Abstract
Introduction
Recent studies suggested that p95HER2, the NH2-terminally truncated form of human epidermal growth factor receptor 2 (HER2), could confer resistance to monoclonal antibodies against HER2 (HER2-mab). The aim of this study was to investigate the role of p95HER2 according to HER2 gene copy number (GCN) and HER2 mutation in non-small cell lung cancer (NSCLC).
Methods
The study included 447 resected NSCLC patients evaluated for P95HER2 status by immunofluorescence. Data were correlated with HER2 GCN evaluated by fluorescence in situ hybridization (FISH) and HER2 mutations. Tumors were scored as positive for p95HER2 expression if any cytoplasmic staining was detected.
Results
P95HER2 was successfully evaluated in 431 patients and was positive (p95HER2+) in 33 (7.6%) cases. HER2 GCN was evaluable in 439 patients, and increased GCN (at least four copies in at least 40% cells) was found in 60 cases, of which 22 (5.0%) displayed gene amplification (GA). Among the 22 patients with HER2 amplification, only one resulted P95HER2+. To further investigate whether the receptor is truncated in presence of gene mutation, in addition to the study cohort, we analyzed p95HER2 status in eight NSCLC samples harboring HER2 mutation, and only one case resulted p95HER2+. In the whole population, p95HER2− patients had numerically higher risk of death than p95HER2+ (hazard ratio = 1.4, p = 0.2). No difference in survival was observed between patients with or without HER2 GA (median 38 versus 41 months, p = 0.46). HER2 GA was significantly associated with EGFR and MET GA, with no effect on survival.
Conclusions
HER2 truncation and HER2 increased GCN are not prognostic in resected NSCLC. P95HER2 is a very rare event in individuals displaying HER2 gene amplification or mutation.
Keywords: p95HER2, HER2, MET, Non-small cell lung cancer
Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths worldwide, and in 2011 it is estimated that it will be responsible for more deaths than colorectal cancer, breast cancer, and prostate cancer combined.1 For patients with advanced disease, chemotherapy with third-generation platinum-based doublets represented the standard of care until recently, when major breakthroughs in the knowledge of cancer biology permitted the development of numerous targeted therapies for NSCLC treatment. The epidermal growth factor receptor (EGFR) is a prototypical member of the EGFR family that also includes HER2/neu (Erb-B2), HER3 (Erb-B3), and HER4 (Erb-B4).2 These receptors represent interesting therapeutic targets because they play a critical role in cancer proliferation and survival.
Anti-EGFR agents and particularly small molecules interfering with the tyrosine kinase activity of the intracellular domain of EGFR represent today an important option against NSCLC, particularly in individuals harboring activating EGFR mutations.3 HER2 has strong kinase activity but has no identified ligand. HER2 overexpression or amplification correlates with poor prognosis in several malignancies including breast and ovarian cancers.4,5 Nevertheless, in lung cancer, the role of HER2 overexpression and amplification, reported in 20% and <10% of cases, respectively,6 remains controversial. A recent meta-analysis showed that HER2 overexpression but not amplification was a poor prognostic factor in lung cancer, particularly in small cell lung cancer, in adenocarcinoma, and in early-stage NSCLC.7
Anti-HER2 strategies, and particularly the monoclonal antibody trastuzumab, are effective for HER2 overexpression or amplified breast and gastric cancers when used in combination with cytotoxic agents.8,9 By contrast, clinical trials using anti-HER2 monoclonal antibodies (HER2-mab) in NSCLC patients have reported modest or disappointing results.10–15 In a phase II study, Herbst et al.11 evaluated the efficacy of single-agent pertuzumab, a monoclonal antibody that binds HER2’s dimerization domain and inhibits HER2 signaling. Among the 43 enrolled patients, no responses were seen, indicating lack of efficacy of the drug at least as single agent in unselected population. Nevertheless, preclinical data and anecdotic clinical data showed that HER2-mab or tyrosine kinase inhibitors could be effective in presence of HER2 gene mutations.16,17
Amino terminally truncated carboxyl terminal fragments of HER2, collectively known as p95HER2, are frequently found in HER2-expressing breast cancer cell lines and tumors.18 The biologic function of p95HER2 has not been fully characterized, although overexpression of p95HER2 has been shown to lead to growth of tumor xenografts in nude mice.19 In breast cancer, p95HER2 is present in approximately 11% of HER2 overexpressing tumors and it is responsible for trastuzumab resistance.20
Based on these data we hypothesized that, despite the fact that ~5% of NSCLC display HER2 gene amplification, the lack of efficacy of HER2-mab could be largely related to p95HER2 truncation. The aim of this study was to assess whether p95HER2 truncation is an event occurring in NSCLC and how such event was associated with HER2 gene copy number (GCN) and presence of HER2 mutations.
PATIENT CHARACTERISTICS AND METHODS
Cohort
This study was conducted in a cohort of 447 NSCLC patients previously analyzed for MET (mesenchymal-epithelial transition factor) GCN by fluorescence in situ hybridization (FISH).21 As previously described (Table 1), the majority of patients were male (83.4%), former (51.9%) or current (35.1%) smokers, with moderately or poorly differentiated tumors (grade II and III); the median age was 66 years; all patients received radical surgery, with evidence of pathological stage III in 34.7% and stage IV in 6% of cases; and a total of 161 patients were EGFR FISH positive and 48 patients were considered as MET FISH positive according to the previously described criteria.21,22 No postoperative therapy was delivered to patients in stage I–II, and only a minority of patients with stage III disease (18 cases) received adjuvant platinum-based chemotherapy. Patients with stage III disease and pathological evidence of N2 disease (n = 101) received postoperative mediastinal radiotherapy. With a median follow-up of 40 months, in the whole population, overall survival was 43.9 months. The criteria used for patient selection included availability of tumor tissue from primary lung cancer and survival data. The study was approved by the local Ethics Committee and was conducted in accordance with ethical principles stated in the most recent version of the Declaration of Helsinki or the applicable guidelines on good clinical practice, whichever represented the greater protection of the individuals.
TABLE 1.
Patient Characteristics
| Characteristics | Total | % |
|---|---|---|
| Total | 447 | |
| Median age, yr (range) | 66 (33–86) | |
| Gender | ||
| Male | 373 | 83.4 |
| Female | 74 | 16.6 |
| Smoking history | ||
| Never | 40 | 8.9 |
| Former | 232 | 51.9 |
| Current | 157 | 35.1 |
| Unknown | 18 | 4.1 |
| Histology | ||
| Adenocarcinoma + BAC | 241 | 53.9 |
| Squamous cell carcinoma | 139 | 31.1 |
| Othera | 67 | 15.0 |
| Pathological stage | ||
| I | 166 | 37.1 |
| II | 99 | 22.2 |
| IIIb | 155 | 34.7 |
| IV | 27 | 6.0 |
| Grade | ||
| I | 32 | 7.2 |
| II | 255 | 57.3 |
| III | 158 | 35.2 |
| Not defined | 2 | 0.3 |
| EGFR FISH+ | 161 | 42.8c |
| MET FISH+ | 48 | 11.1d |
Other histology included 7 patients with large cell carcinoma, 28 with neuroendocrine differentiation and undifferentiated NSCLC.
Stage III included 101 cases in stage IIIA and 54 cases in stage IIIB.
EGFR FISH analysis was performed in 376 cases.
MET FISH analysis was performed in 435 cases.
EGFR, epidermal growth factor receptor; FISH, fluorescent in situ hybridization; MET, mesenchymal-epithelial transition factor; BAC, bronchioloalveolar carcinoma.
Tissue Microarray
Paraffin-embedded tumor specimens were used to construct a tissue microarray (TMA) with 600 μm diameter cores. Three tissue cores represented each patient. An adhesive-coated tape system (Instrumedics, Hackensack, NJ) was used for sectioning the tumor array blocks at 4 μm.
p95HER2 Immunofluorescence and HER2 Immunohistochemistry
Immunohistochemistry (IHC) was performed on 3 μm tissue sections from the TMA blocks. Immunoreaction was displayed using the avidin-biotin-peroxidase complex method. Peroxidase activity was visualized with diaminobenzidine. Counterstaining was performed with hematoxylin. The negative controls were carried out by omitting the primary antibodies. Immunostaining was done using a Benchmark immunostainer (Ventana Medical System, Inc., Tucson, AZ). In all cases, the immunohistochemical evaluation was performed independently by two pathologists (G.A. and G.F.) who were blind to the clinicopathological characteristics and treatment of the patients. In all the discordant cases, a mutual agreement was eventually reached. Sections were incubated with HER2/neu antibody (ready to use for the Ventana automated slide stainer) according to the protocol of the manufacture (Ventana Medical System, Inc). Each slide was evaluated and scored on a 0 to 3 scale, following uniform guidelines developed for breast cancer testing.23 No staining was 0, 1+ indicated faint partial membrane staining in more than 10% of the tumor cells, 2+ indicated weak to moderate complete membrane staining in more than 10% of the tumor cells, and 3+ indicated moderate to strong complete membrane staining in more than 10% of the tumor cells.
The expression of NH2-terminally truncated form of HER2 (p95Her2) was evaluated by immunofluorescence as previously described.20,24 Analysis was performed on two sequential 3-μm tissue sections from the TMA blocks. After deparaffinization, the slides were incubated in 10 mM citrate buffer (pH 6.0) in a heated (95–99°C) water bath for 40 minutes for antigen retrieval. After rinse with dH2O and phosphate buffered saline (PBS), the sections were immersed in a PBS/4% BSA solution for 10 minutes to block nonspecific binding. Then, one section of all tumors was incubated at room temperature with a mouse monoclonal antibody to the intracellular domain of HER2 (clone CB11, Biogenex, San Ramon, CA) diluted 1:20 for 60 minutes, and another sequential section of all tumors was incubated with a mouse monoclonal antibody to the extracellular domain of HER2 (clone CBE1, Novocastra, UK) diluted 1:10 for 120 minutes. Both sections were then incubated with a rabbit polyclonal cytoplasmic anticytokeratin (Pan) (Invitrogen, Carlsbad, CA) diluted 1:50 for 30 minutes. After rinse with PBS, the sections were incubated with Alexa Fluor 555 antimouse IgG (Cell Signaling Technology, Beverly, MA) diluted 1:500 for 30 minutes to detect HER2 antibodies and with Alexa Fluor 488 antirabbit IgG diluted 1:500 for 30 minutes to detect cytokeratin antibody (Cell Signaling Technology). Then sections were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (Abbot Molecular Inc., Des Plaines, IL). If any cytoplasmic staining was detected with CB11 anti-HER2 antibody, tumors were scored as positive for p95HER2 expression.20 Cytoplasmic staining was confirming both with the colocalization of CB11 anti-HER2 antibody with the anticytokeratin antibody that showed yellow signal (red and green overlapping) and with the comparison with the pure membrane staining observed with the CBE1 anti-HER2 antibody. Staining was evaluated using Olympus BX-61 fluorescence microscope (Olympus America, Center Valley, PA) by two pathologists (G.A. and G.F.) who were blind to the clinicopathological characteristics and treatment of the patients.
HER2 FISH
Unstained 4-μm sections from each of the three TMA were submitted to dual-target, dual-color FISH assays using the PathVysion HER-2 DNA probe Kit (Vysis/Abbott Laboratories), which includes the LSI HER-2 SpectrumOrange and the CEP 17 SpectrumGreen probes. The FISH assays were performed according to protocol previously described,6,22 including pretreatment with 2×SSC at 75°C and digestion with Proteinase K for 7 to 15 minutes each, code-naturation at 85°C for 15 minutes, hybridization for approximately 36 hours, and rapid posthybridization washes with 2×SCC/0.4 NP40. Signals were enumerated in 30 tumor nuclei per core, using epifluorescence microscope with single interference filter sets for green (FITC), red (Texas red), and blue (DAPI) as well as dual (red/green) and triple (blue, red, green) band pass filters. Analysis was performed independently by two observers (Y.G.C. and M.V.G.) blinded to the patients’ clinical characteristics. Results were analyzed according to FISH criteria previously reported.25 When heterogeneous results were detected among the three tested cores, the core with the highest result was used to represent the patient in the statistic analyses. For documentation, images were captured using a charge-coupled device camera and merged using dedicated software (CytoVision, AI, Santa Clara, CA).
HER2 Mutation Analysis
Genomic DNA was extracted from tumors and normal lung tissues according to standard procedures. Genetic analysis of the HER2 gene was performed by polymerase chain reaction amplification of exons 19 and 20 with flanking intronic sequences followed by high-resolution melting analysis and sequencing. The primers for polymerase chain reaction amplification were previously reported.26 Tumor DNA samples found to be positive by high-resolution melting analysis were reamplified in duplicates, using the same experimental conditions, purified, and subjected to bidirectional dye-terminator sequencing with the same primers employed for amplification. Sequencing fragments were detected by capillary electrophoresis using the ABI Prism 3100 DNA analyzer (Applied Biosystems). Sequence chromatograms were analyzed by Mutation Surveyor 2.61 (SoftGenetics, State College, PA) followed by manual review.
Statistical Analyses
Kaplan-Meier analyses were performed to compare survival curves of subgroups of patients in different conditions.27 A log-rank test was used to test differences between survival and progression curves. Linear univariate and multivariate regression models were used to select relevant variables, and some statistics of each model were calculated such as odds ratio and relative risk with their respective confidence intervals. Levels of the response in the regression model were converted to a dichotomous variable where necessary. All the analyses were performed on Matlab (The MathWorks, Inc., Natick, MA) in house-built routines.
RESULTS
p95HER2
A total of 431 cases were fully evaluated for HER2 expression by IHC and for p95HER2 by immunofluorescence. HER2 overexpression (HER2 IHC positive) was found in 141 cases (32.7%) including 33 patients (7.7%) expressing p95HER2 (Figure 1). In the whole population, HER2 IHC positive status was not associated with patient survival (data not shown). As illustrated in Table 2, p95HER2 status was not associated with any clinical or biological characteristic except for histology. Among the 33 p95HER2+ cases, 26 (78.7%) had a nonadenocarcinoma histology (p = 0.001). Interestingly, presence of p95HER2 was detected in only one patient with HER2 gene amplification. To further investigate whether the receptor is truncated in presence of gene mutation, in addition to the study cohort, we analyzed p95HER2 status in eight NSCLC samples harboring HER2 mutation. In this small cohort of gene mutant NSCLC, one case resulted p95HER2+. As shown in Figure 2, in the whole population, p95HER2− patients had a shorter survival than p95HER2+ (41.3 months versus not reached, p = 0.21; hazard ratio: 1.41, 95% confidence interval: 1.08–1.82) even if the difference was not statistically significant. No difference in survival was observed in any subgroup of patients, as illustrated in Table 3.
FIGURE 1.
p95HER2 immunofluorescence. Immunofluorescence assay for detection of p95HER2 (top = p95HER2 negative case, bottom = p95HER2+ case). HER2 intracellular domain staining (red) colocalizes with cytokeratin staining (green) only in p95HER2+ tumors. Colocalization appears as a yellow (red and green merged) signal.
TABLE 2.
p95HER2 and Association with Clinical and Biological Characteristics
| Characteristic | p95HER2+, N (%) | p95HER2−, N (%) | p |
|---|---|---|---|
| All | 33 (7.7) | 398 (92.3) | |
| Female | 2 (2.9) | 68 (97.1) | 0.12 |
| Male | 31 (8.6) | 330 (91.4) | |
| Never-smokers | 0 (0) | 40 (100) | 0.09 |
| Smokers (former + current) | 33 (8.9) | 341 (91.1) | |
| Adenocarcinoma | 7 (3.2) | 210 (96.8) | 0.001 |
| Other histology | 26 (12.1) | 188 (87.9) | |
| Grade I–II | 19 (7.1) | 249 (92.9) | 0.68 |
| Grade III | 12 (8.2) | 134 (91.8) | |
| Stage I–II | 24 (9.2) | 237 (90.8) | 0.26 |
| Stage III–IV | 9 (6.0) | 140 (94.0) | |
| EGFR FISH+ | 11 (7.0) | 145 (93.0) | 0.4 |
| EGFR FISH− | 20 (9.5) | 190 (90.5) | |
| MET FISH+ | 3 (6.7) | 42 (93.3) | 0.72 |
| MET FISH− | 30 (8.2) | 336 (91.8) | |
| HER2 FISH+ | 1 (1.7) | 57 (98.3) | 0.1 |
| HER2 FISH− | 31 (8.5) | 336 (91.5) |
EGFR, epidermal growth factor receptor; FISH, fluorescent in situ hybridization; MET, mesenchymal-epithelial transition factor; HER2, human epidermal growth factor receptor 2.
FIGURE 2.
Survival in non-small cell lung cancer according to p95HER2. In the study population, p95HER2− patients had shorter survival (41.3months, n = 398) than p95HER+ patients (not reached, n = 33). The difference was not statistically significant (p = 0.21).
TABLE 3.
Survival in p95HER2 Positive and Negative Patients According to Clinical and Biological Characteristics
| Characteristic | p95HER2+, N (mo) | p95HER2−, N (mo) | p | Hazard Ratio |
|---|---|---|---|---|
| All | 33 (NR) | 398 (41.3) | 0.21 | 1.41 (1.09–1.83) |
| Female | 2 (51.0) | 68 (35.0) | 0.41 | 1.52 (0.03–86) |
| Male | 31 (NR) | 330 (44.0) | 0.27 | 1.35 (1.02–1.79) |
| Never-smokers | 0 (—) | 40 (61.0) | — | — |
| Smokers (former + current) | 33 (NR) | 341 (41.3) | 0.21 | 1.42 (1.09–1.85) |
| Adenocarcinoma | 7 (17.5) | 210 (38.5) | 0.22 | 0.48 (0.11–1.98) |
| Other histology | 26 (NR) | 188 (44.0) | 0.1 | 1.82 (1.27–2.6) |
| Grade I–II | 19 (NR) | 249 (46.0) | 0.21 | 1.5 (0.95–2.4) |
| Grade III | 12 (35.0) | 134 (32.0) | 0.42 | 0.96 (0.46–2) |
| Stage I–II | 24 (NR) | 237 (NR) | 0.44 | 1.0 (0.69–1.45) |
| Stage III–IV | 9 (8.4) | 140 (18.5) | 0.44 | 2.22 (0.86–5.78) |
| EGFR FISH+ | 11 (NR) | 145 (38.0) | 0.28 | 1.44 (0.66–3.1) |
| EGFR FISH− | 20 (NR) | 190 (45.0) | 0.38 | 1.33 (0.86–2) |
| MET FISH+ | 3 (27.5) | 42 (23.3) | 0.45 | 1.25 (0.05–29.7) |
| MET FISH− | 30 (NR) | 336 (45.0) | 0.24 | 1.46 (1.09–1.94) |
| HER2 FISH+ | 1 (35.0) | 57 (36.5) | 0.48 | — |
| HER2 FISH− | 31 (50.0) | 336 (44.0) | 0.36 | 1.21 (0.92–1.6) |
EGFR, epidermal growth factor receptor; FISH, fluorescent in situ hybridization; MET, mesenchymal-epithelial transition factor; HER2, human epidermal growth factor receptor 2; NR, not reached.
HER2 FISH
Among the 447 patients included in the study, 439 were successfully analyzed by FISH. Using the scoring system adopted in previous studies,6,22,25 HER2 FISH positive status (≥4 HER2 copies in ≥40% of cells) was found in 60 cases (17.6%), including 22 cases (5.0%) with gene amplification, defined according to breast cancer criteria.23 As illustrated in Table 4, HER2 FISH positive status was more frequently observed in patients with adenocarcinoma histology, even if the association was not statistically significant (0.09). No association with any other clinical characteristic was observed. As previously reported by our group,6 HER2 FISH positive status was significantly associated with increased EGFR (p = 0.00004) and MET (p = 0.005) GCN. Interestingly, among the 22 patients with HER2 gene amplification, 15 (68.1%) resulted coamplified for MET gene and 4 (18.1%) coamplified also for EGFR gene. Median survival was 36.0 months in HER2 FISH positive and 42.0 months in HER2 FISH negative (p = 0.48, Figure 3A). No difference in survival was detected even when the observation was confined to patients with HER2 gene amplification (38.0 months in HER2 amplified versus 41.3 in HER2 nonamplified, p = 0.47, Figure 3B). No difference in survival was observed in any subgroup of patients, including the small subgroup of MET FISH positive, as illustrated in Figure 4.
TABLE 4.
HER2 GCN and Association with Clinical and Biological Characteristics
| Characteristic | HER2 FISH+, N (%) | HER2 FISH−, N (%) | p |
|---|---|---|---|
| All | 60 (13.7) | 379 (86.3) | |
| Female | 9 (12.5) | 63 (87.5) | 0.75 |
| Male | 51 (13.9) | 316 (86.1) | |
| Never-smokers | 6 (15.8) | 32 (84.2) | 0.74 |
| Smokers (former + current) | 53 (13.8) | 330 (86.2) | |
| Adenocarcinoma | 36 (16.5) | 183 (83.5) | 0.09 |
| Other histology | 24 (10.9) | 196 (89.1) | |
| Grade I–II | 38 (14.0) | 234 (86.0) | 0.71 |
| Grade III | 19 (12.7) | 131 (87.3) | |
| Stage I–II | 38 (14.6) | 223 (85.4) | 0.62 |
| Stage III–IV | 20 (12.8) | 136 (87.2) | |
| EGFR FISH+ | 38 (23.9) | 121 (76.1) | 0.00004 |
| EGFR FISH− | 18 (8.5) | 194 (91.5) | |
| MET FISH+ | 13 (28.3) | 33 (71.7) | 0.005 |
| MET FISH− | 47 (12.6) | 327 (87.4) |
EGFR, epidermal growth factor receptor; FISH, fluorescent in situ hybridization; MET, mesenchymal-epithelial transition factor; HER2, human epidermal growth factor receptor 2; GCN, gene copy number.
FIGURE 3.
HER2 gene copy number and patient survival. The panel shows overall survival in HER2 FISH positive and HER2 negative patients (defined using the Colorado score). A, Patients with increase of HER2 gene copy number (HER2 FISH+, n = 60) had shorter survival than individuals with no increased HER2 gene copy number (HER2 FISH−, n = 379). Median survival was 36.0 months in HER2 FISH+ and 42.0 months in HER2 FISH−. This difference was not statistically significant (p = 0.48). B, The small subgroup of patients with HER2 gene amplification (HER2 GA, n = 22) had a median survival of 38.0 months versus 41.3 months in the HER2 nonamplified group (HER2 no GA, n = 417). The difference was not statistically significant (p = 0.47).
FIGURE 4.
Survival according to HER2 and MET gene amplification. No difference in survival was detected when HER2 gene copy number assessment was evaluated in the context of MET amplification, defined as a mean MET gene copy number ≥5. The subgroup of patients with MET FISH positive and HER2 gene amplification (MET FISH+/HER2 GA, n = 10) had a median survival of 36 months versus 20.5 months in MET FISH positive and HER2 nonamplified (MET FISH+/HER2 no GA, n = 36). The difference was not statistically significant (p = 0.26).
DISCUSSION
In this study, we investigated whether p95HER2 could be implicated in primary resistance to HER2-mab therapy in NSCLC and observed that this event occur in less than 10% of patients, mainly in absence of HER2 amplification or mutation and has no prognostic effect.
The activity of HER2-mab, including trastuzumab and pertuzumab, has been evaluated in NSCLC in phase II trials with disappointing results.10–15 Although it is possible that the lack of patient selection was responsible for clinical trial failures, it is not possible to exclude that additional biological markers could be responsible for drug resistance even in the presence of the target. In the trial conducted by Gatzmeier et al.,10 where only a small proportion of patients displayed HER2 gene amplification, the small subset of individuals with strong HER2 expression (3+) and HER2 amplified had a prolonged response to trastuzumab therapy. Nevertheless, it is unknown whether this effect was related to trastuzumab or to chemotherapy, considering that HER2 amplification is frequently associated with EGFR gene amplification and mutation, a biological event increasing sensitivity to anti-EGFR agents as well as to chemotherapy.3,6 In the current study, where all patients were previously investigated for EGFR by FISH, a significant association between HER2 and EGFR GCN was observed, confirming our previous observation.6 In the pertuzumab trial, among the 43 treated patients, no response was observed. Moreover, no association of HER2 expression levels, evaluated by IHC, and progression-free survival (PFS) was detected, indicating a modest effect of the drug, at least when used as single agent.11 Other small phase II trials investigating the efficacy of trastuzumab together with chemotherapy reported a response rate comparable to that achievable with a standard chemotherapy alone, suggesting that the drug is ineffective in most of NSCLC.12–15 Overall, these data indicated that monoclonal antibodies against HER2 are not effective in the majority of NSCLC, with a potential but no proven efficacy in presence of HER2 amplification.
HER2 activating mutations are rare events in NSCLC, occurring in approximately 2% of cases and mainly in females and never-smokers.28 During the last years, preclinical investigations revealed that NSCLC cell lines harboring HER2 mutations are sensitive to trastuzumab therapy. Wang et al.29 reported that anti-HER2 agents including trastuzumab inhibited H1718 lung cancer cells containing the exon 20 HER2 mutation. Moreover, our group reported a case of pretreated NSCLC patient with HER2 mutation dramatically responding to the combination of trastuzumab and paclitaxel.16 These data suggested that anti-HER2 therapies could be effective in HER2-mutant NSCLC, and the low incidence of HER2 mutations in unselected NSCLC justify why no sign of efficacy with anti-HER2 monoclonal antibodies was detected in clinical trials. Spanish investigators reported that HER2 truncation (p95HER2) frequently occurs in breast cancer with HER2 amplification and such event is responsible for trastuzumab failure in approximately 10% of breast cancer patients.20 In our study, HER2 truncation only occurred in less than 10% of NSCLC, predominantly in HER2 wild-type and nonamplified. Therefore, the incidence of HER2 truncation in NSCLC is too low for explaining the lack of sensitivity to trastuzumab observed in clinical trials.
In breast cancer, presence of p95HER2 predicts worse prognosis in HER2+ patients.18,30,31 Our analysis, the first conducted in NSCLC and analyzing a large cohort of patients, revealed a modest and no statistically significant difference in survival between individuals with or without HER2 truncation, demonstrating that such event plays no prognostic role in surgically resected NSCLC.
In the second part of our study, the attention was focused on the prognostic role of HER2 GCN. A recent meta-analysis, including 974 patients evaluated by FISH, showed that HER2 GCN was not a prognostic factor in lung cancer.7 Nevertheless, the meta-analysis included seven studies using different scoring systems and reporting discordant results. Herein, we report data from a large cohort of NSCLC surgically treated before adjuvant chemotherapy became a standard approach. These data, obtained without a potential confounding effect of chemotherapy, confirmed that increased HER2 GCN is not prognostic in resected NSCLC. Importantly, no difference in survival was detected even when the analyses were confined to patients with HER2 gene amplification or in the context of increased MET GCN.
An intriguing result of our study was the significant association between EGFR, HER2, and MET GCN. Our data provide further evidence that NSCLC does not depend uniquely on HER2 signaling even in presence of HER2 amplification or copy number gain. Studies on NSCLC and breast cancer cell lines shown complex signaling crosstalk between receptor tyrosine kinases and downstream molecules, in particular EGFR, HER2, HER3, c-Met, and AKT.32,33 EGFR and MET have been shown to crosstalk physically and functionally. EGFR has the ability to act as a scaffolding protein to link MET and c-Src.34 MET is commonly expressed in HER2+ breast cancers and directly implicated in the development of trastuzumab resistance, through sustained HER3-PI3K-AKT signaling. The concept of targeting MET pathway as a strategy to circumvent resistance to anti-EGFR or HER2 agents is very attractive, but it has to be prospectively assessed in clinical trials.32,35
In conclusion, HER2 truncation and increased GCN are not prognostic in resected NSCLC. Although p95HER2 is present in a small fraction of NSCLC, mainly in tumors with HER2 wild-type or nonamplified, it is unlikely that this event is responsible for the lack of efficacy of HER2-mab therapies observed in clinical trials. The strong association of HER2 GCN with EGFR and MET GCN support the association of anti-HER2 agents with other targeted drugs in selected NSCLC.
Acknowledgments
Supported in part by the Italian Association for Cancer Research (AIRC) and Association translational Oncology (AOT).
The authors thank the Department of Pathology of Istituto Clinico Humanitas, Rozzano, Italy, for providing patient tissues and to the Division of Molecular Pathology, Basel, Switzerland, for technical assistance.
Footnotes
Disclosure: Federico Cappuzzo, MD, and Marileila Varella-Garcia, PhD, hold a patent for EGFR FISH. The other authors declare no conflicts of interest.
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