CASE SERIES

RET Rearrangement as a Predictorof Unresponsiveness to Immunotherapy in Non-SmallCell Lung Cancer: Report of Two Cases with Reviewof the Literature

Sara Baglivo . Vienna Ludovini . Riccardo Moretti . Guido Bellezza .

Angelo Sidoni . Fausto Roila . Giulio Metro

Received: April 2, 2020� The Author(s) 2020

ABSTRACT

Patients with epidermal growth factor receptorand anaplastic lymphoma kinase positive non-small cell lung cancer (NSCLC) generallyrespond poorly to treatment with immunecheckpoint inhibitors such as anti-programmedcell death-1 (PD-1) or anti-programmed celldeath ligand-1 (PD-L1) given with or without

anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) drugs. However, the efficacy ofimmunotherapy in patients with oncogene-ad-dicted NSCLC harboring minor drivers, such asfusions in the rearranged during transfection(RET) gene, is still unclear. Here we describe twopatients with RET-positive advanced NSCLCwith PD-L1 expression C 50% who developedprogressive disease during first-line treatmentwith the anti-PD-1 agent pembrolizumab. Inparticular, while patient 2 was immediatelyswitched to treatment with a selective RETinhibitor within the setting of a clinical trial,patient 1 responded to cytotoxic chemotherapydelivered at the time of progression while onpembrolizumab. These cases of NSCLC are dis-cussed in the context of current literature,which seems to support our observation thatpatients with RET-positive NSCLC are unlikelyto benefit from immunotherapy. Therefore, wesuggest that for RET-positive patients with PD-L1 C 50%, consideration should be given toupfront treatment approaches other than sin-gle-agent immunotherapy, namely selectiveRET inhibitors (if available) or regimensincluding cytotoxic chemotherapy.

Keywords: Hyper-progressive disease;Immunotherapy; Non-small cell lung cancer;PD-L1 C 50%; Pembrolizumab; RET

Digital Features To view digital features for this articlego to https://doi.org/10.6084/m9.figshare.12162030.

S. Baglivo � V. LudoviniLaboratory of Oncology, Medical Oncology, SantaMaria della Misericordia Hospital, AziendaOspedaliera di Perugia, Perugia, Italy

R. MorettiDepartment of Radiology, Santa Maria dellaMisericordia Hospital, Azienda Ospedaliera diPerugia, Perugia, Italy

G. Bellezza � A. SidoniDepartment of Experimental Medicine, Division ofPathology and Histology, University of PerugiaMedical School, Perugia, Italy

F. Roila � G. Metro (&)Medical Oncology, Santa Maria della MisericordiaHospital, Azienda Ospedaliera di Perugia, Perugia,Italye-mail: giulio.metro@yahoo.com

Oncol Ther

https://doi.org/10.1007/s40487-020-00116-2

Key Summary Points

Rearranged during transfection (RET) generearrangements are found inapproximately 2% of patients with non-small cell lung cancer (NSCLC).Importantly, the presence of theserearrangements predicts patient responseto treatment with selective RET inhibitors.

The efficacy of immune checkpointinhibitors in RET-positive NSCLC,however, has not yet been sufficientlyestablished.

We describe two patients with RET-positive advanced NSCLC with anti-programmed cell death-ligand 1 (PD-L1)C 50% who experienced progressivedisease on first-line immunotherapy withpembrolizumab.

Our data, together with data reported inthe literature, suggest that patients withRET-positive NSCLC are unlikely tobenefit from immunotherapy.

Rather, selective RET inhibitors (ifavailable) or cytotoxic chemotherapy(with or without immunotherapy) shouldbe considered as the preferred treatmentoption in this context, regardless of thePD-L1 status of the tumor.

INTRODUCTION

In the last 15 years, advances in the molecularcharacterization of non-small cell lung cancer(NSCLC) have led to the identification of anumber of genetic alterations that config-ure molecularly defined subgroups of oncogene-addicted disease that is strictly dependent onthe presence of a single actionable driver [1].Epidermal growth factor receptor (EGFR)mutation and anaplastic lymphoma kinase(ALK) gene rearrangements were the first onco-genic drivers to be identified, and established

targeted therapies of these rearrangements havebeen established, with excellent results. Morerecently, minor drivers, such as ROS proto-oncogene 1 receptor tyrosine kinase (ROS1)fusions, v-raf murine sarcoma viral oncogenehomolog B1 (BRAF) mutations, human epider-mal growth factor receptor 2 (HER2) mutations,hepatocyte growth factor receptor (MET) genealterations, neurotrophin receptor kinase(NTRK) fusions, and rearranged during trans-fection (RET) fusions, have emerged as otherimportant actionable alterations [1]. In partic-ular, RET rearrangements have been recentlyadded to the list of druggable target pathways,since selective RET inhibitors have shownimpressive clinical activity in early clinical trials[2–4]. However, it is imperative that the onco-logic community know whether therapies suchas chemotherapy and/or immune checkpointinhibition (ICI) with anti-programmed celldeath-1 (PD-1) or anti-programmed cell deathligand-1 (PD-L1), with or without anti-cytotoxicT lymphocyte antigen-4 (CTLA-4) agents, areeffective in treating oncogene-addicted NSCLCin order to select the best treatment strategy.While the unresponsiveness to immunotherapyof EGFR- and ALK-positive NSCLCs has beenwell described, the efficacy of ICI treatment inpatients whose tumor harbors a minor onco-genic driver, such as a RET rearrangement, isstill uncertain [5]. Here, we describe twopatients with RET-positive advanced NSCLCwith PD-L1 expression C 50% who progressedon single-agent immunotherapy with the anti-PD1 agent pembrolizumab administered as first-line treatment. We also discuss these cases inthe context of the current literature.

Written informed consent was provided byboth patients to published their respective casein an anonymous form.

CASE 1

A 59-year-old man, a never smoker, was diag-nosed by trans-bronchial biopsy during bron-choscopy with a TTF1-positive lungadenocarcinoma of the right upper lobe. Thetumor was classified as stage T4 with mediasti-nal infiltration, N2, and M1c for multiple

Oncol Ther

hepatic and bone lesions based on computedtomography (CT) scan of the thorax and abdo-men (Fig. 1a). Magnetic resonance imaging(MRI) of the brain revealed an asymptomaticleft-sided occipital lesion of 8 mm without sur-rounding edema. Immunohistochemistry anal-ysis showed a PD-L1 tumor proportion score(TPS) of 60% (clone 22C3) (Fig. 2a), but nostaining for ALK (clone D5F3) and ROS1 (cloneD4D6). Next generation sequencing (NGS) (IonAmpliseq Colon and Lung Research Panel v.2;Illumina, Inc., San Diego, CA, USA) showed thatthere was no EGFR mutation. Therefore, basedon PD-L1 expression C 50%, the patient wasadvised to start treatment with pembrolizumabmonotherapy at the flat dose of 200 mg intra-venously (i.v.) every 3 weeks. After only onecycle of therapy, however, he complained ofworsening of pre-existing dyspnoea, with bloodchemistry analysis showing mild alterations inthe liver function tests. A new CT scan of thethorax and abdomen showed unequivocal

progressive disease in both the lung (Fig. 1b)and liver. At that time, NGS for fusion tran-scripts was performed (Ion Ampliseq RNAFusion Lung Cancer Research Panel), with theresults being compatible with the presence of aRET rearrangement (KIF5B) (Fig. 2b). As thepatient was experiencing symptomatic progres-sion with possible hyper-progressive disease,treatment with pembrolizumab was stoppeddespite the patient having received only onecycle, and treatment was initiated with thecytotoxic agents carboplatin AUC 6 ? paclitaxel90 mg/m2 i.v. on days 1, 8 and 15, with a cycleof chemotherapy every 3 weeks. After two cyclesthe patient reported an improvement in dysp-noea, while liver function tests showed pro-gressive resolution; consistent with theseresults, a CT scan showed response to treatment(Fig. 1c). Based on his improved clinical condi-tion, the patient was advised to start treatmentwith the selective RET-inhibitor pralsetinib on aonce-daily dosing regimen of 400 mg taken

Fig. 1 a Computed tomography scan showing solid tissuelocalized at the lung hilum that infiltrates the right upperlobe bronchus with concomitant atelectasis of pulmonaryparenchyma. b Progression of the solid tissue with anincrease in atelectasis after one cycle of pembrolizumab.c Reduction in both the size of solid tissue and pulmonary

parenchyma after two cycles of chemotherapy. d Furtherreduction in the size of solid tissue with absence ofatelectasis after 3 weeks of treatment with the RET(rearranged during transfection) inhibitor pralsetinib

Oncol Ther

orally within an expanded access program. Anew CT scan of the thorax and abdomen andbrain MRI performed only 3 weeks after theinitiation of treatment with pralsetinib showednearly complete resolution of the lung lesion(Fig. 1d), partial response in the liver, andreduction in the size of the brain lesion.

CASE 2

A 53-year-old woman, a never smoker, who hadbeen complaining of a dry cough during theprevious 2 months, underwent a CT scan of thethorax and abdomen that revealed the presenceof a lung lesion of the left upper lobe withhomolateral lymph-node enlargement at sta-tion 4L. A MRI of the brain revealed a smallcortical lesion of 7 mm located at the leftparasagittal site with mild edema, for which shewas asymptomatic. Positron emission tomogra-phy/CT revealed no other extra-cranial lesions.An endobronchial ultrasound bronchoscopywith fine needle aspiration of the station 4Llymph node was performed, and the resultswere compatible with TTF1-positive adenocar-cinoma. Immunohistochemistry analysisrevealed the following disease profile: PD-L1

TPS score of 55% (clone 22C3), ALK negative(clone D5F3), and ROS1 negative (clone D4D6).No EGFR mutation was detected by NGS (IonAmpliseq Colon and Lung Research Panel v.2).Based on the high PD-L1 expression the patientwas advised to start first-line treatment withpembrolizumab at the flat dose of 200 mg i.v.every 3 weeks. After one cycle, the patientcomplained of worsening of the cough withonset of occasional hemoptysis, while a new CTscan showed unequivocal progressive disease atboth the primary tumor and mediastinal lymphnode. As in case 1, rapid worsening of clinicalconditions after only one cycle of pem-brolizumab suggested the presence of hyper-progressive disease. At that time, a more com-prehensive mutational analysis of fusion genes(Ion Ampliseq RNA Fusion Lung CancerResearch Panel) revealed the presence of a RETrearrangement (KIF5B). As a result, the patientwas enrolled into a phase 1/2 clinical trialevaluating the selective RET inhibitor pralse-tinib (BLU-667) in RET-positive advancedmalignancies (‘ARROW’ trial, ClinicalTrials.govIdentifier: NCT03037385).

Fig. 2 a High expression (tumor proportion score: 60%)of programmed cell death ligand-1 (PD-L1; clone 22C3)in neoplastic cells; for comparison, see negative staining innormal epithelium (lower left corner). b Next generationsequencing showing the presence of fusion transcript

KIF5B–RET (K15; R12)–COSF1232. The gene rear-rangement involves the fusion of KIF5B exon 15 and RETexon 12

Oncol Ther

DISCUSSION

RET rearrangements are found in approximately2% of patients with NSCLC [2]. If present, theyconfer a survival advantage to the tumorthrough constitutive activation of multipledownstream signaling pathways. RET rear-rangements are more commonly found inyoung patients who have a never/light smokinghistory [2], as was the case for the two RET-positive patients described here. Importantly,knowing the baseline RET status of patientswith newly diagnosed advanced NSCLC is ofparamount importance as selective RET inhibi-tors with impressive clinical efficacy in treatingRET-positive disease have recently becomeavailable. Pralsetinib and selpercatinib are twohighly selective RET inhibitors that haveachieved outstanding and durable responserates of 60–68% in patients with RET-positiveNSCLC [3, 4]. However, the current availabilityof these drugs in the clinical setting is quitelimited, and they are mostly accessible throughparticipation in a clinical trial or throughexpanded access programs.

Against this background, it is important topinpoint the sensitivity of RET-positive NSCLCtumors to treatments that are currently

available, such as cytotoxic chemotherapy orICI with anti-PD-(L)1 as a single agent or incombination with anti-CTLA-4. With regard tocytotoxic treatments, RET-positive tumorsappear to be particularly sensitive to peme-trexed-based chemotherapy, with responsesobserved in 45–63% of patients and medianprogression-free survival ranging from 9.0 to19.0 months [6–8]. However, it is still unclearwhether RET-positive NSCLC is sensitive to ICItreatment. We performed a literature search onthis subject and identified three publishedreports describing the response of RET-positiveNSCLC to ICI treatment administered either asmonotherapy or as dual combination therapy(Table 1) [9–11]. Two of these reports describevery disappointing results for ICI treatment,with responses in only a very few patients. Inline with these findings, both patients in thepresent report experienced hyper-progressivedisease during first-line treatment with the anti-PD1 agent pembrolizumab. When analyzedcumulatively with other studies, we found adisappointing response rate of 10.2% (4/39), astable disease rate of 20.6% (10/39), and a pro-gressive disease rate as high as 64.1% (25/39).Therefore, it is likely that RET-positive tumors,similar to other oncogene-addicted NSCLC

Table 1 Studies reporting on the efficacy of immunotherapy in RET (rearranged during transfection)-positive patients

First authorand year

Immunecheckpointinhibition

No. ofpatientsa

Partialresponse

Stable disease Progressivedisease

Medianprogression-freesurvival

Medianoverallsurvival

Offin et al.

(2019) [9]

Anti-PD-

(L)1 ± anti-

CTLA-4

13 0 5 8 3.4b NR

Mazieres et al.

(2019) [10]

Anti-PD-(L)1 16 1 3 12 2.1 21.3

Guisier et al.

(2020) [11]

Anti-PD-1 8 3 2 3 7.6 NR

Present

report

Anti-PD-1 2 0 0 2 – –

CTLA-4 Cytotoxic T lymphocyte antigen-4, NR not reported, PD-1 programmed cell death-1, PD-L1 programmed celldeath ligand-1a Evaluable for responseb On 16 patients

Oncol Ther

subtypes, belong to a poorly immunogenicsubgroup in which a single mutation is assumedto act as driver of the respective cancer cells inthe context of a poorly immunogenic tumormicroenviroment [12]. In line with thishypothesis, RET-positive NSCLC has been asso-ciated with low levels of tumor mutationalburden (TMB), which is considered a biomarkerof sensitivity to immunotherapy [9]. In fact, bycomparing RET-positive NSCLC subtypes withwild-type RET cancerous tumors, Offin et al.found a significantly lower median TMB forRET-positive cases. However, it is unknown ifother molecular features might have con-tributed to the hyper-progression on pem-brolizumab of our two patients. Recent datasuggest that simultaneous PD-1 and PD-L1expression in tumor cells could induce tumorsuppression, which is antagonized by the use ofanti-PD-(L)1 agents [13, 14]. Therefore, in theabsence of an adaptive immune system, whichcould be the case of patients with RET-positiveNSCLC lesions, anti-PD-(L)1 agents may pro-mote resistance to treatment.

Having said this, the question of whetherPD-L1 status retains significance in determiningsensitivity to ICI treatment in RET-positiveNSCLC patients is relevant, as two competingstandards exist in this setting, either pem-brolizumab alone or pembrolizumab in combi-nation with platinum-based chemotherapy.Importantly, PD-L1 does not appear to beexpressed at high levels in RET-positive NSCLC;in the study by Offin et al., of 26 cases of RET-positive NSCLCs, only 19% (5/26) had PD-L1expression C 50% [9]. Nevertheless, the ques-tion of will PD-L1 status impact the choice offirst-line treatment in RET-positive NSCLClesions remains. With regard to this, the twoRET-positive cases presented herein are inter-esting because, despite the presence of PD-L1levels C 50%, both were both unresponsive totreatment with the single-agent pem-brolizumab. Similarly, progressive disease asbest response was reported by Offin et al. in theonly patient with PD-L1 expression C 50%treated with ICI [9].

In summary, we conclude that knowing theRET status at baseline in newly diagnosedadvanced NSCLC patients is crucial, especially

in those individuals with PD-L1 C 50%. In thelatter case, in countries with limited access to aselective RET-inhibitor, consideration should begiven to the administration of chemotherapywith or without pembrolizumab rather thanpembrolizumab alone, as these tumors are veryunlikely to respond to ICI treatment. In fact,our cases suggest that PD-L1 status seems tohave little predictive value in patients with RET-positive NSCLC as hyper-progressive disease wasnoted with pembrolizumab as the single agentdespite the presence of PD-L1 TPS C 50%. Inaddition, knowing the poor responsiveness ofRET-positive NSCLC to ICI treatment regardlessof PD-L1 status can also help guide drugsequencing for patients who require salvagetreatment at the time of progression on RET-inhibitors. In fact, RET-positive patients whohave not been treated with immunotherapy inthe first-line setting are unlikely to benefit fromICI treatment in later lines.

ACKNOWLEDGEMENTS

We thank the patients for giving their consentto publish their cases.

Funding. No funding or sponsorship wasreceived for this study or publication of thisarticle.

Authorship. All named authors meet theInternational Committee of Medical JournalEditors (ICMJE) criteria for authorship for thisarticle and take the responsibility for theintegrity of the work as a whole, and have giventheir approval for this version to be published.

Authorship Contributions. Fausto Roila andGiulio Metro were responsible for the concep-tion of the work, data collection, and manu-script writing. Sara Baglivo, Vienna Ludovini,Guido Bellezza, Angelo Sidoni, providedmolecular data of the patients. Riccardo Morettiprovided radiographic material. All authors readand gave their approval for the manuscript to bepublished in its final version.

Oncol Ther

Disclosures. Sara Baglivo, Vienna Ludovini,Riccardo Moretti, Guido Bellezza, AngeloSidoni, Fausto Roila, Giulio Metro have nothingto disclose.

Compliance with Ethics Guidelines. Writ-ten informed consent was provided from bothpatients for publishing their cases in ananonymous form.

Data Availability. All data generated oranalyzed during this study are included in thispublished article.

Open Access. This article is licensed under aCreative Commons Attribution-NonCommer-cial 4.0 International License, which permitsany non-commercial use, sharing, adaptation,distribution and reproduction in any mediumor format, as long as you give appropriate creditto the original author(s) and the source, providea link to the Creative Commons licence, andindicate if changes were made. The images orother third party material in this article areincluded in the article’s Creative Commonslicence, unless indicated otherwise in a creditline to the material. If material is not includedin the article’s Creative Commons licence andyour intended use is not permitted by statutoryregulation or exceeds the permitted use, youwill need to obtain permission directly from thecopyright holder. To view a copy of this licence,visit http://creativecommons.org/licenses/by-nc/4.0/.

REFERENCES

1. Dong J, Li B, Lin D, Zhou Q, Huang D. Advances intargeted therapy and immunotherapy for non-small cell lung cancer based on accurate moleculartyping. Front Pharmacol. 2019;10:230.

2. Subbiah V, Yang D, Velcheti V, Drilon A, Meric-Bernstam F. State-of-the-art strategies for targetingRET-dependent cancers. J Clin Oncol. 2020;38:1209–21.

3. Gainor JF, Lee DH, Curigliano G, et al. Clinicalactivity and tolerability of BLU-667, a highly potentand selective RET inhibitor in patients (pts) with

advanced RET-fusion? non-small cell lung cancer(NSCLC). J Clin Oncol. 2019;37:abstract 9008.

4. Drilon A, Oxnard G, Wirth L, et al. Registrationalresults of LIBRETTO-001: a phase 1/2 trial of LOXO-292 in patients with RET fusion-positive lung can-cers. J Thorac Oncol. 2019;14:abstract S6–S7.

5. Metro G, Di Maio M. Cons: should immunotherapybe incorporated in the treatment of oncogene-dri-ven lung cancer? Transl Lung Cancer Res.2018;7(Suppl 3):S294–S296296.

6. Drilon A, Bergagnini I, Delasos L, et al. Clinicaloutcomes with pemetrexed-based systemic thera-pies in RET-rearranged lung cancers. Ann Oncol.2016;27:1286–91.

7. Lee J, Ku BM, Shim JH, et al. Characteristics andoutcomes of RET-rearranged Korean non-small celllung cancer patients in real-world practice. Jpn JClin Oncol. 2020;50:594–601.

8. Shen T, Pu X, Wang L, et al. Association betweenRET fusions and the efficacy of pemetrexed-basedchemotherapy for advanced NSCLC patients inChina: a multicentre retrospective study. Clin LungCancer. 2020. https://doi.org/10.1016/j.cllc.2020.02.006.

9. Offin M, Guo R, Wu SL, et al. Immunophenotypeand response to immunotherapy of RET-rearrangedlung cancers. JCO Precis Oncol. 2019;3. https://doi.org/10.1200/PO.18.00386.

10. Mazieres J, Drilon A, Lusque A, et al. Immunecheckpoint inhibitors for patients with advancedlung cancer and oncogenic driver alterations:results from the IMMUNOTARGET registry. AnnOncol. 2019;30:1321–8.

11. Guisier F, Dubos-Arvis C, Vinas F, et al. Efficacy andsafety of anti-PD-1 immunotherapy in patients withadvanced non small cell lung cancer with BRAF,HER2 or MET mutation or RET-translocation. GFPC01-2018. J Thorac Oncol. 2020;15:628–36.

12. Mhanna L, Guibert N, Milia J, Mazieres J. When toconsider immune checkpoint inhibitors in onco-gene-driven non-small cell lung cancer? Curr TreatOptions Oncol. 2019;20:60.

13. Du S, Du S, McCall N, Park K, et al. Blockade oftumor-expressed PD-1 promotes lung cancergrowth. Oncoimmunology. 2018;7:e1408747.

14. Wang X, Yang X, Zhang C, et al. Tumor cellintrinsic PD-1 receptor is a tumor suppressor andmediates resistance to PD-1 blockade therapy. ProcNatl Acad Sci USA. 2020;117:6640–50.

Oncol Ther