamorte, Louie, Isabelle Royal, Monica Naujokas, and Morag Park. 2002. "Crk Adapter Proteins Promote an Epithelial-Mesenchymal-like Transition and Are Required for HGF-Mediated Cell Spreading and Breakdown of Epithelial Adherens Junctions." *Molecular Biology of the Cell* 13 (5): 1449-61. https://doi.org/10.1091/mbc.01-10-0477.

Landi, Lorenza, Rita Chiari, Marcello Tiseo, Federica D'Incà, Claudio Dazzi, Antonio Chella, Angelo Delmonte, et al. 2019. "Crizotinib in MET Deregulated or ROS1 Rearranged Pretreated Non-Small-Cell Lung Cancer (METROS): A Phase II, Prospective, Multicentre, Two-Arms Trial." *Clinical Cancer Research : An Official Journal of the American Association for Cancer Research*, August, clincanres.0994.2019. https://doi.org/10.1158/1078-0432.CCR-19-0994.

Lantuejoul, Sylvie, Lénaïg Mescam-Mancini, Anne Mcleer-Florin, R É, and M É S U M M. 2012. "Réarrangements Du Gène ALK Dans Les Cancers Du Poumon Non à Petites Cellules ALK Gene Rearrangement in Non-Small-Cell Lung Carcinoma." http://www.edimark.fr/Front/frontpost/getfiles/18975.pdf.

Lee, Jae Ho, San Uk Han, Hyeseong Cho, Barbara Jennings, Bernard Gerrard, Michael Dean, Laura Schmidt, Berton Zbar, and George F. Vande Woude. 2000. "A Novel Germ Line Juxtamembrane Met Mutation in Human Gastric Cancer." *Oncogene* 19 (43): 4947-53. https://doi.org/10.1038/sj.onc.1203874.

Lee, Nayeon, Junghun Lee, Sang Hwan Lee, Subin Kim, and Sunyoung Kim. 2018. "Disproportionately High Levels of HGF Induce the Degradation of the C-Met Receptor through the Proteasomal Degradation Pathway." *Biochemical and
*Biophysical Research Communications* 505 (3): 925-30. https://doi.org/10.1016/j.bbrc.2018.09.127.

Lee, Yu-Ru, Ming Chen, and Pier Paolo Pandolfi. 2018. "The Functions and Regulation of the PTEN Tumour Suppressor: New Modes and Prospects." *Nature Reviews Molecular Cell Biology* 19 (9): 547-62. https://doi.org/10.1038/s41580-018-0015-0.

Lefebvre, J, G Muharram, C Leroy, Z Kherrouche, R Montagne, G Ichim, S Tauszig-Delamasure, et al. 2013. "Caspase-Generated Fragment of the Met Receptor Favors Apoptosis via the Intrinsic Pathway Independently of Its Tyrosine Kinase Activity." *Cell Death and Disease* 4 (10): e871. https://doi.org/10.1038/cddis.2013.377.

Lefebvre, Jonathan, Frédéric Ancot, Catherine Leroy, Ghaffar Muharram, Arnaud Lemière, and David Tulasne. 2012. "Met Degradation: More than One Stone to Shoot a Receptor Down." *The FASEB Journal* 26 (4): 1387-99. https://doi.org/10.1096/fj.11-197723.

Li, An-Na, Jinji Yang, Xu-Chao Zhang, Zhou Zhang, Jian Su, Lan-Ying Gou, Yu Bai, et al. 2017. "Acquired MET Y1248H and D1246N Mutations Mediate Resistance to MET Inhibitors in Non-Small Cell Lung Cancer." *Clinical Cancer Research*, April, clincanres.3273.2016. https://doi.org/10.1158/1078-0432.CCR-16-3273.

Li, Bob T., Ronglai Shen, Darren Buonocore, Zachary T. Olah, Ai Ni, Michelle S. Ginsberg, Gary A. Ulaner, et al. 2018. "Ado-Trastuzumab Emtansine for Patients with HER2-Mutant Lung Cancers: Results from a Phase II Basket Trial." In *Journal of Clinical Oncology*, 36:2532-37. American Society of Clinical Oncology. https://doi.org/10.1200/JCO.2018.77.9777.

Li, D M, and H Sun. 1997. "TEP1, Encoded by a Candidate Tumor Suppressor Locus, Is a Novel Protein Tyrosine Phosphatase Regulated by Transforming Growth Factor Beta." *Cancer Research* 57 (11): 2124-29. http://www.ncbi.nlm.nih.gov/pubmed/9187108.

Li, J, C Yen, D Liaw, K Podsypanina, S Bose, S I Wang, J Puc, et al. 1997. "PTEN, a Putative Protein Tyrosine Phosphatase Gene Mutated in Human Brain, Breast, and Prostate Cancer." *Science (New York, N.Y.)* 275 (5308): 1943-47. http://www.ncbi.nlm.nih.gov/pubmed/9072974.

Liang, Hongge, and Mengzhao Wang. 2020. "MET Oncogene in Non-Small Cell Lung Cancer: Mechanism of MET Dysregulation and Agents Targeting the HGF/c-Met Axis." *OncoTargets and Therapy*. Dove Medical Press Ltd. https://doi.org/10.2147/OTT.S231257.

Lianidou, Evi, and Klaus Pantel. 2019. "Liquid Biopsies." *Genes Chromosomes and Cancer*. Blackwell Publishing Inc. https://doi.org/10.1002/gcc.22695.

Lin, Jenny C., Monica Naujokas, Hong Zhu, Serge Nolet, and Morag Park. 1998. "Intron-Exon Structure of the MET Gene and Cloning of an Alternatively-Spliced Met Isoform Reveals Frequent Exon-Skipping of a Single Large Internal Exon." *Oncogene* 16 (7): 833-42. https://doi.org/10.1038/sj.onc.1201599.

Lin, Jessica J., Viola W. Zhu, Satoshi Yoda, Beow Y. Yeap, Alexa B. Schrock, Ibiayi Dagogo-Jack, Nicholas A. Jessop, et al. 2018. "Impact of EML4-ALK Variant on Resistance Mechanisms and Clinical Outcomes in ALK-Positive Lung Cancer." *Journal of Clinical Oncology* 36 (12): 1199-1206. https://doi.org/10.1200/JCO.2017.76.2294.

Lin, Jessica J, Gregory J Riely, and Alice T Shaw. 2017. "Targeting ALK: Precision Medicine Takes on Drug Resistance." *Cancer Discovery* 7 (2): 137-55. https://doi.org/10.1158/2159-8290.CD-16-1123.

Lin, Jessica J, and Alice T Shaw. n.d. "Resisting Resistance: Targeted Therapies in Lung Cancer." https://doi.org/10.1016/j.trecan.2016.05.010.

Liu, Pingyu, Yijun Wang, and Xin Li. 2019. "Targeting the Untargetable KRAS in Cancer Therapy." *Acta Pharmaceutica Sinica B*. Chinese Academy of Medical Sciences. https://doi.org/10.1016/j.apsb.2019.03.002.

Liu, Qian, Shengnan Yu, Weiheng Zhao, Shuang Qin, Qian Chu, and Kongming Wu. 2018. "EGFR-TKIs Resistance via EGFR-Independent Signaling Pathways." *Molecular Cancer* 17 (1): 53. https://doi.org/10.1186/s12943-018-0793-1.

Liu, Shuying, Funda Meric-Bernstam, Napa Parinyanitikul, Bailiang Wang, Agda K. Eterovic, Xiaofeng Zheng, Mihai Gagea, et
al. 2015. "Functional Consequence of the MET-T1010I Polymorphism in Breast Cancer." *Oncotarget* 6 (5): 2604-14. https://doi.org/10.18632/oncotarget.3094.

Liu, Xuewen, Yuxia Jia, Mark B Stoopler, Yufeng Shen, Haiying Cheng, Jinli Chen, Mahesh Mansukhani, Sanjay Koul, Balazs Halmos, and Alain C Borczuk. 2016. "Next-Generation Sequencing of Pulmonary Sarcomatoid Carcinoma Reveals High Frequency of Actionable MET Gene Mutations." *Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology* 34 (8): 794-802. https://doi.org/10.1200/JCO.2015.62.0674.

Liu, Yao, Qin Zhang, Chuanli Ren, Yanbing Ding, Guangfu Jin, Zhibin Hu, Yaochu Xu, and Hongbing Shen. 2012. "A Germline Variant N375S in MET and Gastric Cancer Susceptibility in a Chinese Population." *Journal of Biomedical Research* 26 (5): 315-18. https://doi.org/10.7555/JBR.26.20110087.

Liu, Yingjie, Nibedita Chattopadhyay, Shan Qin, Charles Szekeres, Tetyana Vasylyeva, Zhen X. Mahoney, Mary Taglienti, et al. 2009. "Coordinate Integrin and C-Met Signaling Regulate Wnt Gene Expression during Epithelial Morphogenesis." *Development* 136 (5): 843-53. https://doi.org/10.1242/dev.027805.

Liu, Youhua. 1999. "Hepatocyte Growth Factor Promotes Renal Epithelial Cell Survival by Dual Mechanisms." *American Journal of Physiology - Renal Physiology* 277 (4 46-4). https://doi.org/10.1152/ajprenal.1999.277.4.f624.

Longati, P., A. Bardelli, C. Ponzetto, L. Naldini, and P. M. Comoglio. 1994. "Tyrosines1234-1235 Are Critical for Activation of the Tyrosine Kinase Encoded by the MET Proto-Oncogene (HGF Receptor)." *Oncogene* 9 (1): 49-57. https://moh-it.pure.elsevier.com/en/publications/tyrosinessup1234-1235sup-are-critical-for-activation-of-the-tyros.

Lotan, Tamara L., Bora Gurel, Siobhan Sutcliffe, David Esopi, Wennuan Liu, Jianfeng Xu, Jessica L. Hicks, et al. 2011. "PTEN Protein Loss by Immunostaining: Analytic Validation and Prognostic Indicator for a High Risk Surgical Cohort of Prostate Cancer Patients." *Clinical Cancer Research* 17 (20): 6563-73. https://doi.org/10.1158/1078-0432.CCR-11-1244.

Lovly, Christine M, Nerina T McDonald, Heidi Chen, Sandra Ortiz-Cuaran, Lukas C Heukamp, Yingjun Yan, Alexandra Florin, et al. 2014. "Rationale for Co-Targeting IGF-1R and ALK in ALK Fusion-Positive Lung Cancer." *Nature Medicine* 20 (9): 1027-34. https://doi.org/10.1038/nm.3667.

Lu, Shun, Jian Fang, Xingya Li, Lejie Cao, Jianying Zhou, Qisen Guo, Zongan Liang, et al. 2020. "Phase II Study of Savolitinib in Patients (Pts) with Pulmonary Sarcomatoid Carcinoma (PSC) and Other Types of Non-Small Cell Lung Cancer (NSCLC) Harboring MET Exon 14 Skipping Mutations (METex14+)." *Journal of Clinical Oncology* 38 (15_suppl): 9519-9519. https://doi.org/10.1200/jco.2020.38.15_suppl.9519.

Ludovini, Vienna, Fortunato Bianconi, Lorenza Pistola, Rita Chiari, Vincenzo Minotti, Renato Colella, Dario Giuffrida, et al. 2011. "Phosphoinositide-3-Kinase Catalytic Alpha and KRAS Mutations Are Important Predictors of Resistance to Therapy with Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Patients with Advanced Non-Small Cell Lung Cancer." *Journal of Thoracic Oncology* 6 (4): 707-15. https://doi.org/10.1097/JTO.0b013e31820a3a6b.

Lynch, Thomas J., Daphne W. Bell, Raffaella Sordella, Sarada Gurubhagavatula, Ross A. Okimoto, Brian W. Brannigan, Patricia L. Harris, et al. 2004. "Activating Mutations in the Epidermal Growth Factor Receptor Underlying Responsiveness of Non-Small-Cell Lung Cancer to Gefitinib." *New England Journal of Medicine* 350 (21): 2129-39. https://doi.org/10.1056/nejmoa040938.

Ma, Patrick C., Gautam Maulik, James Christensen, and Ravi Salgia. 2003. "C-Met: Structure, Functions and Potential for Therapeutic Inhibition." *Cancer and Metastasis Reviews*. Cancer Metastasis Rev. https://doi.org/10.1023/A:1023768811842.

Ma, Patrick C, Ramasamy Jagadeeswaran, Simha Jagadeesh, Maria S Tretiakova, Vidya Nallasura, Edward A Fox, Mark Hansen, et al. 2005. "Functional Expression and Mutations of C-Met and Its Therapeutic Inhibition with SU11274 and Small Interfering RNA in Non-Small Cell Lung Cancer." *Cancer Research* 65 (4): 1479-88.
https://doi.org/10.1158/0008-5472.CAN-04-2650.

Ma, Patrick C, Takashi Kijima, Gautam Maulik, Edward A Fox, Martin Sattler, James D Griffin, Bruce E Johnson, and Ravi Salgia. 2003. "C-MET Mutational Analysis in Small Cell Lung Cancer: Novel Juxtamembrane Domain Mutations Regulating Cytoskeletal Functions." *Cancer Research* 63 (19): 6272-81.
http://www.ncbi.nlm.nih.gov/pubmed/14559814.

Malanga, Donatella, Marianna Scrima, Carmela De Marco, Fernanda Fabiani, Nicola De Rosa, Silvia de Gisi, Natalia Malara, et al. 2008. "Activating E17K Mutation in the Gene Encoding the Protein Kinase AKT in a Subset of Squamous Cell Carcinoma of the Lung." *Cell Cycle* 7 (5): 665-69. https://doi.org/10.4161/cc.7.5.5485.

Maroun, Christiane R., Monica A. Naujokas, and Morag Park. 2003. "Membrane Targeting of Grb2-Associated Binder-1 (Gab1) Scaffolding Protein through Src Myristoylation Sequence Substitutes for Gab1 Pleckstrin Homology Domain and Switches an Epidermal Growth Factor Response to an Invasive Morphogenic Program." *Molecular Biology of the Cell* 14 (4): 1691-1708. https://doi.org/10.1091/mbc.E02-06-0352.

Marquardt, Jens U., Daekwan Seo, Luis E. Gómez-Quiroz, Koichi Uchida, Matthew C. Gillen, Mitsuteru Kitade, Pal Kaposi-Novak, Elizabeth A. Conner, Valentina M. Factor, and Snorri S. Thorgeirsson. 2012. "Loss of C-Met Accelerates Development of Liver Fibrosis in Response to CCI 4 Exposure through Deregulation of Multiple Molecular Pathways." *Biochimica et Biophysica Acta - Molecular Basis of Disease* 1822 (6): 942-51.
https://doi.org/10.1016/j.bbadis.2012.02.012.

Mazieres, J., A. Drilon, A. Lusque, L. Mhanna, A. B. Cortot, L. Mezquita, A. A. Thai, et al. 2019. "Immune Checkpoint Inhibitors for Patients with Advanced Lung Cancer and Oncogenic Driver Alterations: Results from the IMMUNOTARGET Registry." *Annals of Oncology* 30 (8): 1321-28. https://doi.org/10.1093/annonc/mdz167.

McCoach, Caroline E., Anh T. Le, Katherine Gowan, Kenneth Jones, Laura Schubert, Andrea Doak, Adriana Estrada-Bernal, et al. 2018. "Resistance Mechanisms to Targeted Therapies in ROS1⁺ and ALK⁺ Non-Small Cell Lung Cancer." *Clinical Cancer Research*, April. https://doi.org/10.1158/1078-0432.CCR-17-2452.

Mellor, Paul, Levi A Furber, Jennifer N K Nyarko, and Deborah H Anderson. 2012. "Multiple Roles for the P85α Isoform in the Regulation and Function of PI3K Signalling and Receptor Trafficking." *The Biochemical Journal* 441 (1): 23-37.
https://doi.org/10.1042/BJ20111164.

Mezquita, Laura, and David Planchard. 2018. "The Role of Brigatinib in Crizotinib-Resistant Non-Small Cell Lung Cancer." *Cancer Management and Research* 10: 123-30. https://doi.org/10.2147/CMAR.S129963.

Mhanna, Laurent, Nicolas Guibert, Julie Milia, and Julien Mazieres. 2019. "When to Consider Immune Checkpoint Inhibitors in Oncogene-Driven Non-Small Cell Lung Cancer?" *Current Treatment Options in Oncology* 20 (7): 60.
https://doi.org/10.1007/s11864-019-0652-3.

Miled, N., Y. Yan, W.-C. Hon, O. Perisic, M. Zvelebil, Y. Inbar, D. Schneidman-Duhovny, H. J. Wolfson, J. M. Backer, and R. L. Williams. 2007. "Mechanism of Two Classes of Cancer Mutations in the Phosphoinositide 3-Kinase Catalytic Subunit." *Science* 317 (5835): 239-42. https://doi.org/10.1126/science.1135394.

Mills, Jamie N., Alex C. Rutkovsky, and Antonio Giordano. 2018. "Mechanisms of Resistance in Estrogen Receptor Positive Breast Cancer: Overcoming Resistance to Tamoxifen/Aromatase Inhibitors." *Current Opinion in Pharmacology*.
Elsevier Ltd. https://doi.org/10.1016/j.coph.2018.04.009.

Miyamoto, Shingo, Soichiro Ikushima, Ryu Ono, Nobuyasu Awano, Keisuke Kondo, Yoshiaki Furuhata, Kento Fukumoto, and Toshio Kumasaka. 2016. "Transformation to Small-Cell Lung Cancer as a Mechanism of Acquired Resistance to Crizotinib and Alectinib." *Japanese Journal of Clinical Oncology* 46 (2): 170-73. https://doi.org/10.1093/ijco/hyv173.

Miyawaki, Masayoshi, Hiroyuki Yasuda, Tetsuo Tani, Junko Hamamoto, Daisuke Arai, Kota Ishioka, Keiko Ohgino, et al. 2017. "Overcoming EGFR Bypass Signal-Induced Acquired Resistance to ALK Tyrosine Kinase Inhibitors in ALK-Translocated
Lung Cancer." *Molecular Cancer Research* 15 (1): 106-14. https://doi.org/10.1158/1541-7786.MCR-16-0211.

Mohapatra, Bhopal, Gulzar Ahmad, Scott Nadeau, Neha Zutshi, Wei An, Sarah Scheffe, Lin Dong, et al. 2013. "Protein Tyrosine Kinase Regulation by Ubiquitination: Critical Roles of Cbl-Family Ubiquitin Ligases." *Biochimica et Biophysica Acta - Molecular Cell Research*. NIH Public Access. https://doi.org/10.1016/j.bbamcr.2012.10.010.

Mok, Tony S., Yi-Long Wu, Myung-Ju Ahn, Marina C. Garassino, Hye R. Kim, Suresh S. Ramalingam, Frances A. Shepherd, et al. 2017. "Osimertinib or Platinum-Pemetrexed in EGFR T790M-Positive Lung Cancer." *New England Journal of Medicine* 376 (7): 629-40. https://doi.org/10.1056/NEJMoa1612674.

Mok, Tony S., Yi-Long Wu, Sumitra Thongprasert, Chih-Hsin Yang, Da-Tong Chu, Nagahiro Saijo, Patrapim Sunpaweravong, et al. 2009. "Gefitinib or Carboplatin-Paclitaxel in Pulmonary Adenocarcinoma." *New England Journal of Medicine* 361 (10): 947-57. https://doi.org/10.1056/nejmoa0810699.

Mok, Tony, David Spigel, Enriqueta Felip, Filippo deMarinis, Myung-Ju Ahn, Harry J M Groen, Heather A Wakelee, et al. 2015. "ASCEND-2: A Single-Arm, Open-Label, Multicenter Phase II Study of Ceritinib in Adult Patients (Pts) with ALK-Rearranged (ALK+) Non-Small Cell Lung Cancer (NSCLC) Previously Treated with Chemotherapy and Crizotinib (CRZ)." *Journal of Clinical Oncology* 33 (15_suppl): 8059. https://doi.org/10.1200/jco.2015.33.15_suppl.8059.

Moro-Sibilot, D., N. Cozic, M. Pérol, J. Otto, J. Mazieres, P. Souquet, R. Bahleda, et al. 2018a. "OA12.03 Activity of Crizotinib in MET or ROS1 Positive (+) NSCLC: Results of the AcSé Trial." *Journal of Thoracic Oncology* 13 (10): S348. https://doi.org/10.1016/j.jtho.2018.08.301.

— —. 2018b. "OA12.03 Activity of Crizotinib in MET or ROS1 Positive (+) NSCLC: Results of the AcSé Trial." *Journal of Thoracic Oncology* 13 (10): S348. https://doi.org/10.1016/j.jtho.2018.08.301.

Moumen, Anice, Salvatore Patané, Almudena Porras, Rosanna Dono, and Flavio Maina. 2007. "Met Acts on Mdm2 via MTOR to Signal Cell Survival during Development." *Development* 134 (7): 1443-51. https://doi.org/10.1242/dev.02820.

Mukai, Shoichiro, Tsuyoshi Fukushima, Daiji Naka, Hiroyuki Tanaka, Yukio Osada, and Hiroaki Kataoka. 2008. "Activation of Hepatocyte Growth Factor Activator Zymogen (pro-HGFA) by Human Kallikrein 1-Related Peptidases." *FEBS Journal* 275 (5): 1003-17. https://doi.org/10.1111/j.1742-4658.2008.06265.x.

Mur, Emilie Bousquet, Sara Bernardo, Laura Papon, Maicol Mancini, Eric Fabbrizio, Marion Goussard, Irene Ferrer, et al. 2020. "Notch Inhibition Overcomes Resistance to Tyrosine Kinase Inhibitors in EGFR-Driven Lung Adenocarcinoma." *Journal of Clinical Investigation* 130 (2): 612-24. https://doi.org/10.1172/JCI126896.

Nakamura, Toshikazu, Katsuhiko Nawa, and Akira Ichihara. 1984. "Partial Purification and Characterization of Hepatocyte Growth Factor from Serum of Hepatectomized Rats." *Biochemical and Biophysical Research Communications* 122 (3): 1450-59. https://doi.org/10.1016/0006-291X(84)91253-1.

Nakayama, Mizuho, Katsuya Sakai, Atsuko Yamashita, Takahiro Nakamura, Yoshinori Suzuki, and Kunio Matsumoto. 2013. "Met/HGF Receptor Activation Is Regulated by Juxtamembrane Ser985 Phosphorylation in Hepatocytes." *Cytokine* 62 (3): 446-52. https://doi.org/10.1016/j.cyto.2013.04.006.

Naldini, L., L. Tamagnone, E. Vigna, M. Sachs, G. Hartmann, W. Birchmeier, Y. Daikuhara, H. Tsubouchi, F. Blasi, and P.M. Comoglio. 1992. "Extracellular Proteolytic Cleavage by Urokinase Is Required for Activation of Hepatocyte Growth Factor/Scatter Factor." *The EMBO Journal* 11 (13): 4825-33. https://doi.org/10.1002/j.1460-2075.1992.tb05588.x.

Naldini, L., K. M. Weidner, E. Vigna, G. Gaudino, A. Bardelli, C. R.P. Ponzetto Narsimhan, G. Hartmann, et al. 1991. "Scatter Factor and Hepatocyte Growth Factor Are Indistinguishable Ligands for the MET Receptor." *EMBO Journal* 10 (10): 2867-78. https://doi.org/10.1002/j.1460-2075.1991.tb07836.x.

Naldini, L., K.M. Weidner, E. Vigna, G. Gaudino, A. Bardelli, C. Ponzetto, R.P. Narsimhan, G. Hartmann, R. Zarnegar, and G.K. Michalopoulos. 1991. "Scatter Factor and Hepatocyte Growth Factor Are Indistinguishable Ligands for the MET
Receptor.” *The EMBO Journal* 10 (10): 2867–78. https://doi.org/10.1002/j.1460-2075.1991.tb07836.x.

Neel, Dana S., and Trever G. Bivona. 2017. "Resistance Is Futile: Overcoming Resistance to Targeted Therapies in Lung Adenocarcinoma." *Npj Precision Oncology* 1 (1): 3. https://doi.org/10.1038/s41698-017-0007-0.

Ng, Terry L., and D. Ross Camidge. 2016. "Intracranial Efficacy of Crizotinib versus Chemotherapy in PROFILE 1014: Shining a Light on Central Nervous System Endpoints in Clinical Trials." *Translational Cancer Research* 3 (5).
https://doi.org/10.21037/9463.

Niemann, Catherin, Volker Brinkmann, Eva Spitzer, Guido Hartmann, Martin Sachs, Helga Naundorf, and Walter Birchmeier. 1998. "Reconstitution of Mammary Gland Development in Vitro: Requirement of c- Met and c-ErbB2 Signaling for Branching and Alveolar Morphogenesis." *Journal of Cell Biology* 143 (2): 533–45.
https://doi.org/10.1083/jcb.143.2.533.

Nishino, Tomoyoshi, Hiroko Hisha, Naoki Nishino, Masakazu Adachi, and Susumu Ikehara. 1995. "Hepatocyte Growth Factor as a Hematopoietic Regulator." *Blood* 85 (11): 3093–3100.
https://doi.org/10.1182/blood.v85.11.3093.bloodjournal85113093.

Noonan, Sinéad A., Lynne Berry, Xian Lu, Dexiang Gao, Anna E. Barón, Patrick Chesnut, Jamie Sheren, et al. 2016. "Identifying the Appropriate FISH Criteria for Defining MET Copy Number-Driven Lung Adenocarcinoma through Oncogene Overlap Analysis." *Journal of Thoracic Oncology* 11 (8): 1293–1304.
https://doi.org/10.1016/j.jtho.2016.04.033.

Nuga, Oluwademilade, Ana deCarvalho, Yuling Meng, Laura Hasselbach, Kevin Nelson, Susan Irtenkauf, and Andrea Transou. 2018. "Abstract 2519: Identification of Novel MET Fusion Transcript Amplification in Glioblastoma." In *Cancer Research*, 78:2519–2519. American Association for Cancer Research (AACR). https://doi.org/10.1158/1538-7445.am2018-2519.

Okada, Koutaroh, Mitsugu Araki, Takuya Sakashita, Biao Ma, Ryo Kanada, Noriko Yanagitani, Atsushi Horiike, et al. 2019. "Prediction of ALK Mutations Mediating ALK-TKIs Resistance and Drug Re-Purposing to Overcome the Resistance." *EBioMedicine* 41 (March): 105–19. https://doi.org/10.1016/j.ebiom.2019.01.019.

Ou, Sai-Hong Ignatius, Michele Azada, David J. Hsiang, June M. Herman, Tatiana S. Kain, Christina Siwak-Tapp, Cameron Casey, et al. 2014. "Next-Generation Sequencing Reveals a Novel NSCLC ALK F1174V Mutation and Confirms ALK G1202R Mutation Confers High-Level Resistance to Alectinib (CH5424802/RO5424802) in ALK-Rearranged NSCLC Patients Who Progressed on Crizotinib." *Journal of Thoracic Oncology* 9 (4): 549–53.
https://doi.org/10.1097/JTO.0000000000000094.

Ou, Sai-Hong Ignatius, Joel Greenbowe, Ziad U. Khan, Michele C. Azada, Jeffrey S. Ross, Phil J. Stevens, Siraj M. Ali, Vincent A. Miller, and Barbara Gitlitz. 2015. "I1171 Missense Mutation (Particularly I1171N) Is a Common Resistance Mutation in ALK-Positive NSCLC Patients Who Have Progressive Disease While on Alectinib and Is Sensitive to Ceritinib." *Lung Cancer* 88 (2): 231–34. https://doi.org/10.1016/j.lungcan.2015.02.005.

Ou, Sai-Hong Ignatius, and Misako Nagasaka. 2020. "A Catalog of 5' Fusion Partners in ROS1-Positive NSCLC Circa 2020." *JTO Clinical and Research Reports* 1 (3): 100048. https://doi.org/10.1016/j.jtocrr.2020.100048.

Ou, Sai-Hong Ignatius, Lauren Young, Alexa B. Schrock, Adrienne Johnson, Samuel J. Klempner, Viola W. Zhu, Vincent A. Miller, and Siraj M. Ali. 2017. "Emergence of Preexisting MET Y1230C Mutation as a Resistance Mechanism to Crizotinib in NSCLC with MET Exon 14 Skipping." *Journal of Thoracic Oncology* 12 (1): 137–40.
https://doi.org/10.1016/j.jtho.2016.09.119.

Oxnard, G. R., J. C.H. Yang, H. Yu, S. W. Kim, H. Saka, L. Horn, K. Goto, et al. 2020. "TATTON: A Multi-Arm, Phase Ib Trial of Osimertinib Combined with Selumetinib, Savolitinib, or Durvalumab in EGFR-Mutant Lung Cancer." *Annals of Oncology* 31 (4): 507–16. https://doi.org/10.1016/j.annonc.2020.01.013.
Oxnard, Geoffrey R., Mireille Cantarini, Paul Frewer, George Hawkins, Jane Peters, Paul Howarth, Ghada F. Ahmed, et al. 2019. “ SAVANNAH: A Phase II Trial of Osimertinib plus Savolitinib for Patients (Pts) with EGFR -Mutant, MET -Driven (MET +), Locally Advanced or Metastatic Non-Small Cell Lung Cancer (NSCLC), Following Disease Progression on Osimertinib. ” *Journal of Clinical Oncology* 37 (15_suppl): TPS9119–TPS9119. https://doi.org/10.1200/jco.2019.37.15_suppl.tps9119.

Paik, Paul K., Enriqueta Felip, Remi Veillon, Hiroshi Sakai, Alexis B. Cortot, Marina C. Garassino, Julien Mazieres, et al. 2020. “ Tepotinib in Non-Small-Cell Lung Cancer with MET Exon 14 Skipping Mutations .” *New England Journal of Medicine*, May. https://doi.org/10.1056/nejmoa2004407.

Paik, Paul K., Enriqueta Felip, Remi Veillon, Jürgen Scheele, and Rolf Bruns. 2017. "Phase II Trial of the C-Met Inhibitor Tepotinib in Advanced Lung Adenocarcinoma with MET Exon 14 Skipping Mutations." *Journal of Clinical Oncology* 35 (15_suppl): e20541-e20541. https://doi.org/10.1200/JCO.2017.35.15_suppl.e20541.

Park, K., J. Zhou, D.-W. Kim, A.R. Ahmad, R.A. Soo, R. Bruns, J. Straub, et al. 2019. “Tepotinib plus Gefitinib in Patients with MET-Amplified EGFR-Mutant NSCLC: Long-Term Outcomes of the INSIGHT Study.” *Annals of Oncology* 30 (November): ix159. https://doi.org/10.1093/annonc/mdz437.003.

Park, M., M. Dean, K. Kaul, M. J. Braun, M. A. Gonda, and G. Vande Woude. 1987. "Sequence of MET Protooncogene CDNA Has Features Characteristic of the Tyrosine Kinase Family of Growth-Factor Receptors." *Proceedings of the National Academy of Sciences of the United States of America* 84 (18): 6379-83. https://doi.org/10.1073/pnas.84.18.6379.

Passaro, A., A. Prelaj, A. Pochesci, G. Spitaleri, G. Rossi, E. Del Signore, C. Catania, and F. de Marinis. 2017. "Brigatinib for the Treatment of ALK-Positive Advanced Non-Small Cell Lung Cancer Patients." *Drugs of Today* 53 (8): 435. https://doi.org/10.1358/dot.2017.53.8.2676119.

Paumelle, Rejane, David Tulasne, Zoulika Kherrouche, Serge Plaza, Catherine Leroy, Sylvie Reveneau, Bernard Vandenbunder, Veronique Fafeur, David Tulashe, and Syline Reveneau. 2002. "Hepatocyte Growth Factor/Scatter Factor Activates the ETS1 Transcription Factor by a RAS-RAF-MEK-ERK Signaling Pathway." *Oncogene* 21 (15): 2309-19. https://doi.org/10.1038/sj.onc.1205297.

Paz-Ares, Luis, Alexander Luft, David Vicente, Ali Tafreshi, Mahmut Gümüş, Julien Mazières, Barbara Hermes, et al. 2018. "Pembrolizumab plus Chemotherapy for Squamous Non-Small-Cell Lung Cancer." *The New England Journal of Medicine*, September. https://doi.org/10.1056/NEJMoa1810865.

Paz-Ares, Luis, E. H. Tan, K. O'Byrne, L. Zhang, V. Hirsh, M. Boyer, J. C.H. Yang, et al. 2017. "Afatinib versus Gefitinib in Patients with EGFR Mutation-Positive Advanced Non-Small-Cell Lung Cancer: Overall Survival Data from the Phase IIb LUX-Lung 7 Trial." *Annals of Oncology* 28 (2): 270-77. https://doi.org/10.1093/annonc/mdw611.

Pelicci, G., S. Giordano, Z. Zhen, A. E. Salcini, L. Lanfrancone, A. Bardelli, G. Panayotou, et al. 1995. "The Motogenic and Mitogenic Responses to HGF Are Amplified by the Shc Adaptor Protein." *Oncogene* 10 (8): 1631-38. https://mohit.pure.elsevier.com/en/publications/the-motogenic-and-mitogenic-responses-to-hgf-are-amplified-by-the.

Pento, Joseph Thomas. 2017. "Monoclonal Antibodies for the Treatment of Cancer." *Anticancer Research*. International Institute of Anticancer Research. https://doi.org/10.21873/anticanres.12040.

Peschard, Pascal, Noboru Ishiyama, Tong Lin, Stan Lipkowitz, and Morag Park. 2004. "A Conserved DpYR Motif in the Juxtamembrane Domain of the Met Receptor Family Forms an Atypical C-Cbl/Cbl-b Tyrosine Kinase Binding Domain Binding Site Required for Suppression of Oncogenic Activation." *Journal of Biological Chemistry* 279 (28): 29565-71. https://doi.org/10.1074/jbc.M403954200.

Peters, Solange, D. Ross Camidge, Alice T. Shaw, Shirish Gadgeel, Jin S. Ahn, Dong-Wan Kim, Sai-Hong I. Ou, et al. 2017. "Alectinib versus Crizotinib in Untreated ALK-Positive Non-Small-Cell Lung Cancer." *New England Journal of Medicine*, June, NEJMoa1704795. https://doi.org/10.1056/NEJMoa1704795.
Petrelli, Annalisa, Giorgio F. Gilestro, Stefania Lanzardo, Paolo M. Comoglio, Nicola Migone, and Silvia Giordano. 2002. "The Endophilin-CIN85-Cbl Complex Mediates Ligand-Dependent Downregulation of c-Met." *Nature* 416 (6877): 187-90.
https://doi.org/10.1038/416187a.

Planchard, D., S. Popat, K. Kerr, S. Novello, E. F. Smit, C. Faivre-Finn, T. S. Mok, et al. 2018. "Metastatic Non-Small Cell Lung Cancer: ESMO Clinical Practice Guidelines for Diagnosis, Treatment and Follow-Up." *Annals of Oncology* 29 (October): iv192-237. https://doi.org/10.1093/annonc/mdy275.

Planchard, David, Egbert F Smit, Harry J M Groen, Julien Mazieres, Benjamin Besse, Åslaug Helland, Vanessa Giannone, et al. 2017. "Dabrafenib plus Trametinib in Patients with Previously Untreated BRAFV600E-Mutant Metastatic Non-Small-Cell Lung Cancer: An Open-Label, Phase 2 Trial." *The Lancet Oncology* 18 (10): 1307-16.
https://doi.org/10.1016/S1470-2045(17)30679-4.

Pons-Tostivint, Elvire, Benoît Thibault, and Julie Guillermet-Guibert. 2017. "Targeting PI3K Signaling in Combination Cancer Therapy." *Trends in Cancer* 3 (6): 454-69. https://doi.org/10.1016/j.trecan.2017.04.002.

Ponzetto, C, A Bardelli, F Maina, P Longati, G Panayotou, R Dhand, M D Waterfield, and P M Comoglio. 1993. "A Novel Recognition Motif for Phosphatidylinositol 3-Kinase Binding Mediates Its Association with the Hepatocyte Growth Factor/Scatter Factor Receptor." *Molecular and Cellular Biology* 13 (8): 4600-4608.
https://doi.org/10.1128/mcb.13.8.4600.

Ponzetto, Carola, Alberto Bardelli, Zhu Zhen, Flavio Maina, Paolo dalla Zonca, Silvia Giordano, Andrea Graziani, George Panayotou, and Paolo M. Comoglio. 1994. "A Multifunctional Docking Site Mediates Signaling and Transformation by the Hepatocyte Growth Factor/Scatter Factor Receptor Family." *Cell* 77 (2): 261-71. https://doi.org/10.1016/0092-8674(94)90318-2.

Prat, Maria, Radha P. Narsimhan, Tiziana Crepaldi, M. Rita Nicotra, Pier G. Natali, and Paolo M. Comoglio. 1991. "The Receptor Encoded by the Human C-MET Oncogene Is Expressed in Hepatocytes, Epithelial Cells and Solid Tumors." *International Journal of Cancer* 49 (3): 323-28. https://doi.org/10.1002/ijc.2910490302.

Prunotto, Chiara, Tiziana Crepaldi, Paolo E. Forni, Alessandro Ieraci, Robert G. Kelly, Shahragim Tajbakhsh, Margaret Buckingham, and Carola Ponzetto. 2004. "Analysis of Mlc-LacZ Met Mutants Highlights the Essential Function of Met for Migratory Precursors of Hypaxial Muscles and Reveals a Role for Met in the Development of Hyoid Arch-Derived Facial Muscles." *Developmental Dynamics* 231 (3): 582-91. https://doi.org/10.1002/dvdy.20177.

Pylayeva-Gupta, Yuliya, Elda Grabocka, and Dafna Bar-Sagi. 2011. "RAS Oncogenes: Weaving a Tumorigenic Web." *Nature Reviews Cancer*. Nat Rev Cancer. https://doi.org/10.1038/nrc3106.

Qiao, Huan, and Christine M Lovly. 2016. "Cracking the Code of Resistance across Multiple Lines of ALK Inhibitor Therapy in Lung Cancer." *Cancer Discovery* 6 (10): 1084-86. https://doi.org/10.1158/2159-8290.CD-16-0910.

Qin, Shukui, Stephen Lam Chan, Wattana Sukeepaisarnjaroen, Guohong Han, Su Pin Choo, Virome Sriuranpong, Hongming Pan, et al. 2019. "A Phase II Study of the Efficacy and Safety of the MET Inhibitor Capmatinib (INC280) in Patients with Advanced Hepatocellular Carcinoma." *Therapeutic Advances in Medical Oncology* 11.
https://doi.org/10.1177/1758835919889001.

Raiborg, Camilla, Tor Erik Rusten, and Harald Stenmark. 2003. "Protein Sorting into Multivesicular Endosomes." *Current Opinion in Cell Biology*. Elsevier Ltd. https://doi.org/10.1016/S0955-0674(03)00080-2.

Ramalingam, S S, Y Cheng, C Zhou, Y Ohe, F Imamura, B C Cho, M-C Lin, et al. 2018. "LBA50Mechanisms of Acquired Resistance to First-Line Osimertinib: Preliminary Data from the Phase III FLAURA Study." *Annals of Oncology* 29 (suppl_8). https://doi.org/10.1093/annonc/mdy424.063.

Ramalingam, Suresh S., Johan Vansteenkiste, David Planchard, Byoung Chul Cho, Jhanelle E. Gray, Yuichiro Ohe, Caicun Zhou, et al. 2020. "Overall Survival with Osimertinib in Untreated, EGFR-Mutated Advanced NSCLC." *New England Journal of Medicine*.
*Journal of Medicine* 382 (1): 41–50. https://doi.org/10.1056/nejmoa1913662.

Reck, Martin, Delvys Rodríguez-Abreu, Andrew G. Robinson, Rina Hui, Tibor Csőszi, Andrea Fülöp, Maya Gottfried, et al. 2016. "Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer." *New England Journal of Medicine* 375 (19): 1823-33. https://doi.org/10.1056/nejmoa1606774.

Recondo, Gonzalo, Magda Bahcall, Liam F. Spurr, Jianwei Che, Biagio Ricciuti, Giulia C. Leonardi, Ying Chun Lo, et al. 2020. "Molecular Mechanisms of Acquired Resistance to MET Tyrosine Kinase Inhibitors in Patients with MET Exon 14-Mutant NSCLC." *Clinical Cancer Research* 26 (11): 2615-25. https://doi.org/10.1158/1078-0432.CCR-19-3608.

Renzo, M. F. Di, M. Olivero, T. Martone, A. Maffe, P. Maggiora, A. De Stefani, G. Valente, S. Giordano, G. Cortesina, and P. M. Comoglio. 2000. "Somatic Mutations of the MET Oncogene Are Selected during Metastatic Spread of Human HNSC Carcinomas." *Oncogene* 19 (12): 1547-55. https://doi.org/10.1038/sj.onc.1203455.

Rodon, Jordi, Giuseppe Curigliano, Jean Pierre Delord, Wael Harb, Analia Azaro, Yu Han, Celine Wilke, Valerie Donnet, Dalila Sellami, and Thaddeus Beck. 2018. "A Phase Ib, Open-Label, Dose-Finding Study of Alpelisib in Combination with Paclitaxel in Patients with Advanced Solid Tumors." *Oncotarget* 9 (60): 31709-18.
https://doi.org/10.18632/oncotarget.25854.

Rodrigues, G A, and M Park. 1993. "Dimerization Mediated through a Leucine Zipper Activates the Oncogenic Potential of the Met Receptor Tyrosine Kinase." *Molecular and Cellular Biology* 13 (11): 6711-22.
https://doi.org/10.1128/mcb.13.11.6711.

Rodriguez-Viciana, Pablo, Patricia H. Warne, Ritu Dhand, Bart Vanhaesebroeck, Ivan Gout, Michael J. Fry, Michael D. Waterfield, and Julian Downward. 1994. "Phosphatidylinositol-3-OH Kinase Direct Target of Ras." *Nature* 370 (6490): 527-32. https://doi.org/10.1038/370527a0.

Romond, Edward H., Edith A. Perez, John Bryant, Vera J. Suman, Charles E. Geyer, Nancy E. Davidson, Elizabeth Tan-Chiu, et al. 2005. "Trastuzumab plus Adjuvant Chemotherapy for Operable HER2-Positive Breast Cancer." *New England Journal of Medicine* 353 (16): 1673-84. https://doi.org/10.1056/nejmoa052122.

Rosell, Rafael, Enric Carcereny, Radj Gervais, Alain Vergnenegre, Bartomeu Massuti, Enriqueta Felip, Ramon Palmero, et al. 2012. "Erlotinib versus Standard Chemotherapy as First-Line Treatment for European Patients with Advanced EGFR Mutation-Positive Non-Small-Cell Lung Cancer (EURTAC): A Multicentre, Open-Label, Randomised Phase 3 Trial." *The Lancet Oncology* 13 (3): 239-46. https://doi.org/10.1016/S1470-2045(11)70393-X.

Roskoski, Robert. 2015. "A Historical Overview of Protein Kinases and Their Targeted Small Molecule Inhibitors." *Pharmacological Research*. Academic Press. https://doi.org/10.1016/j.phrs.2015.07.010.

Rotow, Julia K, Philippe Gui, Wei Wu, Victoria M Raymond, Richard B Lanman, Frederic J Kaye, Nir Peled, et al. 2018. "Co-Occurring Genetic Alterations in the RAS Pathway Promote Resistance to MET Inhibitor Treatment in Non-Small Cell Lung Cancer with a MET Exon 14 Skipping Mutation." *BioRxiv*, July, 374181. https://doi.org/10.1101/374181.

Rubin, Jeffrey S., Donald P. Bottaro, and Stuart A. Aaronson. 1993. "Hepatocyte Growth Factor/Scatter Factor and Its Receptor, the c-Met Proto-Oncogene Product." *BBA - Reviews on Cancer*. https://doi.org/10.1016/0304-419X(93)90015-5.

Sabari, Joshua K., Fernando Santini, Isabella Bergagnini, W. Victoria Lai, Kathryn C. Arbour, and Alexander Drilon. 2017. "Changing the Therapeutic Landscape in Non-Small Cell Lung Cancers: The Evolution of Comprehensive Molecular Profiling Improves Access to Therapy." *Current Oncology Reports*. Current Medicine Group LLC 1.
https://doi.org/10.1007/s11912-017-0587-4.

Salgia, Ravi. 2017. "MET in Lung Cancer: Biomarker Selection Based on Scientific Rationale." *Molecular Cancer Therapeutics* 16 (4): 555-65. https://doi.org/10.1158/1535-7163.MCT-16-0472.

Sasaki, T., J. Koivunen, A. Ogino, M. Yanagita, S. Nikiforow, W. Zheng, C. Lathan, et al. 2011. "A Novel ALK Secondary
Mutation and EGFR Signaling Cause Resistance to ALK Kinase Inhibitors." *Cancer Research* 71 (18): 6051-60. https://doi.org/10.1158/0008-5472.CAN-11-1340.

Sasaki, T., K. Okuda, W. Zheng, J. Butrynski, M. Capelletti, L. Wang, N. S. Gray, et al. 2010. "The Neuroblastoma-Associated F1174L ALK Mutation Causes Resistance to an ALK Kinase Inhibitor in ALK-Translocated Cancers." *Cancer Research* 70 (24): 10038-43. https://doi.org/10.1158/0008-5472.CAN-10-2956.

Scheffler, Matthias, Marc Bos, Masyar Gardizi, Katharina König, Sebastian Michels, Jana Fassunke, Carina Heydt, et al. 2015. "&lt;I&gt;PIK3CA&lt;I&gt;;I&gt;Mutations in Non-Small Cell Lung Cancer (NSCLC): Genetic Heterogeneity, Prognostic Impact and Incidence of Prior Malignancies." *Oncotarget* 6 (2): 1315-26. https://doi.org/10.18632/oncotarget.2834.

Schelter, Florian, Julia Kobuch, Marcia L. Moss, J. David Becherer, Paolo M. Comoglio, Carla Boccaccio, and Achim Krüger. 2010. "A Disintegrin and Metalloproteinase-10 (ADAM-10) Mediates DN30 Antibody-Induced Shedding of the Met Surface Receptor." *Journal of Biological Chemistry* 285 (34): 26335-40. https://doi.org/10.1074/jbc.M110.106435.

Schiller, Joan H., David Harrington, Chandra P. Belani, Corey Langer, Alan Sandler, James Krook, Junming Zhu, and David H. Johnson. 2002. "Comparison of Four Chemotherapy Regimens for Advanced Non-Small-Cell Lung Cancer." *New England Journal of Medicine* 346 (2): 92-98. https://doi.org/10.1056/nejmoa011954.

Schmidt, Claudia, Friedhelm Bladt, Stefanie Goedecke, Volker Brinkmann, Wolfgang Zschiesche, Melanie Sharpe, Ermanno Gherardi, and Carmen Birchmeler. 1995. "Scatter Factor/Hepatocyte Growth Factor Is Essential for Liver Development." *Nature* 373 (6516): 699-702. https://doi.org/10.1038/373699a0.

Schmidt, Laura, Fuh-Mei Duh, Fan Chen, Takeshi Kishida, Gladys Glenn, Peter Choyke, Stephen W. Scherer, et al. 1997. "Germline and Somatic Mutations in the Tyrosine Kinase Domain of the MET Proto-Oncogene in Papillary Renal Carcinomas." *Nature Genetics* 16 (1): 68-73. https://doi.org/10.1038/ng0597-68.

Schram, Alison M., Matthew T. Chang, Philip Jonsson, and Alexander Drilon. 2017. "Fusions in Solid Tumours: Diagnostic Strategies, Targeted Therapy, and Acquired Resistance." *Nature Reviews Clinical Oncology*. Nature Publishing Group. https://doi.org/10.1038/nrclinonc.2017.127.

Schrock, Alexa B., Garrett M. Frampton, James Suh, Zachary R. Chalmers, Mark Rosenzweig, Rachel L. Erlich, Balazs Halmos, et al. 2016. "Characterization of 298 Patients with Lung Cancer Harboring MET Exon 14 Skipping Alterations." *Journal of Thoracic Oncology* 11 (9): 1493-1502. https://doi.org/10.1016/j.jtho.2016.06.004.

Schuler, M., R. Berardi, W. T. Lim, M. de Jonge, T. M. Bauer, A. Azaro, M. Gottfried, et al. 2020. "Molecular Correlates of Response to Capmatinib in Advanced Non-Small-Cell Lung Cancer: Clinical and Biomarker Results from a Phase I Trial." *Annals of Oncology* 31 (6): 789-97. https://doi.org/10.1016/j.annonc.2020.03.293.

Sequist, L. V., B. A. Waltman, D. Dias-Santagata, S. Digumarthy, A. B. Turke, P. Fidias, K. Bergethon, et al. 2011. "Genotypic and Histological Evolution of Lung Cancers Acquiring Resistance to EGFR Inhibitors." *Science Translational Medicine* 3 (75): 75ra26-75ra26. https://doi.org/10.1126/scitranslmed.3002003.

Sequist, Lecia V., Ji Youn Han, Myung Ju Ahn, Byoung Chul Cho, Helena Yu, Sang We Kim, James Chih Hsin Yang, et al. 2020. "Osimertinib plus Savolitinib in Patients with EGFR Mutation-Positive, MET-Amplified, Non-Small-Cell Lung Cancer after Progression on EGFR Tyrosine Kinase Inhibitors: Interim Results from a Multicentre, Open-Label, Phase 1b Study." *The Lancet Oncology* 21 (3): 373-86. https://doi.org/10.1016/S1470-2045(19)30785-5.

Sequist, Lecia V, Belinda A Waltman, Dora Dias-Santagata, Subba Digumarthy, Alexa B Turke, Panos Fidias, Kristin Bergethon, et al. 2011. "Genotypic and Histological Evolution of Lung Cancers Acquiring Resistance to EGFR Inhibitors." *Science Translational Medicine* 3 (75): 75ra26. https://doi.org/10.1126/scitranslmed.3002003.

Shaw, A T, G J Riely, Y -J Bang, D -W Kim, D R Camidge, B J Solomon, M Varella-Garcia, et al. 2019. "Crizotinib in ROS1-Rearranged Advanced Non-Small-Cell Lung Cancer (NSCLC): Updated Results, Including Overall Survival, from PROFILE 1001." *Annals of Oncology* 30 (7): 1121-26. https://doi.org/10.1093/annonc/mdz131.
Shaw, Alice T., Todd M. Bauer, Filippo de Marinis, Enriqueta Felip, Yasushi Goto, Geoffrey Liu, Julien Mazieres, et al. 2020. "First-Line Lorlatinib or Crizotinib in Advanced ALK-Positive Lung Cancer." *New England Journal of Medicine* 383 (21): 2018-29. https://doi.org/10.1056/NEJMoa2027187.

Shaw, Alice T., Enriqueta Felip, Todd M. Bauer, Benjamin Besse, Alejandro Navarro, Sophie Postel-Vinay, Justin F. Gainor, et al. 2017. "Lorlatinib in Non-Small-Cell Lung Cancer with ALK or ROS1 Rearrangement: An International, Multicentre, Open-Label, Single-Arm First-in-Man Phase 1 Trial." *The Lancet Oncology* 18 (12): 1590-99. https://doi.org/10.1016/S1470-2045(17)30680-0.

Shaw, Alice T., Luc Friboulet, Ignaty Leshchiner, Justin F. Gainor, Simon Bergqvist, Alexei Brooun, Benjamin J. Burke, et al. 2016. "Resensitization to Crizotinib by the Lorlatinib ALK Resistance Mutation L1198F." *New England Journal of Medicine* 374 (1): 54-61. https://doi.org/10.1056/NEJMoa1508887.

Shaw, Alice T., Dong-Wan Kim, Kazuhiko Nakagawa, Takashi Seto, Lucio Crinó, Myung-Ju Ahn, Tommaso De Pas, et al. 2013. "Crizotinib versus Chemotherapy in Advanced ALK-Positive Lung Cancer." *New England Journal of Medicine* 368 (25): 2385-94. https://doi.org/10.1056/NEJMoa1214886.

Shaw, Alice T., Jean-François Martini, Benjamin Besse, Todd M. Bauer, Chia-Chi Lin, Ross A. Soo, Gregory J. Riely, et al. 2018. "Abstract CT044: Efficacy of Lorlatinib in Patients (Pts) with Advanced ALK-Positive Non-Small Cell Lung Cancer (NSCLC) and ALK Kinase Domain Mutations." *Cancer Research* 78 (13 Supplement): CT044-CT044. https://doi.org/10.1158/1538-7445.am2018-ct044.

Shaw, Alice T, Enriqueta Felip, Todd M Bauer, Benjamin Besse, Alejandro Navarro, Sophie Postel-Vinay, Justin F Gainor, et al. 2017. "Lorlatinib in Non-Small-Cell Lung Cancer with ALK or ROS1 Rearrangement: An International, Multicentre, Open-Label, Single-Arm First-in-Man Phase 1 Trial." *The Lancet Oncology* 18 (12): 1590-99. https://doi.org/10.1016/S1470-2045(17)30680-0.

Shaw, Alice T, Leena Gandhi, Shirish Gadgeel, Gregory J Riely, Jeremy Cetnar, Howard West, D Ross Camidge, et al. 2016. "Alectinib in ALK-Positive, Crizotinib-Resistant, Non-Small-Cell Lung Cancer: A Single-Group, Multicentre, Phase 2 Trial." *The Lancet Oncology* 17 (2): 234-42. https://doi.org/10.1016/S1470-2045(15)00488-X.

Shaw, Alice T, Dong-Wan Kim, Ranee Mehra, Daniel S W Tan, Enriqueta Felip, Laura Q M Chow, D Ross Camidge, et al. 2014. "Ceritinib in ALK-Rearranged Non-Small-Cell Lung Cancer." *N Engl j Med* 37013 (27). https://doi.org/10.1056/NEJMoa1311107.

Shaw, Alice T, Tae Min Kim, Lucio Crinò, Cesare Gridelli, Katsuyuki Kiura, Geoffrey Liu, Silvia Novello, et al. 2017. "Ceritinib versus Chemotherapy in Patients with ALK-Rearranged Non-Small-Cell Lung Cancer Previously given Chemotherapy and Crizotinib (ASCEND-5): A Randomised, Controlled, Open-Label, Phase 3 Trial." *The Lancet. Oncology* 18 (7): 874-86. https://doi.org/10.1016/S1470-2045(17)30339-X.

Shaw, Alice Tsang, Pasi A. Janne, Benjamin Besse, Benjamin J. Solomon, Fiona Helen Blackhall, D. Ross Camidge, Tony Mok, et al. 2016. "Crizotinib vs Chemotherapy in ALK + Advanced Non-Small Cell Lung Cancer (NSCLC): Final Survival Results from PROFILE 1007." *Journal of Clinical Oncology* 34 (15_suppl): 9066-9066. https://doi.org/10.1200/jco.2016.34.15_suppl.9066.

Shepherd, Frances A., José Rodrigues Pereira, Tudor Ciuleanu, Eng Huat Tan, Vera Hirsh, Sumitra Thongprasert, Daniel Campos, et al. 2005. "Erlotinib in Previously Treated Non-Small-Cell Lung Cancer." *New England Journal of Medicine* 353 (2): 123-32. https://doi.org/10.1056/NEJMoa050753.

Shi, Yuankai, Joseph Siu Kie Au, Sumitra Thongprasert, Sankar Srinivasan, Chun Ming Tsai, Mai Trong Khoa, Karin Heeroma, Yohji Itoh, Gerardo Cornelio, and Pan Chyr Yang. 2014. "A Prospective, Molecular Epidemiology Study of EGFR Mutations in Asian Patients with Advanced Non-Small-Cell Lung Cancer of Adenocarcinoma Histology (PIONEER)." *Journal of Thoracic Oncology* 9 (2): 154-62. https://doi.org/10.1097/JTO.000000000000033.
Smit, Egbert F., Kazuhiko Nakagawa, Misako Nagasaka, Enriqueta Felip, Yasushi Goto, Bob T. Li, Jose Maria Pacheco, et al. 2020. "Trastuzumab Deruxtecan (T-DXd; DS-8201) in Patients with HER2-Mutated Metastatic Non-Small Cell Lung Cancer (NSCLC): Interim Results of DESTINY-Lung01." *Journal of Clinical Oncology* 38 (15_suppl): 9504-9504.
https://doi.org/10.1200/jco.2020.38.15_suppl.9504.

Soda, Manabu, Young Lim Choi, Munehiro Enomoto, Shuji Takada, Yoshihiro Yamashita, Shunpei Ishikawa, Shin-Ichiro Fujiwara, et al. n.d. "Identification of the Transforming EML4-ALK Fusion Gene in Non-Small-Cell Lung Cancer." https://doi.org/10.1038/nature05945.

Solomon, B, A Shaw, S Ou, B Besse, E Felip, T Bauer, R Soo, et al. 2017. "OA 05.06 Phase 2 Study of Lorlatinib in Patients with Advanced ALK+/ROS1+ Non-Small-Cell Lung Cancer." *Journal of Thoracic Oncology* 12 (11): S1756.
https://doi.org/10.1016/j.jtho.2017.09.351.

Solomon, Benjamin J., Tony Mok, Dong-Wan Kim, Yi-Long Wu, Kazuhiko Nakagawa, Tarek Mekhail, Enriqueta Felip, et al. 2014. "First-Line Crizotinib versus Chemotherapy in ALK-Positive Lung Cancer." *New England Journal of Medicine* 371 (23): 2167-77. https://doi.org/10.1056/NEJMoa1408440.

Solomon, Benjamin J, Cai Cun Zhou, Alexander Drilon, Keunchil Park, Jürgen Wolf, Yasir Elamin, Hannah M Davis, et al. 2020. "Phase III Study of Selpercatinib vs Chemotherapy +/- Pembrolizumab in Untreated RET Positive Non-Small-Cell Lung Cancer." *Future Oncology*, November. https://doi.org/10.2217/fon-2020-0935.

Solomon, Benjamin, Marileila Varella-Garcia, and D Ross Camidge. 2009. "ALK Gene Rearrangements: A New Therapeutic Target in a Molecularly Defined Subset of Non-Small Cell Lung Cancer." *Journal of Thoracic Oncology* 4 (12): 1450-54.
https://doi.org/10.1097/JTO.0b013e3181c4dedb.

Song, Min Sup, Leonardo Salmena, and Pier Paolo Pandolfi. 2012. "The Functions and Regulation of the PTEN Tumour Suppressor." *Nature Reviews Molecular Cell Biology* 13 (5): 283-96. https://doi.org/10.1038/nrm3330.

Soria, Jean-Charles, Ho-Young Lee, Janet I Lee, Luo Wang, Jean-Pierre Issa, Bonnie L Kemp, Diane D Liu, Jonathan M Kurie, Li Mao, and Fadlo R Khuri. 2002. "Lack of PTEN Expression in Non-Small Cell Lung Cancer Could Be Related to Promoter Methylation." *Clinical Cancer Research : An Official Journal of the American Association for Cancer Research* 8 (5): 1178-84. http://www.ncbi.nlm.nih.gov/pubmed/12006535.

Soria, Jean-Charles, Yuichiro Ohe, Johan Vansteenkiste, Thanyanan Reungwetwattana, Busyamas Chewaskulyong, Ki Hyeong Lee, Arunee Dechaphunkul, et al. 2018. "Osimertinib in Untreated EGFR-Mutated Advanced Non-Small-Cell Lung Cancer." *New England Journal of Medicine* 378 (2): 113-25. https://doi.org/10.1056/nejmoa1713137.

Soria, Jean-Charles, Daniel S W Tan, Rita Chiari, Yi-Long Wu, Luis Paz-Ares, Juergen Wolf, Sarayut L Geater, et al. 2017. "First-Line Ceritinib versus Platinum-Based Chemotherapy in Advanced ALK-Rearranged Non-Small-Cell Lung Cancer (ASCEND-4): A Randomised, Open-Label, Phase 3 Study." *The Lancet* 389 (10072): 917-29.
https://doi.org/10.1016/S0140-6736(17)30123-X.

Sos, Martin L, Mirjam Koker, Barbara A Weir, Stefanie Heynck, Rosalia Rabinovsky, Thomas Zander, Jens M Seeger, et al. 2009. "PTEN Loss Contributes to Erlotinib Resistance in EGFR-Mutant Lung Cancer by Activation of Akt and EGFR." *Cancer Research* 69 (8): 3256-61. https://doi.org/10.1158/0008-5472.CAN-08-4055.

Stamos, Jennifer, Robert A. Lazarus, Xiaoyi Yao, Daniel Kirchhofer, and Christian Wiesmann. 2004. "Crystal Structure of the HGF β-Chain in Complex with the Sema Domain of the Met Receptor." *EMBO Journal* 23 (12): 2325-35.
https://doi.org/10.1038/sj.emboj.7600243.

Stoker, Michael, Ermanno Gherardi, Marion Perryman, and Julia Gray. 1987. "Scatter Factor Is a Fibroblast-Derived Modulator of Epithelial Cell Mobility." *Nature* 327 (6119): 239-42. https://doi.org/10.1038/327239a0.

Suzawa, Ken, Michael Offin, Daniel Lu, Christopher Kurzatkowski, Morana Vojnic, Roger S. Smith, Joshua K. Sabari, et al. 2019. "Activation of KRAS Mediates Resistance to Targeted Therapy in MET Exon 14-Mutant Non-Small Cell Lung
Cancer." *Clinical Cancer Research* 25 (4): 1248-60. https://doi.org/10.1158/1078-0432.CCR-18-1640.

Suzawa, Ken, Michael Offin, Daniel Lu, Christopher Kurzatkowski, Morana Vojnic, Roger S Smith, Joshua K Sabari, et al. 2018. "Activation of KRAS Mediates Resistance to Targeted Therapy in MET Exon 14 Mutant Non-Small Cell Lung Cancer." *Clinical Cancer Research : An Official Journal of the American Association for Cancer Research*, October, clincanres.1640.2018. https://doi.org/10.1158/1078-0432.CCR-18-1640.

Take, Hitoshi, Shinji Watanabe, Kazuyo Takeda, Zu Xi Yu, Nobuhisa Iwata, and Sachiko Kajigaya. 2000. "Cloning and Characterization of a Novel Adaptor Protein, CIN85, That Interacts with c-Cbl." *Biochemical and Biophysical Research Communications* 268 (2): 321-28. https://doi.org/10.1006/bbrc.2000.2147.

Takeda, Hiromasa, Nagio Takigawa, Kadoaki Ohashi, Daisuke Minami, Itaru Kataoka, Eiki Ichihara, Nobuaki Ochi, Mitsune Tanimoto, and Katsuyuki Kiura. 2013. "Vandetanib Is Effective in EGFR-Mutant Lung Cancer Cells with PTEN Deficiency." *Experimental Cell Research* 319 (4): 417-23. https://doi.org/10.1016/j.yexcr.2012.12.018.

Tani, Tetsuo, Hiroyuki Yasuda, Junko Hamamoto, Aoi Kuroda, Daisuke Arai, Kota Ishioka, Keiko Ohgino, et al. 2016. "Activation of EGFR Bypass Signaling by TGFα Overexpression Induces Acquired Resistance to Alectinib in ALK-Translocated Lung Cancer Cells." *Molecular Cancer Therapeutics* 15 (1): 162-71. https://doi.org/10.1158/1535-7163.MCT-15-0084.

Tchekmedyian, Nishan, Siraj M Ali, Vincent A Miller, and Eric B Haura. 2016. "Acquired ALK L1152R Mutation Confers Resistance to Ceritinib and Predicts Response to Alectinib." *Journal of Thoracic Oncology : Official Publication of the International Association for the Study of Lung Cancer* 11 (7): e87-8. https://doi.org/10.1016/j.jtho.2016.03.018.

Teis, D., and L. A. Huber. 2003. "The Odd Couple: Signal Transduction and Endocytosis." *Cellular and Molecular Life Sciences*. Cell Mol Life Sci. https://doi.org/10.1007/s00018-003-3010-2.

Tempest, P. R., M. R. Stratton, and C. S. Cooper. 1988. "Structure of the Met Protein and Variation of Met Protein Kinase Activity among Human Tumour Cell Lines." *British Journal of Cancer* 58 (1): 3-7. https://doi.org/10.1038/bjc.1988.150.

Tovar, Elizabeth A., and Carrie R. Graveel. 2017. "MET in Human Cancer: Germline and Somatic Mutations." *Annals of Translational Medicine*. AME Publishing Company. https://doi.org/10.21037/atm.2017.03.64.

Toyokawa, Gouji, Eiko Inamasu, Shinichiro Shimamatsu, Tsukihisa Yoshida, Kaname Nosaki, Fumihiko Hirai, Masafumi Yamaguchi, Takashi Seto, Mitsuhiro Takenoyama, and Yukito Ichinose. 2015. "Identification of a Novel ALK G1123S Mutation in a Patient with ALK-Rearranged Non-Small-Cell Lung Cancer Exhibiting Resistance to Ceritinib." *JTO Acquisition* 10 (7): e55-57. https://doi.org/10.1097/JTO.0000000000000509.

Tsao, Anne S., Xi Ming Tang, Bradley Sabloff, Lianchun Xiao, Hisayuki Shigematsu, Jack Roth, Margaret Spitz, Waun Ki Hong, Adi Gazdar, and Ignacio Wistuba. 2006. "Clinicopathologic Characteristics of the EGFR Gene Mutation in Non-Small Cell Lung Cancer." *Journal of Thoracic Oncology* 1 (3): 231-39. https://doi.org/10.1016/S1556-0864(15)31573-2.

TULASNE, DAVID, REJANE PAUMELLE, CATHERINE LEROY, SYLVIE REVENEAU, BERNARD VANDENBUNDER, and VERONIQUE FAFEUR. 2002. "Involvement of RAS-ERK Signaling in Multiple Biological Responses to HGF/SF." *Annals of the New York Academy of Sciences* 973 (1): 105-8. https://doi.org/10.1111/j.1749-6632.2002.tb04615.x.

VanderLaan, Paul A., Deepa Rangachari, Susan M. Mockus, Vanessa Spotlow, Honey V. Reddi, Joan Malcolm, Mark S. Huberman, Loren J. Joseph, Susumu S. Kobayashi, and Daniel B. Costa. 2017. "Mutations in TP53, PIK3CA, PTEN and Other Genes in EGFR Mutated Lung Cancers: Correlation with Clinical Outcomes." *Lung Cancer* 106 (April): 17-21. https://doi.org/10.1016/j.lungcan.2017.01.011.

Verret, B., J. Cortes, T. Bachelot, F. Andre, and M. Arnedos. 2019. "Efficacy of PI3K Inhibitors in Advanced Breast Cancer." *Annals of Oncology : Official Journal of the European Society for Medical Oncology*. NLM (Medline). https://doi.org/10.1093/annonc/mdz381.
Verstreken, Patrik, Ole Kjaerulff, Thomas E. Lloyd, Richard Atkinson, Yi Zhou, Ian A. Meinertzhagen, and Hugo J. Bellen. 2002. "Endophilin Mutations Block Clathrin-Mediated Endocytosis but Not Neurotransmitter Release." *Cell* 109 (1): 101-12. https://doi.org/10.1016/S0092-8674(02)00688-8.

Verstreken, Patrik, Tong Wey Koh, Karen L. Schulze, R. Grace Zhai, P. Robin Hiesinger, Yi Zhou, Sunil Q. Mehta, Yu Cao, Jack Roos, and Hugo J. Bellen. 2003. "Synaptojanin I Is Recruited by Endophilin to Promote Synaptic Vesicle Uncoating." *Neuron* 40 (4): 733-48. https://doi.org/10.1016/S0896-6273(03)00644-5.

Wang, Q., P. Liu, J. M. Spangle, T. Von, T. M. Roberts, N. U. Lin, I. E. Krop, E. P. Winer, and J. J. Zhao. 2016. "PI3K-P110α Mediates Resistance to HER2-Targeted Therapy in HER2+, PTEN-Deficient Breast Cancers." *Oncogene* 35 (27): 3607-12. https://doi.org/10.1038/onc.2015.406.

Wang, Samantha X.Y., Bing M. Zhang, Heather A. Wakelee, Michael Z. Koontz, MingGui Pan, Maximilian Diehn, Christian A. Kunder, and Joel W. Neal. 2019. "Case Series of MET Exon 14 Skipping Mutation-Positive Non-Small-Cell Lung Cancers with Response to Crizotinib and Cabozantinib." *Anti-Cancer Drugs* 30 (5): 537-41. https://doi.org/10.1097/CAD.0000000000000765.

Wang, Wen xian, Chunwei Xu, Yu Chen, You-cai Zhu, Yueping Liu, Hong Wang, Wu Zhuang, et al. 2018. "MET Gene Fusions in Non-Small Cell Lung Cancer (NSCLC) in the Chinese Population: A Multicenter Study." *Journal of Clinical Oncology* 36 (15_suppl): e13539-e13539. https://doi.org/10.1200/jco.2018.36.15_suppl.e13539.

Waqar, Saiama Naheed, Mary Weber Redman, Susanne M. Arnold, Fred R. Hirsch, Philip C. Mack, Lawrence Howard Schwartz, David R. Gandara, et al. 2019. "Phase II Study of ABBV-399 (Process II) in Patients with C-MET Positive Stage IV/Recurrent Lung Squamous Cell Cancer (SCC): LUNG-MAP Sub-Study S1400K (NCT03574753)." *Journal of Clinical Oncology* 37 (15_suppl): 9075-9075. https://doi.org/10.1200/jco.2019.37.15_suppl.9075.

Weidner, K. Michael, Jürgen Behrens, Joel Vandekerckhove, and Walter Birchmeier. 1990. "Scatter Factor: Molecular Characteristics and Effect on the Invasiveness of Epithelial Cells." *Journal of Cell Biology* 111 (5): 2097-2108. https://doi.org/10.1083/jcb.111.5.2097.

Westover, D., J. Zugazagoitia, B. C. Cho, C. M. Lovly, and L. Paz-Ares. 2018. "Mechanisms of Acquired Resistance to First-and Second-Generation EGFR Tyrosine Kinase Inhibitors." *Annals of Oncology*. Oxford University Press. https://doi.org/10.1093/annonc/mdx703.

Wolf, J, T Seto, J-Y Han, N Reguart, E B Garon, H J M Groen, D S Tan, et al. 2018. "LBA52Results of the GEOMETRY Mono-1 Phase II Study for Evaluation of the MET Inhibitor Capmatinib (INC280) in Patients (Pts) with METΔex14 Mutated Advanced Non-Small Cell Lung Cancer (NSCLC)." *Annals of Oncology* 29 (suppl_8). https://doi.org/10.1093/annonc/mdy424.090.

Wolf, Juergen, Tobias R. Overbeck, Ji-Youn Han, Maximilian Hochmair, Filippo de Marinis, Kadoaki Ohashi, Egbert F. Smit, et al. 2020. "Capmatinib in Patients with High-Level MET -Amplified Advanced Non-Small Cell Lung Cancer (NSCLC): Results from the Phase 2 GEOMETRY Mono-1 Study." *Journal of Clinical Oncology* 38 (15_suppl): 9509-9509. https://doi.org/10.1200/jco.2020.38.15_suppl.9509.

Wu, Shang-Gin, Yih-Leong Chang, Chong-Jen Yu, Pan-Chyr Yang, and Jin-Yuan Shih. 2016. "The Role of PIK3CA Mutations among Lung Adenocarcinoma Patients with Primary and Acquired Resistance to EGFR Tyrosine Kinase Inhibition." *Scientific Reports* 6 (1): 35249. https://doi.org/10.1038/srep35249.

Wu, Yi-Long, Masahiro Tsuboi, Jie He, Thomas John, Christian Grohe, Margarita Majem, Jonathan W. Goldman, et al. 2020. "Osimertinib in Resected EGFR-Mutated Non-Small-Cell Lung Cancer." *New England Journal of Medicine* 383 (18): 1711-23. https://doi.org/10.1056/nejmoa2027071.

Wu, Yi Long, Ying Cheng, Xiangdong Zhou, Ki Hyeong Lee, Kazuhiko Nakagawa, Seiji Niho, Fumito Tsuji, et al. 2017. "Dacomitinib versus Gefitinib as First-Line Treatment for Patients with EGFR-Mutation-Positive Non-Small-Cell Lung
Cancer (ARCHER 1050): A Randomised, Open-Label, Phase 3 Trial.” *The Lancet Oncology* 18 (11): 1454–66.
https://doi.org/10.1016/S1470-2045(17)30608-3.

Wu, Yi Long, Li Zhang, Dong Wan Kim, Xiaoqing Liu, Dae Ho Lee, James Chih Hsin Yang, Myung Ju Ahn, et al. 2018. “Phase Ib/II Study of Capmatinib (INC280) Plus Gefitinib after Failure of Epidermal Growth Factor Receptor (EGFR) Inhibitor Therapy in Patients with EGFR-Mutated, MET Factor-Dysregulated Non-Small-Cell Lung Cancer.” *Journal of Clinical Oncology* 36 (31): 3101–9. https://doi.org/10.1200/JCO.2018.77.7326.

Xu, Tao, Hongxiang Wang, Xiaoquan Huang, Weiqing Li, Qilin Huang, Yong Yan, and Juxiang Chen. 2018. “Gene Fusion in Malignant Glioma: An Emerging Target for Next-Generation Personalized Treatment.” *Translational Oncology*.
Neoplasia Press, Inc. https://doi.org/10.1016/j.tranon.2018.02.020.

Xu, Tian-Xiao, Si-Fan Yu, Meng Ma, Jie Dai, Zhi-Hong Chi, Lu Si, Xi-Nan Sheng, Chuan-Liang Cui, Yan Kong, and Jun Guo. 2018. “CMET-N375S Germline Mutation Is Associated with Poor Prognosis of Melanoma in Chinese Patients.” *Translational Cancer Research* 7 (2): 248–56. https://doi.org/10.21037/19570.

Yakes, F. Michael, Jason Chen, Jenny Tan, Kyoko Yamaguchi, Yongchang Shi, Peiwen Yu, Fawn Qian, et al. 2011. “Cabozantinib (XL184), a Novel MET and VEGFR2 Inhibitor, Simultaneously Suppresses Metastasis, Angiogenesis, and Tumor Growth.” *Molecular Cancer Therapeutics* 10 (12): 2298–2308. https://doi.org/10.1158/1535-7163.MCT-11-0264.

Yamamoto, H., H. Shigematsu, M. Nomura, W. W. Lockwood, M. Sato, N. Okumura, J. Soh, et al. 2008. “PIK3CA Mutations and Copy Number Gains in Human Lung Cancers.” *Cancer Research* 68 (17): 6913–21. https://doi.org/10.1158/0008-5472.CAN-07-5084.

Yan, S. Betty, Victoria L. Peek, Rose Ajamie, Sean G. Buchanan, Jeremy R. Graff, Steven A. Heidler, Yu-Hua Hui, et al. 2013. “LY2801653 Is an Orally Bioavailable Multi-Kinase Inhibitor with Potent Activity against MET, MST1R, and Other Oncoproteins, and Displays Anti-Tumor Activities in Mouse Xenograft Models.” *Investigational New Drugs* 31 (4): 833–44. https://doi.org/10.1007/s10637-012-9912-9.

Yan, Sau-Chi Betty, Suzane L. Um, Victoria L. Peek, Jennifer R. Stephens, Wei Zeng, Bruce W. Konicek, Ling Liu, Volker Wacheck, and Richard A. Walgren. 2017. “Abstract 528: Evaluation of Single Agent Merestinib (LY2801653) or Emibetuzumab (LY2875358) and the Combination in a Xenograft Tumor Model Bearing MET Exon 14 Skipping.” In *Molecular and Cellular Biology, Genetics*, 77:528–528. American Association for Cancer Research.
https://doi.org/10.1158/1538-7445.AM2017-528.

Yang, J.-, J. Fang, Y. Shu, J. Chang, G. Chen, J. He, W. Li, et al. 2017. “OA 09.06 A Phase Ib Trial of Savolitinib plus Gefitinib for Chinese Patients with EGFR-Mutant MET-Amplified Advanced NSCLC.” *Journal of Thoracic Oncology* 12 (11): S1769. https://doi.org/10.1016/j.jtho.2017.09.379.

Yano, Seiji, Wei Wang, Qi Li, Kunio Matsumoto, Haruko Sakurama, Takahiro Nakamura, Hirokazu Ogino, et al. 2008. “Hepatocyte Growth Factor Induces Gefitinib Resistance of Lung Adenocarcinoma with Epidermal Growth Factor Receptor-Activating Mutations.” *Cancer Research* 68 (22): 9479–87. https://doi.org/10.1158/0008-5472.CAN-08-1643.

Yeh, Iwei, Thomas Botton, Eric Talevich, A. Hunter Shain, Alyssa J. Sparatta, Arnaud De La Fouchardiere, Thaddeus W. Mully, et al. 2015. “Activating MET Kinase Rearrangements in Melanoma and Spitz Tumours.” *Nature Communications* 6 (May): 7174. https://doi.org/10.1038/ncomms8174.

Yoda, Satoshi, Jessica J Lin, Michael S Lawrence, Benjamin J Burke, Luc Friboulet, Adam Langenbucher, Leila Dardaei, et al. 2018. “Sequential ALK Inhibitors Can Select for Lorlatinib-Resistant Compound ALK Mutations in ALK-Positive Lung Cancer.” *Cancer Discovery*, April, CD-17-1256. https://doi.org/10.1158/2159-8290.CD-17-1256.

Yoo, Seol Bong, Xianhua Xu, Hyun Ju Lee, Sanghoon Jheon, Choon-Taek Lee, Gheeyoung Choe, and Jin-Haeng Chung. 2011.
"Loss of PTEN Expression Is an Independent Poor Prognostic Factor in Non-Small Cell Lung Cancer." *The Korean Journal of Pathology* 45 (4): 329. https://doi.org/10.4132/KoreanJPathol.2011.45.4.329.

Yu, Helena A., Ken Suzawa, Emmet Jordan, Ahmet Zehir, Ai Ni, Ryan Kim, Mark G. Kris, et al. 2018. "Concurrent Alterations in EGFR-Mutant Lung Cancers Associated with Resistance to EGFR Kinase Inhibitors and Characterization of MTOR as a Mediator of Resistance." *Clinical Cancer Research* 24 (13): 3108-18. https://doi.org/10.1158/1078-0432.CCR-17-2961.

Zhang, Jianming, Priscilla L. Yang, and Nathanael S. Gray. 2009. "Targeting Cancer with Small Molecule Kinase Inhibitors." *Nature Reviews Cancer*. Nature Publishing Group. https://doi.org/10.1038/nrc2559.

Zhang, Sen, Sara Nadworny, Scott D. Wardwell, Lindsey Eichinger, Biplab Das, Emily Y. Ye, J. Graeme Hodgson, and Victor M. Rivera. 2015. "Abstract 781: The Potent ALK Inhibitor AP26113 Can Overcome Mechanisms of Resistance to First- and Second-Generation ALK TKIs in Preclinical Models." *Cancer Research* 75 (15 Supplement): 781-781. https://doi.org/10.1158/1538-7445.AM2015-781.

Zhang, Yu Wen, Ling Mei Wang, Richard Jove, and George F. Vande Woude. 2002. "Requirement of Stat3 Signaling for HGF/SF-Met Mediated Tumorigenesis." *Oncogene* 21 (2): 217-26. https://doi.org/10.1038/sj.onc.1205004.

Zhang, Yu, Jinping Yin, and Feng Peng. 2017. "Acquired Resistance to Crizotinib in Advanced Lung Adenocarcinoma with MET Exon 14 Skipping." *Lung Cancer (Amsterdam, Netherlands)* 113 (November): 69-71. https://doi.org/10.1016/j.lungcan.2017.09.006.

Zhou, Caicun, Yi-Long Wu, Gongyan Chen, Jifeng Feng, Xiao-Qing Liu, Changli Wang, Shucai Zhang, et al. 2011. "Erlotinib versus Chemotherapy as First-Line Treatment for Patients with Advanced EGFR Mutation-Positive Non-Small-Cell Lung Cancer (OPTIMAL, CTONG-0802): A Multicentre, Open-Label, Randomised, Phase 3 Study." *The Lancet*. *Oncology* 12 (8): 735-42. https://doi.org/10.1016/S1470-2045(11)70184-X.

Zhu, Chengpei, Liangcai Wu, Yanling Lv, Jinxia Guan, Xue Bai, Jianzhen Lin, Tingting Liu, et al. 2019. "The Fusion Landscape of Hepatocellular Carcinoma." *Molecular Oncology* 13 (5): 1214-25. https://doi.org/10.1002/1878-0261.12479.

Zhu, Qingqing, Ping Zhan, Xinlin Zhang, Tangfeng Lv, and Yong Song. 2015. "Clinicopathologic Characteristics of Patients with ROS1 Fusion Gene in Non-Small Cell Lung Cancer: A Meta-Analysis." *Translational Lung Cancer Research* 4 (3): 300-309. https://doi.org/10.3978/j.issn.2218-6751.2015.05.01.

Zou, Helen Y., Luc Friboulet, David P. Kodack, Lars D. Engstrom, Qiuhua Li, Melissa West, Ruth W. Tang, et al. 2015a. "PF-06463922, an ALK/ROS1 Inhibitor, Overcomes Resistance to First and Second Generation ALK Inhibitors in Preclinical Models." *Cancer Cell* 28 (1): 70-81. https://doi.org/10.1016/j.ccell.2015.05.010.

Zou, Helen Y, Luc Friboulet, David P Kodack, Lars D Engstrom, Qiuhua Li, Melissa West, Ruth W Tang, et al. 2015b. "PF-06463922, an ALK/ROS1 Inhibitor, Overcomes Resistance to First and Second Generation ALK Inhibitors in Preclinical Models." *Cancer Cell* 28 (1): 70-81. https://doi.org/10.1016/j.ccell.2015.05.010.
# 9 Annexe

## Annexe 1

Figure extraite de l'article « Ligand-dependent oncogene addiction of the MET exon14 skipping ; » ; soumission prévue en janvier 2021 » (A) Expérience de tumorigenèse in vivo sur souris hHGFki-SCID ; injection des lignées 16HBE6WT et 16HBE-Ex14 (B) Expérience de tumorigenèse in vivo sur souris hHGFki-SCID et NSG contrôle, injection de la lignée 16HBE-Ex14 (C) Efficacité de l'OMO-1 (inhibiteur MET) sur la croissance in-vivo de la lignée 16HBE-Ex14 dans les souris hHGFki-SCID.

### A.

<table><thead><tr><td></td><td>% of positive xenograft in KI-HGF mice</td><td>Mean volume (mm3)</td></tr></thead><tbody><tr><td>16HBE-WT</td><td>14 % (1/7)</td><td>47 +/- 0</td></tr><tr><td>16HBE-Ex14</td><td>100 % (7/7)</td><td>171 +/- 112</td></tr></tbody></table>

Comparé au récepteur sauvage, METex14 est un oncogene efficace en présence d'HGF.

### B.

<table><thead><tr><td></td><td>% of positive 16HBE-Ex14 xenograft</td><td>Mean volume (mm3)</td></tr></thead><tbody><tr><td>Control NSG mice</td><td>35 (9/26)</td><td>8 +/- 4</td></tr><tr><td>KI-HGF mice</td><td>86 (38/44)</td><td>50 +/- 71</td></tr></tbody></table>

<figure><img src="image_40.png" /></figure>

Le récepteur muté METex14 est un oncogène faible en présence de stimulation par son ligand

### C.

<figure><img src="image_41.png" /></figure>

Efficacité du TKI-MET OMO-1 pour inhiber la croissance tumorale in vivo
<figure><img src="image_42.png" /></figure>

# Optimization of Routine Testing for *MET* Exon 14 Splice Site Mutations in NSCLC Patients

<figure><img src="image_43.png" /></figure>

Check for updates

Clotilde Descarpentrries, PharmD,ª Frédéric Leprêtre, PhD,ᵇ

Fabienne Escande, PharmD, PhD,ª Zoulika Kherrouche, PhD,ᶜ Martin Figeac, PhD,ᵇ

Shéhérazade Sebda, MSc,ᵇ Simon Baldacci, MD, PhD,ᶜ Valérie Grégoire,ᵈ

Philippe Jamme, MD,ᶜ Marie-Christine Copin, MD, PhD, c,d David Tulasne, PhD, c

Alexis B. Cortot, MD, PhDc,e,f,\*

Received 18 April 2018; revised 3 August 2018; accepted 14 August 2018

Available online - 6 September 2018

## ABSTRACT

**Introduction:** Genomic alterations affecting splice sites of MNNG HOS transforming gene (MET) exon 14 were recently identified in NSCLC patients. Objective responses to MET tyrosine kinase inhibitors have been reported in these patients. Thus, detection of *MET* exon 14 splice site mutations represents a major challenge. So far, most of these alterations were found by full-exome sequencing or large capture-based next-generation sequencing (NGS) panels, which are not suitable for routine diagnosis.

**Methods:** Aiming to provide a molecular testing method applicable in routine practice, we first developed a fragment-length analysis for detecting deletions in introns flanking *MET* exon 14. Second, we designed an optimized targeted NGS panel called CLAPv1, covering the *MET* exon 14 and flanking regions in addition to the main molecular targets usually covered in genomic testing. In patients with *MET* exon 14 mutations, *MET* gene amplification, gene copy number and *MET* receptor expression were also determined.

**Results:** Among 1514 formalin-fixed paraffin-embedded NSCLC samples, nonoptimized NGS allowed detection of *MET* exon 14 mutations in only 0.3% of the patients, and fragment length analysis detected deletions in 1.1% of the patients. Combined, the optimized CLAPv1 panel and fragment-length analysis implemented for routine molecular testing revealed *MET* exon 14 alterations in 2.2% of 365 additional NSCLC patients. *MET* gene amplification or high gene copy number was observed in 6 of 30 patients (20%) harboring *MET* exon 14 mutations.

**Conclusions:** These results show that optimized targeted NGS and fragment-length analysis improve detection of *MET* alterations in routine practice.

© 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

**Keywords:** *MET*; Receptor tyrosine kinase; Next-generation sequencing; Lung cancer; Splice site

## Introduction

Targeted therapies have recently changed the care of patients with cancers displaying well-defined oncogene-addiction-causing alterations. Several such genomic

<footnote>ªDepartment of Biochemistry and Molecular Biology, Hormonology Metabolism Nutrition Oncology, CHU Lille, Lille, France</footnote>

<footnote>ᵇLille University, Functional and Structural Platform, CHU Lille, Lille, France</footnote>

<footnote>ᶜLille University, CNRS, Institut Pasteur de Lille, Mechanisms of Tumorigenesis and Targeted Therapies, Lille, France</footnote>

<footnote>ᵈLille University, Pathology Department, CHU Lille, Lille, France</footnote>

<footnote>ᶜLille University, Thoracic Oncology Department, CHU Lille, Lille, France</footnote>

<footnote>*Corresponding author.</footnote>

<footnote>Drs. Descarpentrries and Leprêtre contributed equally to this work.</footnote>

<footnote>**Disclosure:** Dr. Descarpentrries has received personal fees from Astra Zeneca and Novartis Pharma SAS; and has received nonfinancial support from Astra Zeneca, Novartis Pharma SAS, Roche SAS, Boehringer Ingelheim France, and Pfizer. Dr. Escande has received personal fees and nonfinancial support from Astra Zeneca, Boehringer, and Roche. Dr. Kherrouche has received grants from Canceropole Nord-ouest, ANR Metropolit, Ligue Contre le Cancer, INCA, and Merck. Dr. Baldacci has received personal fees from Astra Zeneca, Roche, and Glaxo Smith Kline, Lilly, and Pfizer. Dr. Jamme has received grants from Canceropole Nord-ouest, ANR Metropolis, Ligue Contre le Cancer, INCA, and Merck; and has received personal fees from Chugai Pharma and Boehringer. Dr. Tulasne has received grants from Canceropole Nord-ouest, Agence Nationale pour la recherche ANR, Ligue Nationals Contre le Cancer, Institut National du Cancer INCa, Merck, and Roche. Dr. Cortot has received grants from Canceropole Nord-ouest, Agence National pour la recherche ANR, Ligue Nationale Contre le Cancer, Institut National du Cancer INCa, and Merck; and has received personal fees from Astra Zeneca, Bristol Myers Squibb, MSD, Roche, Novartis, Pfizer, and Takeda. The remaining authors declare no conflict of interest.</footnote>

<footnote>**Address for correspondence:** Alexis Cortot, MD, PhD, Thoracic Oncology Department, Hopital Calmette, CHU Lille, F-59000 Lille, France. E-mail: alexis.cortot@chru-lille.fr</footnote>

<footnote>© 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.</footnote>

<footnote>ISSN: 1556-0864</footnote>

<footnote>https://doi.org/10.1016/j.jtho.2018.08.2023</footnote>
alterations have been evidenced, including mutations, translocations, and gene amplifications. In NSCLC, muta- tions occurring in the kinase domain of EGFR or BRAF and rearrangements involving the tyrosine kinase re- ceptor ALK receptor tyrosine kinase (ALK) or ROS1 define unique subsets of patients who benefit from tyrosine kinase inhibitors (TKIs). Point mutations, in- sertions, and deletions are usually investigated by next- generation sequencing (NGS), with the help of commer- cial panels covering the main oncogenes amenable to targeted therapies. However, the spectrum of targetable molecular alterations is constantly expanding with the regular discovery of novel driver alterations in onco- genes and tumor suppressor genes. Recent publications describe a set of novel mutations located within splice sites of MNNG HOS transforming gene (MET) exon 14, causing constitutive activation of the MET receptor.

MET is a receptor tyrosine kinase produced pre-
dominantly in cells of epithelial origin. It is activated by
its stromal ligand, the hepatocyte growth factor, also
called scatter factor.¹ Hepatocyte growth factor and MET
are essential to embryonic development and tissue
regeneration in adults.²⁻⁹ Amplification of the MET gene,
leading to its massive expression and ligand-
independent activation, has been reported in 1% to 5%
of gastric and lung cancers.¹⁰⁻¹² MET kinase domain
mutations have also been described in various cancer
types, including renal and lung cancers.¹³ In a recent
study, mutations at MET exon 14 splice sites were found
in approximately 3% of the profiled lung tumors.¹⁴ To
date, more than 130 different mutations altering splice
sites of MET exon 14 have been described. These mu-
tations include point mutations, deletions, insertions,
and complex mutations (indels), all of which affect
conserved sequences of splice donor or acceptor sites. At
the 5' end of the exon, most of the alterations are de-
letions, insertions, and indels affecting the splice
acceptor site, the branch point, or the polyprimidine
tract. At the 3' end, most are point mutations affecting
the splice donor site.¹⁵ All these alterations are thought
to induce in-frame exon 14 skipping, which leads to
deletion of the juxtamembrane domain of the MET re-
ceptor. This juxtamembrane domain is a well-known
negative regulatory domain containing at least three
regulatory sites: (1) a ligand-dependent phosphorylated
tyrosine residue (Y1003) involved in receptor degrada-
tion through recruitment of the ubiquitin ligase CBL
proto-oncogene (CBL), (2) a phosphorylated serine res-
idue (S985) involved in downregulation of MET kinase
activity, and (3) a caspase cleavage site involved in MET
degradation during apoptosis. Thus, MET exon 14
splicing and subsequent juxtamembrane domain skip-
ping result in constitutive MET activation (for a review
see Cortot et al.¹⁶).

Importantly, several case reports describe objective
responses to MET-targeting TKIs in patients with such
mutations, and preliminary results of a phase I clinical
trial reported a 39% objective response rate with cri-
zotinib.¹⁵,¹⁷⁻²⁷ Thus, identifying NSCLC patients
harboring *MET* exon 14 splice site mutations has become
an important goal. Given the expected low rate of such
mutations, clinicians need a sensitive routine method for
detecting them to identify all patients liable to benefit
from MET TKI treatment.

MET exon 14 splice site mutations have been found mainly by whole-exome sequencing, large-hybrid-capture-based NGS, or Sanger sequencing of MET.¹⁴,¹⁵,²⁸⁻³⁰ Such technologies are not applicable in routine practice because of high cost, the need to use large tumor samples, or a lack of sensitivity. In contrast, targeted NGS, now widely implemented in molecular laboratories, allows comprehensive routine molecular profiling of tumor samples enabling the detection of known altered oncogenes and tumor suppressors. Commercially available panels, however, are not suitable for detecting the wide range of *MET* exon 14 splice site mutations because of the great diversity and specific localization of these alterations.³¹

Here we describe a method for detecting *MET* exon 14 splice site mutations. It can be implemented in routine practice and is based on combining fragment-length analysis with optimized targeted NGS.

Materials and Methods

Tumor Samples and DNA/RNA Extraction

Formalin-fixed paraffin-embedded (FFPE) samples
from non-small cell lung tumors were received in our
laboratory between October 2015 and December 2017,
in the context of daily practice. Before molecular anal-
ysis, a hematoxylin-and-eosin-stained slide was pre-
pared from each tumor and examined by a pathologist.
The pathologist evaluated the tumor cell content. If
easily feasible, macrodissection was performed to
enhance the tumor cell content. DNA was extracted from
FFPE tumor blocks. Briefly, 10-µm-thick sections were
cut from each block. After removing the paraffin with
Deparaffinization Solution (Qiagen, Courtaboeuf,
France), DNA was extracted with the QIAamp FFPE tis-
sue kit (Qiagen) according to the manufacturer's in-
structions. Extracted DNA was quantified with the
Quant-it PicoGreent dsDNA Assay Kit (Thermofisher
Scientific, Courtaboeuf, France) on a Xenius XC spectro-
fluorometer (Safas, Monaco). Immunohistochemistry
(IHC) (for MET expression) and fluorescence in situ
hybridization (FISH) (for detection of MET gene ampli-
fication) were performed on 4-µm-thick FFPE sections.
RNA was extracted from three 20-µm-thick sections of
each available FFPE tumor block. This was done with the Total RNA FFPE tissue kit (Macherey-Nagel, Inc., Bethlehem, Pennsylvania) according to the manufacturer's instructions.

## NGS Panel Design

The panel designed for FFPE samples was developed with the AmpliSeq designer software (Thermofisher Scientific) with white glove assistance to cover some positions. These targets were partially selected from the Ion AmpliSeq Colon and Lung Research Panel V2, and the panel was optimized to cover the whole *MET* exon 14 to detect most of the described variants. Additional targets included partial or entire exons of the following selected genes: ACVR1, AKT1, ALK, BRAF, CTNNB1, EGFR, ERBB2, ERBB4, FGFR1, FGFR2, FGFR3, GNA11, GNAQ, GNAS, H3F3A, H3F3B, HIST1H3B, HIST1H3C, HRAS, IDH1, IDH2, KRAS, KIT, MAP2K1, MET, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, SMAD4, and TP53. The designed panel named CLAPv1 is provided in Supplementary Table S3.

## Library Preparation

AmpliSeq libraries were prepared with the Ion AmpliSeq Library Kit 2.0, the Ion Xpress Barcode Adapter Kit, and primers (Ion AmpliSeq custom panel or Colon and Lung V2) (Thermofisher Scientific). Ten nanograms of each DNA sample were used as template to prepare the library according to the manufacturer's instructions. Quality control and quantification of all libraries were performed on the Agilent 2200 (Agilent Technologies, Inc., Wilmington, Delaware) Tapestation using the High Sensitivity D1000 ScreenTape assay. After normalization, the libraries were pooled. Emulsion polymerase chain reaction (PCR) was used to clonally amplify the obtained library pool, diluted to 65 µmol/L. Emulsion PCR, enrichment, and chip loading were performed on an Ion Chef instrument with the Ion P Hi-C Chef Kit or the Ion 54 Kit-Chef (Thermofisher Scientific). Samples were sequenced using Ion P1 chips on the Ion Proton System or Ion 540 chips on the Ion S5XL system (Thermofisher Scientific). Forty-eight barcoded samples were loaded per chip, that is, 45 patient tumor samples and 3 control samples. A positive control, a negative control, and a nontemplate control were analyzed on each run to ensure the analytical performance of the assay. Data were analyzed with Torrent Suite Software v.5.2.2 (Thermofisher Scientific). Variant calling was performed with optimized settings (available on demand). The variants obtained were annotated with the Variant Effect Predictor.³² Control sample analysis and quality metrics were checked on each sample to avoid false results: mean read length greater than 108 bp and number of variants less than 35. If these quality metrics were not achieved, the

sample was considered not interpretable. The lower limit of detection for our NGS assays was approximately 5% mutated DNA (i.e., 10% tumor cells). If a sample contained less than 10% tumor cells and no mutation on any driver gene, it was considered not interpretable (risk of false-negative results). For a wild-type (WT) call, a minimum of 500× was required.

## IHC

For MET IHC staining, the Ultra automated slide processing system (Ventana Roche, Tucson, Arizona) was used according to the manufacturer's protocol, with rabbit monoclonal primary antibody against MET (CONFIRM Anti-Total MET, clone SP44, Ventana), as previously described.³³

## FISH

Gene copy number (GCN) and *MET* gene amplification were determined by FISH as previously described.³⁴ FFPE tissue sections were hybridized with SPEC Zytolight Dual Color probe *MET/CEN7q* (Zytovision ref Z-2087-200 GmbH, Bremerhaven, Germany) according to the manufacturer's instructions.

Evaluation of *MET* GCN was performed on the whole slide for each sample. Signals were counted in at least 100 nonoverlapping nuclei per slide, with an epifluorescence microscope (Olympus BX63) and an automated imaging application (BioView, Abbott, Berkshire, United Kingdom). The following data were recorded for each sample: mean *MET* GCN per cell and the *MET/CEN7* ratio.

Tumors were classified according to the *MET* amplification status, which provides the clearest definition of a *MET*-GCN-gain-addicted state. This status was defined as follows: high-level amplification, when the *MET/CEN7* ratio was greater than or equal to 5 or when 10% or more of the tumor cells displayed clusters or 15 or more copies of the *MET* gene; intermediate-level amplification when the *MET/CEN7* ratio was greater than 2.2 but less than 5; low-level amplification when the *MET/CEN7* ratio was 1.8 or more but less than or equal to 2.2. All other tumors were classified as negative.³⁵

## Fragment-Length Analysis

Intron sequences flanking exon 14 of *MET* were amplified with two primer pairs in one tube. The primer pair for *MET1* was: *MET1_F* 5'-CATGAGTTCTGGG CACTGG, *MET1_R* 5'-6-FAM-CTTCTGGAAAAGTAGCTCGG and the primer pair for *MET2* was: *MET2_F* 5'-TAAGTGCCCGAAGT GTAAGC, *MET2_R* 5'-HEX-CAA CAATGTCACAACCCACTG. PCR was performed in a 25-µL final volume containing 1 × Multiplex Master Mix (Qiagen), 0.4 µmol/L *MET1* primer pair, 0.2 µmol/L *MET2*
primer pair, and 5 to 25 ng DNA. The cycling conditions were: 95°C for 15 minutes, followed by 33 cycles of 95°C for 30 seconds, 62°C for 30 seconds, 72°C for 30 seconds, and a final extension of 72°C for 10 minutes. Fluorescent amplified products were separated on an ABI Prism 3130XL analyzer (Thermofisher Scientific). Data were interpreted with GeneMapper software version 4.1 (Thermofisher Scientific) followed by visual inspection. The lower limit of detection of the fragment analysis assay was approximately 10% mutated DNA (i.e., 20% tumor cells).

## Real-Time Reverse Transcribed-PCR and Melting Curves

Total cDNA was reverse-transcribed from total RNA with random hexamers, using the High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Foster City, California). Two primer pairs in exons flanking exon 14 of *MET* were designed to generate fragments of different sizes and melting temperatures for wild type *MET* and *MET* displaying exon 14 skipping. The primer pair A was: MET3: 5'-GGTTTTTCCTGTGGCTGAAA, MET4: 5'-GGCATGAACCGTCTGAGAT and the primer pair B was: MET5: 5'-CCAGATCAGAATTTCACAGG and MET4. Fragments were amplified with Fast SYBR Green mix (Applied Biosystems) in an Mx3005P instrument (Agilent), as described by the supplier. The cycling conditions were: 95°C for 5 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and a final cycle 95°C for 1 minute, 55°C for 30 seconds and 95°C for 30 seconds. The dissociation curves of the amplified products were analyzed with MxPro software version 4.10. The PCR products were separated on 2.0% agarose gels and visualized under ultraviolet light.

## Copy Number Analysis of the NGS Data

The count assigned to each amplicon for each sample was computed from bam files with bedtools coverage [bedtools], taking into account only reads showing a 95% overlap with the amplicon.³⁶ Log read counts were normalized on the basis of the GC percentage and size of each amplicon, the Guanine Cytosine (GC) percentages of the forward and reverse primers, and also the GC percentage of a 150-kb window around the amplicon as in [WACA].³⁷ We also normalized the read count with respect to the derivative log ratio spread (DLRS) introduced by Agilent Technologies for comparative genomic hybridization arrays to take DNA quality into account. All normalizations were performed with a multiple linear model in one step. The log2 ratio of each amplicon for each sample was computed as the log2 of the number obtained by dividing the normalized read count for a particular sample by the median read count of all

samples. Segmentation of the log2 ratio was performed with the CNA package of R, implementing the circular binary segmentation method.³⁸ We only kept segments with a mean log2 ratio above max (1.5 × DLRS, 1) or below min (-1.5 × DLRS, -0.5) and encompassing at least 3 amplicons.

## Results

### Optimization of Targeted NGS and Fragment Analysis for Detecting MET Exon 14 Splice Site Mutations

Between November 2015 and August 2017, 1826 FFPE tumor samples from 1722 NSCLC patients were received in our laboratory for molecular screening as part of its routine practice. All samples were tested with the "Colon&Lung V2" NGS panel. For 208 of the 1722 patients (12%), the sequencing data were not interpretable because of poor DNA quality or insufficient quantity (138 patients) or a low number of tumor cells (70 patients). Among the 1514 remaining patients, the main mutations detected were *KRAS* (36.2%), *EGFR* (13.1%), *BRAF* (4%), and *ERBB2* (0.3%) mutations (Fig. 1A). *MET* exon 14 splice site mutations were detected in 4 patients (0.3%) (Fig. 1A, Supplementary Table 1A).

To optimize detection of the wide range of *MET* exon 14 splice site mutations, we developed two alternative molecular screens, one based on fragment-length analysis and one on optimized targeted NGS. We set up a fragment-length analysis with primers surrounding either the 5' (225 bp) or the 3' end (142 bp) of exon 14, covering 91% of the previously identified deletions (Fig. 2).¹⁵ Among the 1514 samples screened by fragment-length analysis and yielding interpretable results, 16 *MET* exon 14 splice site mutations (1.1%) were detected, including mutations in both the 5' and 3' ends of exon 14 (Fig. 3A, Supplemental Table 1A). Differences between mutant and WT alleles ranged from 1 to 55 bp. Representative fragment analyses of WT and mutant samples are shown (Supplemental Figs. 1A, B, and C). One complex indel (patient 21) detected with the Colon&Lung V2 panel (mutant allele frequency: 3%) (Supplemental Table 1A) was not picked up by the fragment analysis.

An optimized NGS panel named CLAPv1 (Colon&Lung Adapted Panel Version 1) was derived from the Colon&Lung V2 panel. It contained 182 amplicons ranging from 62 to 154 bases in size (mean, 112 bases) and spanning 12.93 kb of the human genome (hg19) in 2 pools. This CLAPv1 panel was notably designed to better cover *MET* exon 14 splice site mutations, using 3 amplicons respectively covering the 140 bp of exon 14, 97 bp of intron 13, and 36 bp of intron 14. These amplicons thus cover 87% of the *MET* exon 14 alterations previously described (Fig. 2).¹⁵ The CLAPv1 panel was also designed to allow
<figure><img src="image_44.png" /><figcaption>**Figure 1.** Percentage of genomic alterations found in NSCLC patients according to different next-generation sequencing (NGS) panel assays. The pie charts show the percentages of main genomic alterations found by the targeted NGS panels (A) Colon&Lung V2 on 1514 tumor samples and (B) CLAPv1 on 365 tumor samples. WT (wild-type) indicates the absence of any mutation in *EGFR*, *KRAS*, *BRAF*, *ERBB2*, and *MET*.</figcaption></figure>

detection of all main molecular targets previously covered by the Colon&Lung V2 panel, including key genes involved in NSCLC and colorectal cancer, melanoma, stromal gastrointestinal tumors, and glioma.

To assess the efficacy of using the CLAPv1 panel to detect *MET* exon 14 alterations while maintaining accurate detection of all the other targeted genomic regions, we selected 52 samples already known to harbor various mutations, including 20 samples harboring *MET* exon 14 splice site mutations. These 52 samples were sequenced again with CLAPv1. Coverage depth analysis of the first sequencing run showed efficient sequencing with an average of 8510× (Supplemental Fig. 2). Only 3 amplicons among the 182 amplicons had a median coverage depth below 500×, corresponding to *ERBB2* exon 19: AA 761 to 769 (NM_004448.3) and *FGFR3* exons 6 and 9: AA 229 to 247 and 369 to 375 (NM_000142.4) (Supplemental Fig. 2). Eighteen of the 20 *MET* exon 14 splice site mutations were detected with the CLAPv1 panel. The two remaining mutations were two large deletions (50 and 55 bp, patients 14 and 15) preventing primer hybridization (Supplemental Table 1A). Importantly, these two deletions were detected by fragment-length analysis, suggesting that the two techniques are complementary. Furthermore, all known mutations in other genes were confirmed with the CLAPv1 panel (data not shown). For example, 10 *EGFR* mutations with allelic fractions between 3.5% and 88% were picked up.

## Routine Detection of *MET* Exon 14 Mutations in NSCLC Cancer Patients

On the basis of these results, both the CLAPv1 panel and fragment-length analysis were further implemented in routine practice for molecular testing of NSCLC

patients. Among 402 samples analyzed over 5 months, 365 samples (91%) had interpretable sequencing results, among which 8 tumors (2.2%) displayed *MET* exon 14 splice site mutations. All were found with the CLAPv1 panel and 4 of them were also detected by fragment-length analysis (Fig. 3B and Supplemental Tables 1A and B). Other oncogenic drivers detected in this series of patients included 47 *EGFR* mutations (12.9%), 133 *KRAS* mutations (36.4%), 18 *BRAF* mutations (4.9%), and 3 *ERBB2* mutations (0.8%) (Fig. 1B).

Altogether, *MET* exon 14 splice site mutations were found in 30 patients (Fig. 4). The CLAPv1 panel assay detected 26 mutations among the 28 samples tested (93%), and combining fragment-length analysis with the CLAPv1 panel assay allowed detection of 100% of the mutations (Fig. 4). The main clinical characteristics of the 30 *MET*-mutated NSCLC patients are presented in Table 1 (see also Supplemental Table 2 for individual patient data).

## Validation of *MET* Exon 14 Skipping

To assess *MET* exon 14 skipping in the patients displaying splice site mutations, mRNA was extracted from seven tumor samples of six patients and analyzed by SYBR green fluorescence real-time PCR. We designed two sets of specific primers both able to discriminate splicing isoforms on the basis of the melting curves of the PCR products. PCR with the primers set A from a control WT *MET* FFPE sample or from EBC1 cell line having a WT *MET* isoform, amplified a 216 bp product and the melting curve analysis revealed a single peak at 77.4°C. The amplicon size of PCR from Hs746T and H596 cells, showing *MET* exon 14 skipping was 58 bp and the melting curves displayed a separate peak at
<figure><img src="image_45.png" /><figcaption>**Figure 2.** Positions of the amplicons of targeted next-generation sequencing (NGS) panels and fragment analysis products below *MET* exon 14. Exon 14 of *MET* (140 bp located between genomic positions g.1164121902 and g.1164122042 on *Homo sapiens* chromosome 7, GRCh37, GenBank: CM000669.1 or between cDNA positions c.2942 and c.3082 NM_00127500.1) and its flanking intronic regions are represented, with its coverage by the single amplicon of the Colon&lung V2 panel and by the three amplicons of the CLAPv1 panel. Fragment analysis products covering 225 bp of the 5' end and 144 bp of the 3' end of exon 14 are also shown.</figcaption></figure>

approximately 72°C (Supplemental Figs. 3A and C). PCR with the primers set B generated a 286 bp amplicon, from the control WT *MET* FFPE sample or cell line, with a dissociation temperature of 77.3°C and a 128 bp amplicon from the *MET* exon 14 skipping cell lines with a dissociation temperature of 74.8°C (Supplemental Figs. 3B and D). For six patients, paraffin-embedded tumor material was available (for patient 7, two samples were available and thus analyzed) and messenger RNAs were extracted, retrotranscribed and analyzed by PCR. For five patients of six, analysis showed exon 14 skipping with both primers sets. For patient 14, the exon 14 skipping was revealed with the primers set A, whereas primer set B did not allow PCR product detection. These results show that the identified genomic mutations of these six patients lead to *MET* exon 14 skipping. Furthermore, the mutations of eight patients have been

previously reported to induce exon 14 skipping on RNA (Supplemental Table 1) and the remaining 17 mutations were predicted to induce exon 14 skipping based on in silico tools.<sup>14,19,21,23,39</sup> Thus, all identified mutations are anticipated to induce *MET* exon 14 skipping.

MET Expression Level and MET GCN in Tumors
Harboring Exon 14 Splice Site Mutations

To evaluate *MET* expression in patients harboring *MET* exon 14 splice site mutations, IHC staining of *MET* was performed on the available paraffin-embedded tumor slides. Of 30 samples, 25 were available for IHC analysis. The samples were divided into 4 grades, from no to high expression. Two patients did not express *MET* (IHC 0) and 7 patients showed low-level expression (IHC 1+). *MET* overexpression (IHC 2+ and 3+) was found in
A n = 1514

<figure><img src="image_46.png" /></figure>

B n = 365

<figure><img src="image_47.png" /><figcaption>Figure 3. Percentage of tumors with *MET* exon 14 splice site mutations found in NSCLC patients by means of next-generation sequencing (NGS) panel assays and fragment analysis. (A) Percentage of tumor samples displaying *MET* exon 14 mutations as determined on 1514 NSCLC tumor samples by Colon&Lung V2 or fragment-length analysis. (B) Percentage of tumor samples displaying *MET* exon 14 mutations by CLAPv1 and fragment-length analysis combined, as determined on 365 NSCLC tumor samples.</figcaption></figure>

16 of 25 patients (64%) (Fig. 4 and Supplemental Tables 1A and B). Next, we performed FISH analysis on the 25 available paraffin-embedded tumor slides. Three patients displayed *MET* gene amplification (*MET/CEN7* ratio >1.8 or the presence of clusters) and one displayed an increased *MET* gene copy number (>5 copies) but no significant *MET/CEN7* ratio increase. All four patients displayed *MET* overexpression (IHC 2+ or 3+) (Fig. 4 and Supplemental Tables 1A and B).

In parallel, CLAPv1 sequencing data were analyzed to
measure potential *MET* GCN increases on the basis of

read counts. Read count analysis was performed on 23 of
the 25 samples for which FISH results were available. It
revealed two samples (patients 20 and 22) with a sig-
nificant *MET* copy number increase (Fig. 4 and
*Supplemental Tables* 1A and B). According to the FISH
analysis, one of these patients displayed *MET* gene
amplification (patient 20), and the other, a high copy
number without any *MET/CEN7* ratio increase (patient
22). Patient 29 displayed *MET* gene amplification ac-
cording to the FISH analysis, whereas the read count did
not reveal any significant *MET* gene copy number

<figure><img src="image_48.png" /><figcaption>Figure 4. Molecular features of the 30 patients harboring somatic *MET* exon 14 alterations. Synthetic view of the *MET* exon 14 mutations detected (yellow squares) or not (grey squares) by the Colon&Lung V2 panel assay or by fragment-length analysis and CLAPv1 panel. Tumors examined by immunohistochemistry were each attributed one of four scores (0 to 3+) according to their level of *MET* expression. Score 0: no expression; score 3+ maximal expression. Samples displaying *MET* gene amplification (as determined by fluorescence in situ hybridization [FISH]) or an increased copy number (as determined by FISH or by counting reads from the NGS CLAPv1 panel) are indicated by red squares. ND, not determined.</figcaption></figure>