Figures
Fig. 1 The frequency of observed drug resistance mechanisms. The pie chart depicts the prevalence of observed mechanisms of resistance to EGFR TKIs in 37 patients with NSCLC biopsied at the time that resistance was acquired. Pre- and posttreatment specimens were compared and only acquired mechanisms of resistance are depicted. The blue wedge represents resistant cancers that developed the EGFR T790M resistance mutation including a subset that developed concomitant EGFR amplification. The green wedge represents cancers that developed MET amplification, and the red wedge represents cancers that underwent transformation to SCLC. The yellow wedge represents cancers that developed PIK3CA mutations, and the orange wedge represents one patient who had both SCLC transformation and acquisition of a PIK3CA mutation.
Fig. 2 Acquired genetic amplifications in drug-resistant lung tumors. Amplification of MET and EGFR genes was observed in biopsies of tumors from patients who had acquired resistance to EGFR TKIs. Shown are FISH analyses that detect the MET gene (green), EGFR gene (red), and the control CEP7 gene (aqua). (A) The pretreatment specimen from patient 19 (left panel) shows a normal MET copy number but significant EGFR amplification; the drug-resistant posttreatment specimen (right panel) from the same patient exhibits acquired MET amplification but normal EGFR copy number. (B) Patient 13 demonstrated an acquired EGFR amplification in the drug-resistant posttreatment specimen (right panel) compared to the pretreatment specimen (left panel).
Fig. 3 Drug resistance and transformation of NSCLC to SCLC. The SCLC histological phenotype was observed in five (14%) NSCLC patients who had acquired resistance. Two examples are shown. (A) Patient 23 had an exon 19 deletion in EGFR. (B) Patient 22 carried the L858R mutation in EGFR. The presence of the original activating mutation was confirmed in both pretreatment (pre-Rx) specimens (upper panels) and drug-resistant specimens (lower panels). Hematoxylin and eosin (H&E) (left) and synaptophysin (right) staining are shown for each case. Notably, the pretreatment biopsies (A and B, upper panels) demonstrate adenocarcinoma consisting of cellular growths of atypical glands with (A) or without (B) a cribriform pattern that are negative for synaptophysin. The post-resistance biopsies (A and B, lower panels) demonstrate a SCLC phenotype consisting of nests of small cells with a high nuclear-to-cytoplasmic ratio with (A) or without (B) the classic SCLC-associated finding of “crush artifact,” with positive immunohistochemical staining for synaptophysin. (C) Computed tomography scans of a representative patient (patient 25) with SCLC in the acquired resistance specimen before (above) and after (below) chemotherapy with cisplatin and etoposide (the standard regimen for treating SCLC). Yellow arrows denote right third rib metastases, and white arrows denote left axillary adenopathy.
Fig. 4 EMT and acquired resistance. (A) H1975 cells were cultured in the presence of the irreversible EGFR inhibitor PF00299804 until drug resistance developed, as demonstrated by Syto60 viability assays. (B) Images of the parental and drug-resistant H1975 cells by bright-field microscopy demonstrate that the resistant cells have developed a spindle-like morphology. (C) Parental and resistant H1975 cells were lysed and probed with antibodies against E-cadherin, vimentin, and actin, revealing loss of E-cadherin expression and gain of vimentin expression among drug-resistant H1975 cells. For comparison, HCC827 cells and the derived HCC827 GR6 cell line (HCC827 cells that acquired resistance to gefitinib via MET amplification), which do not undergo an EMT, are shown. (D) Example of a case (patient 28) whose drug-resistant tumor shows evidence of an EMT (top, pretreatment specimens; bottom, drug-resistant posttreatment specimens). Left, H&E staining; middle, staining for vimentin; right, staining for E-cadherin. Notably, the pretreatment cancer had an adenocarcinoma histology (panel 1), does not stain for vimentin (panel 2), and shows preserved membranous staining with E-cadherin (panel 3). The vimentin-positive areas in panel 2 include alveolar macrophages (red circles), inflammatory and stromal cells in fibrovascular cores (black arrows), but not tumor cells lining papillary structures (yellow arrows). The drug-resistant posttreatment specimen has sarcomatoid histology (panel 4), is positive for vimentin (panel 5), and is negative for E-cadherin (panel 6), consistent with an EMT.
Fig. 5 Longitudinal evaluation of patients treated repeatedly with erlotinib. The color-coded boxes to the left of each panel describe the data displayed across the timeline. The tumor burden depicted is not quantitative but represents tumor growth and shrinkage. (A) Patient 12 with a lung adenocarcinoma carrying the L858R EGFR mutation and a mutation in the tumor suppressor p53 had a modest response to first-line chemotherapy. The patient then achieved a more robust and durable response to second-line erlotinib, with near-complete resolution of her lung nodules. After 8 months on an EGFR TKI, she developed resistance with growing bilateral pulmonary nodules. A lung core biopsy revealed an acquired T790M mutation in EGFR. There was no response to chemotherapy and she subsequently developed bone and liver metastases. At that time (after not taking the EGFR inhibitor for 8 months), a second lung core biopsy revealed the L858R EGFR mutation, but no detectable T790M EGFR resistace mutation. The patient responded to erlotinib (in combination with an investigational agent that did not target T790M). (B) Patient 24 had an L858R EGFR-mutant adenocarcinoma that responded markedly to first-line erlotinib for 12 months with resolution of her pleural effusion and pulmonary nodules. After 1 year, there was progression of the largest nodule. Core biopsy of this lesion revealed histological transformation to SCLC that harbored the EGFR L858R mutation and acquired a PIK3CA mutation. She was treated with radiation and chemotherapy. After a 6-month break from all treatment, her pleural effusion reaccumulated and small bilateral pulmonary nodules reappeared. Pleural effusion analysis revealed adenocarcinoma with the L858R EGFR mutation only (no PIK3CA mutation). Her disease responded to a second-line course of erlotinib. After 6 months, bony metastases and an adrenal lesion developed. Assessment of a growing bone metastasis revealed SCLC with both the L858R EGFR and PIK3CA mutations.
Tables
- Table 1
Thirty-seven paired lung tumor biopsies resistant to EGFR inhibitors. EGFR, epidermal growth factor receptor; amp, amplification; del, deletion; Adeno, adenocarcinoma; Adenosquam, adenosquamous; NSCLC, non–small cell lung cancer not otherwise specified; SCLC, small cell lung cancer; CA, carcinoma; EMT, epithelial to mesenchymal transition; TKI, tyrosine kinase inhibitor; Erlo, erlotinib; Gef, gefitinib.
ID# Age Sex EGFR mutation Baseline histology Summary of changes Primary TKI (time on TKI) TKI status at repeat biopsy T790M 1 66 M L858R Adeno T790M Erlo (6 months) Off (2 months) 2 74 F Exon 19 del Adeno T790M Erlo (12 months) On 3 47 F Exon 19 del Adeno T790M Gef (15 months) On 4 60 F Exon 19 del Adeno T790M Erlo (7 months) On 5 57 M L858R Adeno T790M Gef (5+ years) On 6 47 M Exon 19 del Adeno T790M Erlo (12 months) Off (14 months) 7 58 F Exon 19 del Adeno T790M Erlo (3+ years) On 8 69 M L858R Adeno T790M* Erlo (2 years) On 9 58 F G719C, S768I Adeno T790M Erlo (2+ years) On 10 46 F Exon 19 del Adeno T790M Erlo (3 years) Off (3 months) 11 53 F Exon 19 del Adeno T790M Erlo (16 months) On 12 59 F L858R Adeno T790M Erlo (8 months) Off (5 months) T790M + EGFR amp 13 42 M Exon 19 del Adeno T790M, EGFR amp Erlo (5 months) On 14 55 M Exon 19 del Adeno T790M, EGFR amp Erlo (10 months) On 15 37 F Exon 19 del Adeno T790M, EGFR amp Erlo (6 months) On T790M + new, additional mutations 16 88 F Exon 19 del Adeno T790M, β-catenin Erlo (2+ years) On 17 85 M Exon 19 del Adeno T790M, β-catenin Erlo (22 months) On 18 75 F Exon 19 del Adeno T790M, APC† Erlo (18 months) On MET amplification 19 61 M L858R Adenosquam MET amp, loss EGFR amp Erlo (15 months) On 20 76 M L858R Adeno MET amp Erlo (13 months) Off (5 months) Acquired PIK3CA mutation 21 65 M Exon 19 del Adeno PIK3CA acquisition Erlo (21 months) On Histologic transformation (one with acquired PIK3CA mutation) 22 67 F L858R Adeno SCLC transformation Erlo (22 months) On 23 54 F Exon 19 del Adeno SCLC transformation Erlo (3+ years) On 24 56 F L858R Adeno SCLC transformation, PIK3CA Erlo (14 months) On 25 40 F Exon 19 del Adeno SCLC transformation Erlo (2+ years) Off (2 months) 26 61 F L858R Adeno SCLC transformation Erlo (18 months) On 27 66 M L858R Adeno EMT Erlo (11 months) On 28 59 M Exon 20 ins‡ Adeno EMT Gef (11 months) On 29 64 M L858R Adeno Sarcomatoid CA, loss of β-catenin Erlo (11 months) Off (2 weeks) No histological or genetic changes identified 30 62 F L858R Adeno None Erlo (6 months) On 31 52 F Exon 19 del Adeno None Gef (17 months) On 32 58 F Exon 19 del Adeno None Erlo (14 months) On 33 61 F L858R Adeno None Erlo (13 months) On 34 85 F Exon 19 del Adeno None Erlo (6 months) On 35 62 M L858R NSCLC None Gef (3+ years) On 36 56 M L858R Adeno None Erlo (5 months) Off (<2 weeks) 37 51 F Exon 19 del Adeno None Erlo (8 months) On *TP53 mutation suspected to be present, but not confirmed.
†APC mutation confirmed in resistant specimen, but not confirmed to be present in initial biopsy.
‡Exon 20 insertion assay added to SNaPshot for this patient given direct sequencing result from pretreatment sample.
- Table 2
Patients with lung tumors showing an NSCLC to SCLC transformation. ID number refers to the patient number in Table 1. Pre-, pretreatment; Post-, posttreatment/drug-resistant.
Age (years) Gender ID no. Biopsy Histology Synaptophysin Chromogranin CD56 Genotype 67 Female 22 Pre- Adenocarcinoma − − − L858R Post- SCLC + + + L858R 54 Female 23 Pre- Adenocarcinoma − − Weak+ Exon 19 deletion Post- SCLC Strong+ Exon 19 deletion 56 Female 24 Pre- Adenocarcinoma − − L858R Post- SCLC + + L858R, PIK3CA 40 Female 25 Pre- Adenocarcinoma − − − Exon 19 deletion Post- SCLC + − Exon 19 deletion 61 Female 26 Pre- Adenocarcinoma − − − L858R Post- SCLC + + L858R
- Supplementary Material for:
Genotypic and Histological Evolution of Lung Cancers Acquiring Resistance to EGFR Inhibitors
Lecia V. Sequist,* Belinda A. Waltman, Dora Dias-Santagata, Subba Digumarthy, Alexa B. Turke, Panos Fidias, Kristin Bergethon, Alice T. Shaw, Scott Gettinger, Arjola K. Cosper, Sara Akhavanfard, Rebecca S. Heist, Jennifer Temel, James G. Christensen, John C. Wain, Thomas J. Lynch, Kathy Vernovsky, Eugene J. Mark, Michael Lanuti, Anthony J. Iafrate, Mari Mino-Kenudson, Jeffrey A. Engelman*
*To whom correspondence should be addressed. E-mail: lvsequist{at}partners.org (L.V.S.); jengelman{at}partners.org (J.A.E.)
Published 23 March 2011, Sci. Transl. Med. 3, 75ra26 (2011)
DOI: 10.1126/scitranslmed.3002003This PDF file includes:
- Fig. S1. Percent change in measurable disease, by patient.
- Fig. S2. Amplification of the T790M allele in tumors with EGFR amplification.
- Fig. S3. The overall distribution of the genetic and histological findings in the resistant biopsies.
- Table S1. Detailed results for 37 paired specimens with TKI resistance.
- Table S2. Mutations tested in SNaPshot version I.
- Table S3. Results from first autopsy case.
- Table S4. Results from second autopsy case.
