Multiple EGFR Mutations in Patients With Lung Adenocarcinoma

Multiple EGFR Mutations in Patients With Lung Adenocarcinoma

Case Report Germline Mutation of T790M and Dual/Multiple EGFR Mutations in Patients With Lung Adenocarcinoma Yanyan Lou,1 Chad V. Pecot,1 Hai T. Tran...

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Case Report

Germline Mutation of T790M and Dual/Multiple EGFR Mutations in Patients With Lung Adenocarcinoma Yanyan Lou,1 Chad V. Pecot,1 Hai T. Tran,1 Vikki J. DeVito,1 Xi Ming Tang,1,2 John V. Heymach,1 Raja Luthra,3 Ignacio I. Wistuba,1,2 Zhuang Zuo,3 Anne S. Tsao1 Clinical Practice Points  We report a confirmed case of germline EGFR T790M

mutation and a case series of dual/multiple EGFR mutations in patients with lung adenocarcinoma.  Primary or de novo EGFR T790M mutations have been found at a greater frequency in patients with dual or multiple EGFR mutations.

 Germline EGFR T790M mutations are rare but in-

crease lung cancer susceptibility.  Screening the relatives of patients with lung cancer

and germline EGFR T790M mutations could be important, given the increased risk of developing lung cancer.

Clinical Lung Cancer, Vol. 17, No. 2, e5-11 ª 2016 Elsevier Inc. All rights reserved. Keywords: Dual EGFR mutations, T790M germline mutation, Lung adenocarcinoma

Introduction Exon 19 deletions and point mutations in L858R are the most common somatic activating mutations in the epidermal growth factor receptor (EGFR) gene that confer sensitivity to EGFR tyrosine kinase inhibitors (TKIs) in lung cancer.1 However, despite the initial response to EGFR TKIs, all patients will eventually develop resistance. One of the most common mechanisms of resistance is acquisition of a second mutation at exon 20, which causes a T790M substitution.2,3 Although most of these cases have acquired resistance through somatic mutations, a small number of germline Y.L. and C.V.P. contributed equally to this work. Y. Lou is currently at the Division of Hematology/Oncology, Mayo Clinic, Jacksonville, FL. C.V. Pecot is currently at Thoracic Medical Oncology, UNC Lineberger Comprehensive Cancer Center, Chapel Hill, NC. 1

Department of Thoracic and Head and Neck Medical Oncology Department of Translational Molecular Pathology Department of Hematopathology, University of Texas MD Anderson Cancer Center, Houston, TX 2 3

Submitted: Jun 16, 2015; Revised: Oct 29, 2015; Accepted: Nov 10, 2015; Epub: Nov 17, 2015 Address for correspondence: Anne S. Tsao, MD, Department of Thoracic and Head and Neck Medical Oncology, University of Texas MD Anderson Cancer Center, 1400 Holcombe Boulevard, Unit 432, Houston, TX 77030 E-mail contact: [email protected]

1525-7304/$ - see frontmatter ª 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cllc.2015.11.003

EGFR T790M have been reported and are estimated to occur in 1% of nonesmall-cell lung cancer (NSCLC) cases.4-6 These germline EGFR T790M mutations are believed to predispose patients to the development of lung cancer, because preclinical studies have shown the germline T790M mutation to be a weak oncogene that often requires a secondary mutation to potentiate cancer development.5,6 In Asia, a few cases of dual EGFR mutations containing primary de novo T790M substitution before TKI treatment have been described7,8; however, none were identified to be germline mutations. In contrast, thus far, germline EGFR T790M mutations have only been described in white patients with lung cancer.5,6 Several family members of European descent with hereditary bronchoalveolar carcinoma were identified with germline T790M mutations.9 Our group previously reported a case of a 72-year-old patient with a solitary T790M mutation who had a germline T790M mutation in her peripheral blood mononuclear cells (PBMCs).10 Recently, 2 cases of germline T790M mutations were reported in never smoking female white patients in the United States.5,6 In the present brief report, we describe another case of a white female patient with lung adenocarcinoma who had a germline EGFR T790M mutation and concurrent somatic L858R mutation. We also describe a case series of patient demographics and tumor characteristics associated with primary EGFR T790M mutations in patients with NSCLC.

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Pt. No.

Mutation

Age (Years)

Sex

Ethnicity

Pk-y, Quit (Years)

Histologic Type

Stage at Diagnosis

1

G719S [18]; T790M [20]

51

Fe

White

0

AC

IV, T4N2M1

Lungs, skeletal F, LCa (NS); pA, LCa (NS); pU, LCa (NS)

2

[del 19]; T790M [20]

71

Ma

White

0

AC

IV, T2N3M1

Left choroid, skeletal, adrenal

3

T790M [20]; L858R [21]

62

Fe

White

0

BAC

IV, T2N0M1

4

T790M [20]; L858R [21]; [del 19]

53

Fe

White

0

AC

IIIA, T4N0M0

5a

T790M [20]; L858R [21]

34

Fe

White

0

AC

IV, T4N3M1

Met

FH

B, “BCa”

Other Cancers

Germline Mutation

None

NA

None

NA

Bilateral lungs S, LCa (NS); M, SCC of skin LCa (ES); F, LCa (ES)

None

F, LCa (ES); B, Papillary TCa GBA; M, UCa; pU, HNC (ES); mGF, colon Ca; mA, BCa; mC, BCa Skeletal, liver, See family None pulmonary pedigree

NA

No

Yes

Treatment History

TTP With EGFR TKI

Palliative RT; UA lost to FU; EGFR TKI treatment history UA Taxotere, 1.5 mo carboplatin  2 cycles, discontinued for presumed Taxotere reaction; alimta and carboplatin  1, discontinued for carboplatin reaction; alimta  3 cycles; erlotinib  1.5 mo; Gemzar  1 Multiple wedge >5 years resections, followed by erlotinib for >5 years at last FU Carboplatin, No TKI to date Taxotere  6 cycles, stereotactic RT, no evidence of disease to date Erlotinib  1 mo; 1 mo cetuximab, cisplatin, alimta  1 mo

Abbreviations: A ¼ aunt; AC ¼ Adenocarcinoma; B ¼ brother; BAC ¼ bronchoalveolar carcinoma; BCa ¼ breast cancer; BoCa ¼ bone cancer; C ¼ cousin; ES ¼ ever smoker; F ¼ father; Fe ¼ female; FH ¼ family history; FU ¼ follow-up; GBA ¼ glioblastoma; GF ¼ grandfather; GM ¼ grandmother; HNC ¼ head and neck cancer; LCa ¼ lung cancer; M ¼ mother; m ¼ maternal; Ma ¼ male; Met ¼ metastases; NS ¼ never smoker; p ¼ paternal; Pk ¼ pack; Pt. No. ¼ patient number; RT ¼ radiation therapy; S ¼ sister; SCC ¼ squamous cell carcinoma; TCa ¼ thyroid cancer; TKI ¼ tyrosine kinase inhibitor; TTP ¼ time to progression; U ¼ uncle; UA ¼ unavailable; UCa ¼ uterine cancer. a Proband.

Germline EGFR T790M Mutations and EGFR Mutations in Lung Cancer

Clinical Lung Cancer March 2016

Table 1 Summary of Data From Lung Cancer Patients With Dual or Multiple EGFR Mutations Containing Primary EGFR T790M Mutations

Yanyan Lou et al Materials and Methods Patient Selection and Data Collection After institutional review board approval from the MD Anderson Cancer Center, the clinical and demographic data were collected for all patients with lung adenocarcinoma identified as having  2 EGFR mutations from May 2005 to August 2009. In 2 patients identified as having a primary de novo T790M mutation, PBMCs were isolated and assessed for germline EGFR mutation status.

Tumor and Germline Genotyping DNA sequences for EGFR (exons 18-21) extracted from paraffinembedded tissue (NSCLC tumors) or PBMCs (for germline assessment) were amplified using standard polymerase chain reaction (PCR) primers and sequenced. All sequence variants were confirmed by independent PCR amplifications from  2 independent DNA extractions and sequenced in both directions.

Results Frequency of Primary De Novo Dual or Multiple EGFR Mutations in Patients With Lung Adenocarcinoma We evaluated 427 patients with lung adenocarcinoma treated from May 2005 to August 2009 at the MD Anderson Cancer Center Thoracic Clinic. Of these 427 NSCLC patients, 55 were identified with de novo EGFR mutations in their tumor tissue. Twelve patients (2.8%) were found to have either dual or multiple EGFR mutations. Five of these patients had primary de novo T790M mutations. The clinical and demographic information of the patients with primary de novo T790M mutations are listed in Table 1. The information of the patients with dual or multiple EGFR mutations but without T790M mutations are included in

Supplemental Table 1 (available in the online version). All the mutations were tested in the tumor samples, except for patients 4 and 5, whose peripheral blood samples were also available for germline mutation testing.

Germline EGFR T790M Mutation in a White Woman A 34-year-old never-smoking white woman presented to the local emergency room in July 2009 because of a persistent cough. The chest radiograph showed a large right-sided pleural effusion and pathologic examination of the pleural fluid revealed metastatic adenocarcinoma of the lung. A positron emission tomography/ computed tomography scan revealed extensive hypermetabolic activity in the pulmonary parenchymal lymph nodes (right hilar, precarinal, right supraclavicular) and multiple liver and osseous metastases (Figure 1A). At the initial treatment, the EGFR mutation T790M had not yet been identified, and her poor performance status prevented initial chemotherapy administration. Therefore, erlotinib monotherapy was started empirically, with close monitoring. After 1 month of erlotinib treatment, her performance status had improved, but the dyspnea was worse. A repeat computed tomography scan confirmed disease progression (Figure 1B and C). Erlotinib was stopped and replaced with cetuximab, cisplatin, and alimta. However, she was admitted to the hospital after 1 cycle of treatment because of progressive dyspnea. She was unable to tolerate further treatment and was entered into hospice. Sequencing of her tumor specimen obtained before treatment revealed dual mutations of both EGFR T790M and L858R (Figure 2A and B). Mutation analysis of her PBMCs also identified a germline T790M mutation, with equivalent heights of the mutant and wild-type peaks (Figure 2C). The proband’s family pedigree is presented in Figure 3.

Figure 1 Positron Emission Tomography-Computed Tomography (PET-CT) and CT Scans of the Proband Patient With a Germline EGFR T790M Mutation

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Germline EGFR T790M Mutations and EGFR Mutations in Lung Cancer Figure 2 Results From Polymerase Chain Reaction Sequencing on Proband’s Tumor Sample (A, B) and Peripheral Blood Cells (C)

Proband

A Tumor exon 20 mutaƟon C

A

T

C

G

A

C

A

B G

T

C

C

Tumor exon 21 mutaƟon G

G

G

C

G

C/T

G

C

C

PBMC germline T790M mutaƟon

A

C/T

T/G

Abbreviation: PBMC ¼ peripheral blood mononuclear cell.

Discussion The occurrence of germline EGFR T790M mutations is rare and has only been reported in case series.5,6 These cases have been predominantly identified in white women with a history of never smoking and adenocarcinoma histologic features. The findings from our case report are consistent with this clinical presentation. From

the published data, about 73% of lung cancers that arise in patients with germline EGFR T790M mutations also appear to carry second EGFR mutations.5 One hypothesis suggests that a germline T790M mutation by itself might be a weak oncogene that requires a secondary mutation to potentiate cancer development.5 Of the secondary mutations, concurrent L858R mutations have been the most

Figure 3 Pedigree of Family With Germline T790M Mutation. Age, Smoking History, and History of Other Cancers Were Recorded

Never smoker Smoker

75 Lung cancer

83 Lung cancer

50 Breast cancer

80 Mesothelioma

70 Bladder cancer

70

75

41

10

e8

-

Clinical Lung Cancer March 2016

85

75

61

62 PancreaƟc cancer, tesƟcular cancer

Proband 34, Lung cancer

13

64

75

65

63

41

60

42

9

12

51

54

Yanyan Lou et al common.5 To evaluate this, we interrogated our lung cancer database of 427 patient samples that had been genotyped for EGFR mutations. Of the 427 patients, 12 (3%) were found to carry dual or multiple EGFR mutations before EGFR TKI treatment. Also, 5 of these 12 patients had a primary de novo T790M mutation. All the patients with a primary de novo EGFR T790M mutation had a concurrent second mutation. The clinical and demographic features of these 5 patients with a primary EGFR T790M are summarized in Table 1. The proband patient reported in our case report is listed as patient 5 in Table 1. The sequencing electropherogram of the other 4 patients is shown in Figure 4. Patients 1, 2, and 3 had almost identical heights of peak in the mutant and nonmutant alleles, suggesting the likelihood of germline mutations. However, we are unable to confirm the presence of germline mutations owing to the unavailability of blood or normal tissue specimens from these patients. The sequencing data of patient 4 showed different heights of

the peak in the mutant and nonmutant alleles. The peripheral blood sample from patient 4 was available, which did not identify a germline T790M mutation (data not shown). Of the 5 patients with dual mutations containing primary de novo T790M, 4 had a family history of lung cancer, although some of the family members were smokers. Of those 5 patients who had primary de novo EGFR T790M mutations in addition to sensitive EGFR mutations, 3 patients received EGFR TKI treatment (Table 1). Two patients experienced disease progression after 1 to 1.5 months of EGFR TKI treatment. The third patient underwent multiple wedge resections of her cancer and has been stable with good disease control for > 5 years of erlotinib therapy. Although T790M mutations have most commonly developed as a resistance mechanism after TKI treatment, rare cases of de novo T790M mutations have been reported.11-13 Studies have suggested that an EGFR T790M mutation might be present in small populations of tumor cells before EGFR

Figure 4 Results From Polymerase Chain Reaction Sequencing of Additional Patients With a Primary EGFR T790M Mutation in Patients’ Tumor Samples

EX20 PaƟent # 1

PaƟent # 2

C

A

T

C

A

C

A

T

C

A

EX21 C/T

G

C

A

G

C

G

C

A

G

C

G

C

A

C/T

PaƟent # 3

C

A

T

C

A

G

C

T

G

G

G

C

C/T

PaƟent # 4

C

A

T

C

A

G

C/T

g

G

G

C

C

A

G

C

C

A

T/G

C

A

G

C

T

G

G

G

C

g

G

T/G

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Germline EGFR T790M Mutations and EGFR Mutations in Lung Cancer

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TKI treatment and that the tumor cells harboring the T790M mutation are likely to be enriched after drug treatment.12 Consistent with this suggested theory, our data have demonstrated that primary de novo EGFR T790M mutation are present with a high frequency (5 of 12; 42%) in patients carrying dual or multiple EGFR mutations. Several groups in Asia have reported small subsets of patients with NSCLC with dual EGFR mutations.7,8,14 It was estimated that complex mutations occurred in 13% to 18% of Asian patients with EGFR mutations.8,15 Our study found similar findings, demonstrating that 12 of 55 patients (22%) had dual or multiple EGFR mutations. It has been hypothesized that because these dual mutations are found in cis, this confers a “second-hit” growth advantage to the cancer cells.14 In murine models, the expression of EGFR T790M has been reported to induce lung adenocarcinoma, suggesting that this mutation alone can be tumorigenic, although a control with wild-type EGFR overexpression is missing.16 However, mice expressing the EGFR T790M transgene alone developed tumors with much longer latency than the tumors in mice expressing either EGFR L858R plus T790M or EGFR L858R alone.16 Although more definitive studies are required, this is highly suggestive that primary de novo EGFR T790M mutations, whether somatic or germline, can lead to lung adenocarcinoma and that a secondary EGFR mutation further potentiates this and leads to an earlier onset of malignancy.17 From a clinical perspective, several features observed in the cases of germline T790M mutations are interesting. To date, all cases have been reported in white patients, except for 2 cases that were found in patients of East Indian descent.18 From the cases for which the family histories were reported, the onset of lung cancer appears to occur at a younger age in each subsequent generation and appears to be inherited in an autosomal dominant pattern.5,6 Our proband patient was diagnosed with lung cancer at the age of 34 years, and the other case was reported in a 29-year-old woman; these are the 2 youngest patients reported to date.5 This acceleration of disease onset in patients with younger ages in subsequent generations might due to “anticipation.” Anticipation is a phenomenon that has been described in inherited diseases such as Huntington disease and fragile X syndrome, in which the disease arises at younger ages or with augmented severity in subsequent generations. DNA instability in subsequent generations is a likely cause of anticipation. In addition, environmental factors could play a role. Similar to sporadic EGFR mutations, germline T790M mutations are predominantly found in women. This might be explained by the growing body of evidence showing functional interactions between the estrogen and EGFR pathways.19 Studies have suggested that women have more altered activity in DNA repair compared with men, and it is well documented that women who smoke are more prone to KRAS mutations.20 Additional studies are necessary to understand the roles of gender and biology in germline T790M mutation in lung cancer development. EGFR T790M mutation was previously considered to be an acquired resistant mutation to EGFR TKI therapy. However, the findings from our study, along with those from previously published cases, suggest that primary de novo EGFR T790M mutations might occur at a frequency as great as 35% to 42% in patients with dual or multiple EGFR mutations. This supports the theory that patients

Clinical Lung Cancer March 2016

with an EGFR mutation might harbor unseen T790M mutations at presentation, with the resistance mutation only identified after clonal selection occurs with EGFR TKI therapy.4,11,12 As a separate issue, germline EGFR T790M mutations were reported to be present in approximately 50% of all patients with primary EGFR T790M, although this statistic was generated from a very limited number of patients owing to the rarity of the disease.4 However, it is reasonable to consider performing germline testing in all patients with primary de novo EGFR T790M mutations. This could subsequently lead to potentially screening first-degree relatives of patients with lung cancer and germline EGFR T790M mutations and monitoring them, given the increased risk of their developing lung cancer.

Conclusion We report the case of a germline EGFR T790M mutation and a case series of patients with NSCLC and dual/multiple EGFR mutations. A primary de novo EGFR T790M mutation has been found at a greater frequency in patients with dual or multiple EGFR mutations. A sensitive detection method such as next generation sequencing will be helpful in identifying these less-frequent mutations. Although this occurs in a rare population of patients, it is important to identify these patients rapidly and enroll them in clinical trials with third-generation TKIs or EGFR T790M-specific targeted therapy. Additional study is essential to furthering our understanding of this disease and its resistance mechanisms.

Acknowledgments This study was supported by National Institutes of Health (T32 CA 009666) Research Training in Academic Medical Oncology (to Y.L. and C.V.P).

Disclosure The authors have stated that they have no conflicts of interest.

Supplemental data The supplemental data accompanying this article can be found in the online version at http://dx.doi.org/10.1016/j.cllc.2015.11.003.

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Yanyan Lou et al 9. Bell DW, Gore I, Okimot RA, et al. Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR. Nat Genet 2005; 37:1315-6. 10. Prudkin L, Tang X, Wistuba II. Germ-line and somatic presentations of the EGFR T790M mutation in lung cancer. J Thorac Oncol 2009; 4:139-41. 11. Piotrowska Z, Sequist LV. Epidermal growth factor receptor-mutant lung cancer: new drugs, new resistance mechanisms, and future treatment options. Cancer J 2015; 21:371-7. 12. Inukai M, Toyooka S, Ito S, et al. Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer. Cancer Res 2006; 66:7854-8. 13. Ayoola A, Barochia A, Belani K, et al. Primary and acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer: an update. Cancer Invest 2012; 30:433-46. 14. Yokoyama T, Kondo M, Goto Y, et al. EGFR point mutation in non-small cell lung cancer is occasionally accompanied by a second mutation or amplification. Cancer Sci 2006; 97:753-9.

15. Huang SF, Liu HP, Li LH, et al. High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in Taiwan. Clin Cancer Res 2004; 10:8195-203. 16. Regales L, Balak MN, Gong Y, et al. Development of new mouse lung tumor models expressing EGFR T790M mutants associated with clinical resistance to kinase inhibitors. PLoS One 2007; 2:1-10. 17. Chmielecki J, Foo J, Oxnard GR, et al. Optimization of dosing for EGFR-mutant non-small cell lung cancer with evolutionary cancer modeling. Sci Transl Med 2011; 3:90ra59. 18. Girard N, Lou E, Azzoli CG, et al. Analysis of genetic variants in never-smokers with lung cancer facilitated by an Internet-based blood collection protocol: a preliminary report. Clin Cancer Res 2010; 16:755-63. 19. Stabile LP, Lyker JS, Gubish CT, et al. Combined targeting of the estrogen receptor and the epidermal growth factor receptor in non-small cell lung cancer shows enhanced antiproliferative effects. Cancer Res 2005; 65:1459-70. 20. Ben Aissa A, Mach N. [Is lung cancer in women different?]. Rev Med Suisse 2012; 8:1108-11.

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