Abstract

Background

   Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKI)
   acquired resistance was an inevitably events in NSCLC treatment.

Aims

   Intending to overcome the acquired resistance of EGFR‐TKI.

Materials & Methods

   A clinical trial was, we enrolled 12 patients who were slowly
   progressing on first‐generation EGFR‐TKI, and added apatinib when the
   patients got slow progression.

Results

   Seven patients were included in the efficacy analysis. The median PFS2
   of apatinib combined with EGFR‐TKI was 8.2 months (95% CI, 7.3 m‐NA),
   and the total PFS reached 20.9 months (95% CI, 17.3 m‐NA) when plus
   PFS1. All the adverse events were manageable. The median PFS was
   significantly longer for circulating tumor DNA (ctDNA)‐cleared patients
   (8.4 months; 95% CI, 8.2‐NA) than for those ctDNA not cleared
   (7.1 months; 95% CI, 6.9‐NA) (p = 0.0082).

Discussion

   The addition of apatinib did improve the duration of first‐generation
   EGFR‐TKI use, and the duration was better than the first‐line use of
   third‐generation EGFR‐TKI.

Conclusion

   The addition of apatinib when the patients got slow progression after
   initial EGFR‐TKI therapy may be a good treatment option and the side
   effects are controllable. It is possible to monitor treatment efficacy
   using ctDNA.

   Keywords: apatinib, circulating tumor DNA, EGFR‐TKI, NSCLC, safety

1. INTRODUCTION

   The overall 5‐year survival rate of advanced lung cancer is as low as
   18% in the United States.[44] ^1 The use of epidermal growth factor
   receptor tyrosine kinase inhibitors (EGFR‐TKIs) in the clinic improved
   the survival prognosis of lung cancer patients. However, the inevitable
   problem is EGFR‐TKI resistance. Most first generation EGFR‐TKIs, such
   as gefitinib and erlotinib, result in drug resistance by about
   12 months.

   Many mechanisms underlying the first generation of EGFR‐TKI‐acquired
   resistance have been revealed, such as T790M gatekeeper EGFR
   mutations,[45] ^2 MET amplification, STAT3 hyperactivation, activated
   STST3, PIK3CA, conversion to small‐cell lung cancer and dysregulation
   of the cell cycle.[46] ^3 Osimertinib, a third generation EGFR‐TKI, was
   effective in patients with the T790M mutation, but most patients
   develop resistance within 6 months.[47] ^4 Therefore, it remains a
   challenge to overcome acquired resistance to first‐generation targeted
   drugs in lung cancer treatment.

   Apatinib, a novel VEGF receptor 2 TKI, is an anti‐angiogenic drug with
   promising antitumor activity and a well‐tolerated toxicities in several
   malignancies.[48] ^5 In advanced EGFR‐mutant NSCLC, as first‐line
   treatment, apatinib plus gefitinib resulted in a PFS of 13.7 months
   compared with 10.2 months in the gefitinib group.[49] ^6 Although the
   concurrent use of apatinib and EGFR‐TKI improves the PFS, we think that
   perhaps a sequential mode of adding apatinib can achieve a longer PFS.
   In our study, we explored the clinical effectiveness of adding apatinib
   when NSCLC patients got slow progression with EGFR‐TKI.

   In clinical practice, CT imaging and serum carcinoembryonic antigen
   (CEA) are now commonly used to assess response to treatment in
   NSCLC,[50] ^7 , [51]^8 yet these approaches do not fully reflect the
   molecular and pathologic changes that occur in the tumor during
   treatment. Circulating tumor DNA (ctDNA), mainly released by apoptotic
   and necrotic cancer cells, has also been demonstrated to be useful for
   monitoring the treatment efficacy of targeted therapy, chemotherapy,
   and immunotherapy in NSCLC.[52] ^9 , [53]^10 , [54]^11

   In this study, a prospective, single‐arm study was conducted to assess
   the efficacy and safety of apatinib combined with EGFR‐TKI on patients
   who got slow progression after first line EGFR‐TKI treatment.
   Meanwhile, we comprehensively analyzed ctDNA to dynamically monitor the
   efficacy of treatment.

2. PATIENTS AND METHODS

   Patients newly diagnosed with primary lung cancer in the Department of
   Lung Cancer Surgery of the Tianjin Medical University General Hospital
   from July 2018 to March 2021 were recruited. A total of 12 NSCLC
   patients who got slow progression after first line EGFR‐TKI treatment
   were enrolled, then the patients took 250 mg of apatinib per day
   combined with the original EGFR‐TKI until disease progression or
   unacceptable toxicity. Slow progression is defined here as when CEA
   test and imaging examination were performed at 2–4 months' intervals,
   the abnormal CEA level increased on three consecutive tests compared to
   the CEA level of pre‐EGFR‐TKI treatment or target lesions relatively
   increases 0%–20% compared to the previous assessment according to
   RECIST 1.1. Peripheral blood was collected at baseline and 2–4 months'
   intervals after starting combination therapy for CEA and ctDNA
   detection. The Ethical review approval was obtained from Tianjin
   Medical University General Hospital for this study (ID number for IRB
   approval: IRB‐2018‐124‐01). Biological samples and images were acquired
   with the written informed consent of the patient. The detailed patient
   inclusion and exclusion criteria, treatments, study design, outcomes
   measurements and assessments, ctDNA testing by an 825‐gene panel of
   Genetron Co. Ltd. were described in Data [55]S1.

3. RESULTS

3.1. Clinicopathological characteristics of patients at baseline

   As shown in Table [56]S1, the median age of 12 patients including seven
   (58.3%) females and five (41.7%) males were 66 years, ranging from 44
   to 78 years. Most of the patients were non‐smokers (9/12, 75%). Two
   third (66.7%) patients were stage IIIB and the rest were stage IVa or
   IVb. All of them were diagnosed with adenocarcinoma. Two thirds
   patients (8/12) did not have metastasis, except for one patient with
   brain, two with bone and one with pleural metastasis. The first‐line
   EGFR‐TKI drugs patients received included gefitinib, icotinib and
   erlotinib. The median PFS1 was 13.1 months (95% CI, 10.6 m‐ NA),
   ranging from 8.9 to 16.4 months (Table [57]S1; Figure [58]1A).

FIGURE 1.

   FIGURE 1
   [59]Open in a new tab

   Kaplan–Meier analysis of PFS1 of 12 enrolled patients, PFS2 and total
   PFS of seven patients included in the efficacy analysis. (A)
   Kaplan–Meier analysis of PFS in 12 patients before slow progression for
   the first‐line EGFR‐TKI monotherapy (PFS1); (B) Kaplan–Meier analysis
   of PFS in seven patients for apatinib combined with EGFR‐TKI treatment
   (PFS2). (C) Kaplan–Meier analysis of total PFS of the seven patients.
   The median PFS and 95% CI are shown on the graph.

3.2. Treatments and efficacy

   All 12 patients received oral apatinib (250 mg/day) combined with
   initial EGFR‐TKI of which dose were determined by the principal
   investigator. Seven of which were included in efficacy analysis who had
   at least one imaging follow‐up. Five patients were excluded because two
   of them lost follow‐up and three did not comply with the study
   protocol. All seven patients achieved SD. Until the last follow‐up in
   May 2021, all seven patients were still alive. The median PFS2 was
   8.2 months (95% CI, 7.3 m‐NA), when plus the PFS1 for each patient, the
   total PFS reached 20.9 months (95% CI, 17.3 m‐NA) (Figure [60]1B,C).

3.3. Adverse events

   Treatment‐related adverse events (AEs) were presented in [61]Table
   [62]S2. The most frequent AEs were grade 1/2 hypertension (6, 50%),
   hand‐and‐foot syndrome (4, 33.3%), anorexia (3, 25%), fecal occult
   blood (3, 25%), and proteinuria (2, 16.7%), There were no grade 3/4 AEs
   observed, and all of AEs were manageable.

3.4. Genetic profiles and KEGG pathway enrichment of baseline samples

   Plasma were collected from 12 patients prior to apatinib combined with
   EGFR‐TKIs treatment as baseline samples. The genetic profiling showed
   that EGFR (25%), FAT3 (17%), and TP53 (17%) were the three most common
   mutated genes (Figure [63]S2A). Next, KEGG pathway enrichment analysis
   showed that the mutated genes significantly enriched in FoxO and
   endocrine resistance signaling pathway (Figure [64]S2B).

3.5. Serial ctDNA dynamically monitor the efficacy of combination treatment

   Plasma samples were collected from seven patients at 2–4 months
   intervals since starting combination therapy to dynamically monitor the
   changes of ctDNA and CEA. The dynamics of ctDNA variant allele fraction
   (VAF) was shown in Figure [65]2A, five patients: P01, P02, P04, P05,
   and P06, were found to have had at least once ctDNA cleared during
   monitoring. But for patients P07 and P03, ctDNA was always detectable.
   We found that the mPFS of ctDNA‐cleared patients (8.4 months, 95% CI,
   8.2‐NA) was significantly longer than that of ctDNA not cleared
   patients (mPFS 7.1 months, 95% CI, 6.9‐NA, p = 0.0082) (Figure [66]2B).
   The ctDNA of patients P05 and P07 were continued to be monitored after
   disease progression and their ctDNA were both detected, particularly
   there was a large increase of ctDNA VAF for P07 (Figure [67]2A,
   Figure [68]S2). The changes of ctDNA copy number per mL plasma showed
   almost the same as that of ctDNA VAF (Figure [69]S3). For CEA
   monitoring, the concentrations of patients P04 and P06 were always
   within the normal range (<5 ng/mL), while the other patients were
   beyond the normal range (≥5 ng/mL). The mPFS in the normal CEA group
   (9.65 months; 95% CI, 9.3‐NA) was longer than that of the abnormal CEA
   group (7.9 months; 95% CI, 7.3‐NA; p = 0.041) (Figure [70]2C). For
   patient P07, after disease progression, ctDNA increased a lot while CEA
   remained stable, indicating that CEA is not a reliable marker
   reflecting the disease progression.

FIGURE 2.

   FIGURE 2
   [71]Open in a new tab

   Dynamic Analysis of ctDNA VAF and CEA concentration of 7 patients
   included in the efficacy analysis. (A) Dynamic changes of ctDNA VAF for
   each gene (left panel) and the average ctDNA VAF and CEA concentration
   (ng/ml) (right panel) at 2–4 month’ intervals during apatinib and
   EGFR‐TKI combination therapy of each patient. The horizontal axis
   indicates the time since the combination therapy, and the vertical axis
   indicates the variant allele fraction of ctDNA or CEA concentration. A
   red asterisk indicates that an EGFR‐TKI resistant mutation T790M has
   been detected in patient P02. (B) Kaplan–Meier analysis of PFS
   stratified by ctDNA. (C) Kaplan–Meier analysis of PFS stratified by
   CEA. The p value was calculated using a two‐sided log‐rank test.

4. DISCUSSION

   Our study aimed to extend the effective time of first‐line EGFR‐TKI by
   using sequential mode of adding apatinib. Previous studies WJTOG3405,
   NEJ002, and OPTIMAL indicated that the mPFS of first‐generation
   EGFR‐TKI as first‐line treatment for advanced EGFR‐mutated NSCLC was
   only 9–13 months.[72] ^12 , [73]^13 , [74]^14 This limits the clinical
   application of EGFR‐TKI.

   ARTEMIS (CTONG1509), NEJ026, RELAY, and ACTIVE (CTONG 1706) studies
   have demonstrated EGFR‐TKI combined with anti‐angiogenic drugs can
   prolong PFS compared to EGFR‐TKI alone.[75] ^6 , [76]^15 , [77]^16 ,
   [78]^17 Our results showed that adding apatinib prolonged the total
   mPFS to 20.9 months (95% CI, 17.3‐NA), longer than or comparable to the
   PFS of these previous studies. The difference between these previous
   studies and our study is the intervention time of anti‐angiogenic
   drugs. In these previous studies, the anti‐angiogenic drugs were used
   concurrently with EGFR‐TKI, but in our study, the anti‐angiogenic drug
   was added when patients got slow progression after EGFR‐TKI
   monotherapy, that is, a sequential mode. The results suggested although
   the effect of concurrent and sequential modes both extend the PFS of
   EGFR‐TKI, the sequential mode could reduce medical costs and perhaps
   reduce adverse effects of anti‐angiogenic drugs.

   Using the third‐generation EGFR‐TKI as the first‐line treatment options
   is also an approach to extend the effective time of EGFR‐TKI. For
   example, the mPFS of osimertinib was 18.9 months,[79] ^18 and
   aumolertinib was 19.3 months.[80] ^19 It suggested the PFS of the
   third‐generation EGFR‐TKI as first‐line treatment options were
   comparable to the PFS in our study.

   We considered the dose of apatinib in terms of both safety and
   efficacy. In Zhou's clinical trial, apatinib is administered at a dose
   of 500 mg or 750 mg daily.[81] ^20 CTONG1706 study showed that 500 mg
   of apatinib daily combined with gefitinib results in more AEs with
   Grade ≥3 compared to other similar clinical trials.[82] ^6 To determine
   the dose of apatinib, we first tried to treat some cases at a dose of
   250 mg or 500 mg daily after obtaining informed consent, and found that
   250 mg was as effective as 500 mg of apatinib when combined with
   EGFR‐TKI, but it was better tolerated by patients. Thus, we determined
   a dose of 250 mg. During our study, when the AE of patients was severe,
   we adjusted the dose of apatinib in two ways: 5 days a week or once
   every 2 days. If the AE does not abate, it is recommended to
   discontinue. Most patients can adapt to this dose adjustment.

   In our study, we found a significant difference in PFS between the
   ctDNA‐cleared and not cleared groups (Figure [83]2A). Such responsive
   effect of ctDNA has also been reported in several studies. A study of
   ctDNA responsive to targeted therapy in NSCLC revealed that responders
   had nearly complete elimination of ctDNA, while non‐responders showed
   limited changes in ctDNA levels and significantly shorter PFS.[84] ^21
   Similar results were observed in both immunotherapy and chemotherapy
   efficacy monitoring.[85] ^11 , [86]^22

   The main limitation of this study is the small sample size, which may
   lead to analysis bias. But our study conveys valuable information about
   the function of apatinib in delaying EGFR‐TKI resistance and ctDNA
   profiling in monitoring efficacy. First, the addition of apatinib can
   prolong the effective time of the first‐generation EGFR‐TKI, providing
   a reference for solving the problem of TKI resistance in the clinic.
   Second, ctDNA may be a biomarker for monitoring treatment efficacy, and
   as a non‐invasive liquid biopsy, it may play a greater role in terms of
   efficacy monitoring in the future. To get a more reliable conclusion,
   further exploration of large‐scale, multi‐center clinical trials is
   needed.

AUTHOR CONTRIBUTIONS

   Minghui Liu: Data curation (lead); formal analysis (lead); project
   administration (supporting); validation (supporting); writing –
   original draft (lead); writing – review and editing (lead). Xin Li:
   Writing – original draft (supporting); writing – review and editing
   (supporting). Hongbing Zhang: Writing – original draft (equal); writing
   – review and editing (equal). Fan Ren: Resources (equal); software
   (equal). Jinghao Liu: Formal analysis (equal); software (equal).
   Yongwen Li: Data curation (equal); formal analysis (equal). Ming Dong:
   Investigation (equal); software (equal). Honglin Zhao: Software
   (equal); supervision (equal); validation (equal). Song Xu: Data
   curation (equal); investigation (equal); software (equal). Hongyu Liu:
   Formal analysis (equal); investigation (equal); validation (equal). Jun
   Chen: Project administration (lead); writing – original draft
   (supporting).

FUNDING INFORMATION

   This work was supported by the National Natural Science Foundation of
   China (82072595, 82172569, 81773207 and 61973232), Natural Science
   Foundation of Tianjin (19YFZCSY00040 and 19JCYBJC27000), Tianjin Key
   Medical Discipline (Specialty) Construction Project. Tianjin Health
   Science and Technology Project (ZC20179) and Tianjin Municipal
   Education Commission Natural Science Foundation (2019KJ202, 2021KJ221).

CONFLICT OF INTEREST STATEMENT

   All authors disclosed no commercial or financial relationships.

ETHICS STATEMENT

   The Ethical Review Committee of the Tianjin Medical University General
   Hospital approved the study protocol. All enrolled patients had read
   and signed informed consent. The study was conducted according to Good
   Clinical Practice and Helsinki Declaration principles. All biological
   samples, images and patients' information were obtained with patients'
   written informed consent.

CLINICAL TRIAL REGISTRATION

   This study was registered at [87]http://www.chictr.org.cn/, the trial
   registration number (TRN) was ChiCTR1800019185 and the registration
   date was October 30th, 2018.

Supporting information

   Figure S1:
   [88]Click here for additional data file.^ (2.4MB, tif)

   Figure S2:
   [89]Click here for additional data file.^ (1.3MB, tif)

   Figure S3:
   [90]Click here for additional data file.^ (2.1MB, tif)

   Table S1:
   [91]Click here for additional data file.^ (11.7KB, xlsx)

   Table S2:
   [92]Click here for additional data file.^ (10.8KB, xlsx)

   Data S1:
   [93]Click here for additional data file.^ (27.3KB, docx)

ACKNOWLEDGEMENTS