Experimental and Molecular Pathology 98 (2015) 27–32
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Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
Prolonged survival after neoadjuvant chemotherapy related with specific molecular alterations in the patients with nonsmall-cell lung carcinoma Jelena Stojsic a, Tijana Stankovic b, Sonja Stojkovic b, Vedrana Milinkovic b, Jelena Dinic b, Zorica Milosevic b, Zorka Milovanovic c, Nikola Tanic b, Jasna Bankovic b,⁎ a b c
Department of Thoracopulmonary Pathology Service of Pathology, Clinical Centre of Serbia, Koste Todorovica 20/26, 11000 Belgrade, Serbia Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia Institute for Oncology and Radiology of Serbia, University of Belgrade, Pasterova 14, 11000 Belgrade, Serbia
a r t i c l e
i n f o
Article history: Received 13 November 2014 Accepted 18 November 2014 Available online 20 November 2014 Keywords: Non-small cell lung carcinoma Neodjuvant chemotherapy PTEN pERK pAKT
a b s t r a c t Lung cancer is the most common cause of neoplasia-related death worldwide. Accounting for approximately 80% of all lung carcinomas, the non-small cell lung carcinoma (NSCLC) is the most common clinical form with its two predominant histological types, adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Although surgical resection is the most favorable treatment for patients with NSCLC, relapse is still high, so neoadjuvant chemotherapy (NAC) is an accepted treatment modality. In this study we examined whether some of the key molecules associated with the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways could have predictive and prognostic value for the NAC application. To that end we examined the expression status of PTEN, pAKT, pERK and loss of heterozygosity (LOH) of PTEN in two groups of NSCLC patients, those who received and those who did not receive NAC. LOH PTEN and low pERK expression is shown to be correlated with the longest survival of patients with SCC and ADC, respectively, who received NAC. These results point that the application of NAC is beneficial in the NSCLC patients with specific molecular alterations which could further help to improve constant search for the druggable molecular targets used in personalized therapy. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Lung cancer is the most common cause of neoplasia-related death worldwide with a variety of histological types. The non-small cell lung carcinoma (NSCLC), which predominantly comprises of adenocarcinoma (ADC) and squamous cell carcinoma (SCC), is the most common histological subgroup of lung cancer, accounting for approximately 80% of all lung carcinomas. Surgical resection is the most favorable treatment for early-stage NSCLC, although relapse is still high, especially in stages II and III (Win et al., 2008). Management of locally advanced NSCLC represents a challenge, particularly because the role of surgery in this group is controversial in view of the fact that most patients relapse within 3 years from diagnosis, causing 5-year overall survival b10%. Considering the high rate of extrapulmonary relapses, possibly due to the presence of distant micro-metastases at the time of diagnosis, neoadjuvant chemotherapy (NAC) is an accepted treatment modality for patients with NSCLC. Potential disadvantages include the development of medical illness and treatment-related toxicity (Boudaya et al., 2013). Therefore, there is a need for the identification of appropriate molecular markers
⁎ Corresponding author at: Despot Stefan Boulevard 142, 11060 Belgrade, Serbia. E-mail address: [email protected] (J. Bankovic).
http://dx.doi.org/10.1016/j.yexmp.2014.11.010 0014-4800/© 2014 Elsevier Inc. All rights reserved.
which would identify patients who would benefit from neoadjuvant chemotherapy. The PI3K/AKT/mammalian target of rapamycin (mTOR) (PI3K) and the RAS/RAF/MAP kinase–ERK kinase (MEK)/extracellular-signal-regulated kinase (ERK) (MAPK) pathways are frequently deregulated in human cancers, as well as lung cancer, mostly as a result of genetic alterations in their components (De Luca et al., 2012). PI3K/AKT/mTOR signaling pathway has been shown to be involved in the regulation of cell proliferation and apoptosis, and is a key to the initiation and progression of malignancies, enhancing cell survival by the stimulation of cell proliferation and the inhibition of apoptosis (Cantrell, 2001). The main regulator of this pathway, PTEN (phosphatase and tensin homolog deleted on chromosome 10), frequently altered in lung cancer, reduces the downstream activity of AKT, thereby inducing cell-cycle arrest and apoptosis (Hosoya et al., 1999). Another signaling pathway very important in carcinogenesis is RAS/RAF/MEK/ERK and it promotes cell proliferation, angiogenesis, cell differentiation and migration (McKay and Morrison, 2007). In the current study we investigated the expression status of PTEN, pAKT, pERK and loss of heterozygosity (LOH) of PTEN in two groups of NSCLC patients, those who received and those who did not receive NAC. The aim was to evaluate whether these key components of RAS/ RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways could have the predictive and prognostic value for the rational application of NAC.
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J. Stojsic et al. / Experimental and Molecular Pathology 98 (2015) 27–32
2. Materials and methods
were assessed spectrophotometrically. The quality of DNA was verified by electrophoresis.
2.1. Tissue samples 2.3. LOH analysis Paired samples from cancer and adjacent normal lung tissue from 70 patients with NSCLC who underwent surgery, lobectomy or pneumonectomy with regional lymphadenectomy, at Clinic of Thoracic Surgery, Clinical Centre of Serbia, Belgrade, Serbia, were analyzed. Patients were divided into two groups. One group of 35 patients received NAC, while the other did not. All chemotherapy protocols were platinum-based ranging from I to IV cycles. The samples were collected and used after obtaining an informed consent and approval from the Ethics Committee, in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. The tumors were histologically classified according to the latest World Health Organization classification of the lung cancer (Travis et al., 2004) while the pathological stage of the tumors diagnosed before 2010 was reclassified according to the recommendation from 2009 (Travis, 2009; Vallières et al., 2009). Diagnosis of NSCLC, the histological grade and regional lymph node involvement were established by histological examinations of the surgical specimens. The patients' distribution by clinicopathologic parameters is presented in Table 1.
2.2. DNA extraction Specimens obtained from the patients without NAC were frozen in liquid nitrogen, where they were kept until DNA extraction. This DNA was extracted using the phenol/chloroform/isoamyl alcohol method (Sambrook et al., 1989). DNA from paraffin-embedded tumor material from patients with NAC was extracted using a Kappa Express Extract DNA extraction kit (KapaBiosystems, USA) according to the manufacturer's procedure. The concentrations of isolated nucleic acids
Table 1 Clinicopathological parameters of patients with and without NAC. Parameter
Patients with NAC
Patients without NAC
Total Age ≤50 N50 Gender Male Female NSCLC subtype Adenocarcinoma Squamous cell carcinoma Necrosisa n1 n2 n3 Inflammation Level 1 Level 2 Level 3 Histological gradeb g1 g2 g3 Stage I II III Lymph node invasion Positive Negative
35
35
6 29
11 24
26 9
23 12
16 19
15 20
27 5 3
25 6 4
9 20 6
15 17 3
10 15 10
6 24 5
5 15 15
2 12 21
25 9
29 6
NAC — neoadjuvant chemotherapy. a n1, obscure or no necrosis; n2, necrosis in 50% of tumor mass; n3, necrosis in more than 50% of tumor mass. b g1, well differentiated; g2, moderately differentiated; g3, poorly differentiated.
The DNA obtained from the malignant and normal lung tissue from all 70 patients was used to study the LOH of PTEN, tumor suppressor gene. LOH analyses were performed using highly polymorphic microsatellite markers. Five polymorphic microsatellite markers spanning the PTEN gene (D10S579, D10S1765, D10S215, AFM086wg9, and D10S541) were selected to cover deletions at the whole PTEN locus on chromosome 10q23. All forward primers were 5′-labeled with Fam, Vic, Ned, Pet, and Fam fluorescent dyes, respectively. The choice of the microsatellite markers and locus-specific PCR conditions were determined from published sources (Feilotter et al., 1998; Hahn et al., 1999). The PCR products were separated by capillary electrophoresis on an ABI Prism 3130 automated sequencer and sized using GeneScan-500 LIZ size standard (Applied Biosystems). The obtained data were analyzed with the GeneMapper software (Applied Biosystems). The DNA from normal lung tissue adjacent to tumors from the same patient was used as reference. On the one hand, a marker was defined as noninformative (homozygote) when only 1 allelic peak was detected in the DNA sample of the normal lung tissue. On the other hand, a marker was considered informative (heterozygote) when 2 major allelic peaks occurred in a normal specimen. The LOH score for the informative cases was calculated automatically by GeneMapper software according to the following equation: (peak height of normal allele 2)/(peak height of normal allele 1) divided by (peak height of tumor allele 2)/(peak height of tumor allele 1). A sample was considered to be an LOH candidate for particular locus if the ratio values were less than 0.66 and higher than 1.5. 2.4. Immunohistochemistry Tumor samples were fixed in buffered 10% formalin, embedded in paraffin blocks and cut in 3 μm for routine analysis. The following antibodies were used according to manufacturer's instructions: PTEN (1:50, clone: PN37, Invitrogen, USA), pERK (1:100, clone: P44/42MAPL-ERK1/2(137 FS), Cell Signaling, USA) and pAKT (1:40, clone: HCL-l AKT-Phos, Novocastra, Leica Biosystems, USA). Immunostaining was performed by incubating tissue sections with appropriate serum for 30 min at room temperature in humidity chamber, using the streptavidinbiotin technique (LSAB + Kit, Peroxidase Labeling, K0690, DAKO Cytomation, Denmark). Antigen–antibody complexes were visualized with diaminobenzidinehydrochloride (DAB, No. K3468, DAKO Cytomation, Denmark) substrate solution. The cell nuclei were contra-stained with Mayer's hematoxylin. At the same time, tissue samples with appropriate positive immunostaining were used as indicators of the quality of the target retrieval procedure. Positive immunoreactivity of pAKT in epidermis of human skin and PTEN and pERK in regular breast ductal epithelia was used as the internal positive control. Immunohistochemical (IHC) results were independently evaluated by two pathologists (J.S. and Z.M.) on microscope Leica DM2500 (Leica Microsystems, Germany). The immunoreactivity of PTEN was assessed using the semiquantitative method based on the score of percentage of stained cells—cytoplasm/nuclei (P) (0, no immunoreactivity; 1,1–10%; 2, 11–50%; 3, 51–100%) and intensity of staining (SI) (0, no immunoreactivity; 1, reduced staining intensity relative to the corresponding normal cells; 2, same as normal cells staining; 3, increased staining). Since cutoff levels for reduced PTEN expression by immunohistochemical methods have not been defined so far, we used the mean PTEN score as a cutoff point to designate reduced expression (Shoman et al., 2005). Accordingly, PTEN status was defined as follows: low expression if score was ≤ 4; and high expression if score was N4. The pAKT staining was evaluated by an H-score, which was calculated
J. Stojsic et al. / Experimental and Molecular Pathology 98 (2015) 27–32
by multiplying the P by the corresponding SI (1 = weak, 2 = moderate, and 3 = strong), giving a maximum score of 300 (100% × 3). A positive control for pAKT (human skin) was included in each analysis. H-scores N 50 were considered positive (Esteva et al., 2010). The pERK immunoreactivity levels of each case were assessed semi-quantitatively under a light microscope by assessing the average signal intensity (on scale of 0 to 3) and the proportion of cells showing a positive cytoplasmatic stain (0, none; 0.1, less than one tenth; 0.5, less than one half; and 1, greater than one half). The intensity and proportion scores were then multiplied to give the H-score (Al-Haddad et al., 1999). H-score more than 1 was considered as elevated pERK immunoreactivity (Handra-Luca et al., 2003).
2.5. Statistical analysis A statistical analysis was performed using STATISTICA 6.0 software (StatSoft, Inc., Tulsa, USA). The following clinicopathological and genetic parameters were evaluated: histological type, grade, tumor clinicopathologic stage of tumor, lymph node invasion, reactive inflammation, degree of tumor necrosis, PTEN alterations, PTEN, pERK and pAKT expression and patients' outcome. To test correlations between different parameters Fisher exact test and Mann–Whitney U test were used. Survival analyses were performed using the Kaplan and Meier product-limit method; to test the difference in overall survival, the log-rank test was used. The overall survival was calculated from day one after surgery to the last follow-
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up examination or death of the patient. Statistical differences were considered significant when P ≤ 0.05. 3. Results 3.1. LOH analysis of PTEN tumor suppressor gene Loss of heterozygosity of PTEN gene was evaluated by fragment analysis (Fig. 1). In the group of patients without neoadjuvant chemotherapy 19 tumor samples were noninformative for all 5 tested markers. All the rest of the 16 informative cases showed the loss of an allele (45.7%, Table 2). In the group of patients who received the neoadjuvant chemotherapy 32 samples were available for LOH analysis and 16 of them were noninformative for PTEN markers. Among 16 informative cases, which were heterozygous for at least 1 examined locus, 13 showed the loss of an allele (40.6%, Table 2). 3.2. pAKT, pERK and PTEN protein expression According to the established IHC scoring immunohistochemical studies of the patients' samples in the group without NAC were detected: the high level of PTEN expression in 13 (37.1%), of pAKT in 18 (51.4%) and of pERK in 11 (31.4%) out of 35 NSCLC patients (Table 2). The results obtained for the group of patients with NAC were similar with high pAKT in 19 (54.3%) and pERK in 9 (25.7%) out of 35 patients, while PTEN expression was slightly different being high in 7 patients
Fig. 1. Loss of heterozygosity (LOH) analysis of PTEN gene. A) LOH of PTEN detected using D10S579 microsatellite marker in the sample from patient 9 receiving NAC, B) LOH PTEN detected using D10S1765 microsatellite marker in the sample from patient 33 not receiving NAC, C) LOH of PTEN detected using D10S215 microsatellite marker in the sample from patient 21 receiving NAC. Arrows indicate allelic loss in tumor samples.
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Table 2 Gene alterations and immunophenotype in NSCLC tissue samples. Patients
LOH PTEN
High PTEN expression
High pAKT expression
High pERK expression
With NACa Without NAC
13/32 (40.6%) 16/35 (45.7%)
7/35 (20%) 13/35 (37.1%)
19/35 (54.3%) 18/35 (51.4%)
9/35 (25.7%) 11/35 (25.7%)
a
Neoadjuvant chemotherapy.
(20%) out of 35 patients (Table 2). Representative examples of staining intensities for PTEN, pAKT and pERK are shown in Fig. 2. 3.3. LOH PTEN, pAKT, pERK and PTEN expression in relation to clinicopathological parameters The correlation between LOH PTEN, pAKT, pERK and PTEN expression and clinicopathological parameters was assessed regarding two groups of patients, those with and those without NAC. Samples were grouped according to the following clinicopathological parameters: histological type, histological grade, tumor stage, lymph node invasion, degree of tumor necrosis, and reactive inflammation. The number of patients without lymph node invasion who received NAC and had low pAKT expression was significantly higher than the number of those who had high expression of this protein (Table 3, P = 0.01). 3.4. LOH PTEN, pAKT, pERK and PTEN expression and survival rate The overall survival of patients with and without NAC was evaluated in relation to LOH PTEN, pAKT, pERK and PTEN expression in their NSCLC samples (Fig. 3). pERK expression was significant for the survival of the patients with NAC. Namely, patients in this group who had low pERK expression lived significantly longer (Fig. 3A, P = 0.05). Further, all patients were stratified according to the tumor subtype and in those who had SCC and carried LOH PTEN, NAC had favorable effect on survival, i.e. those who received it lived significantly longer than those who did not (Fig. 3B, P = 0.006). Patients who had ADC, low pERK expression and received NAC had the longest survival, followed by patients with low
pERK expression without NAC, patients with high pERK and NAC and finally patient with high pERK and no therapy (Fig. 3C, P = 0.01). 4. Discussion The aim of this study was to evaluate whether key molecules of RAS/ RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways could have the predictive and prognostic value for the rational application of NAC in the treatment of NSCLC. The expression status of PTEN, pAKT, pERK and LOH PTEN in two groups of NSCLC patients, those who received and those who did not receive NAC was evaluated. The expression levels of pAKT, pERK as well as LOH PTEN were similar in both groups of patients, in those who received and in those who did not receive NAC. Only PTEN expression was slightly higher in the group without NAC (37.1% vs. 20%) but with no statistical significance. Similarly, studies that included breast or rectal cancer samples demonstrated the same modality, i.e. low or lost PTEN expression did not correlate with chemotherapy (Yonemori et al., 2009; Barbareschi et al., 2012; Erben et al., 2011). Further investigation included assessment of the correlations of LOH PTEN, pAKT, pERK and PTEN expression and clinicopathological parameters among patients with and without NAC. The obtained results showed that in the group of patients who received NAC and were without lymph node invasion the number of those with low pAKT expression was significantly higher than the number of those with high pAKT expression. Hence, it could be said that chemotherapy lowers the expression of this protein suggesting its beneficial usage in the tumors with activation of PI3K/AKT/mTOR pathway and clear lymph nodes which is usually expected in the early-stage tumors. This is supported by the fact that PI3K and pAKT expression has already
Fig. 2. Immunohistochemical analysis of pAKT, PTEN and pERK expression in non-small cell lung carcinoma. Representative examples of: A) positive antibody controls: pAKT skin (10× objective); PTEN breast tissue (10× objective); pERK breast tissue (10× objective); B) weak nuclear pAKT immunoreactivity (20× objective), weak cytoplasmatic PTEN immunoreactivity (20× objective) and weak cytoplasmatic pERK immunoreactivity (20× objective); C) moderate nuclear pAKT immunoreactivity (20× objective), moderate cytoplasmatic PTEN immunoreactivity (20× objective) and moderate cytoplasmatic pERK immunoreactivity (20× objective); D) strong nuclear pAKT immunoreactivity (20× objective), strong cytoplasmatic PTEN immunoreactivity (20× objective) and strong cytoplasmatic pERK immunoreactivity (20× objective).
J. Stojsic et al. / Experimental and Molecular Pathology 98 (2015) 27–32 Table 3 pAKT expression in patients without lymph node invasion. Variables
NACb Without With a b c
pAKT expression Low
High
NPa (%)
NP (%)
1 (16.7) 8 (88.9)
5 (83.3) 1 (11.1)
P value
0.01c
NP, number of patients per group. Neoadjuvant chemotherapy. Bold indicates statistically significant values, P ≤ 0.05.
been detected in the early stage NSCLC (Al-Saad et al., 2009; David et al., 2004). In addition, Yip P. et al. reported that patients with early-stage NSCLC who expressed higher levels of nuclear pAKT had a poorer prognosis than those with lower levels of expression (Yip et al., 2014). Also, it was shown that down-regulation of pAKT could suppress invasion and migration of osteosarcoma cells (Long et al., 2013). When analyzing patients' survival in the relation to the expression status of PTEN, pAKT, pERK and LOH PTEN, patients in the group with NAC who had low pERK expression lived significantly longer than those with high pERK expression. The investigation on lung ADC cell line A549 also showed that their migration and invasion could be suppressed via PKC-α-ERK1/2-NF-κB pathway (Cheng et al., 2014). Similar results were obtained on osteosarcoma cells, whose growth and invasion were inhibited possibly through inhibition of the MAPK/ERK pathway (Miao et al., 2014). In addition, neoadjuvant therapies administered to skin carcinoma and colorectal adenocarcinoma induced a synergistic cytotoxicity after a prolonged ERK inhibition (Weyergang et al., 2013). This all suggests that low pERK could be an important factor for the prolonged survival of our patients with NSCLC who received NAC regardless of tumor histological type. However, the most interesting results were obtained when the NSCLC samples were grouped according to the histological type. Namely, patients with SCC, LOH PTEN and NAC lived significantly longer than those who did not have this type of treatment. Among the patients with ADC, those who had low pERK expression and received NAC had the longest survival, followed by patients with low pERK expression without chemotherapy, patients with high pERK and chemotherapy and finally patient with high pERK and no therapy. Previous studies reported that the activation of the PI3K– AKT–mTOR signaling pathway inhibits ERK1/2 activation (Milosevic et al., 2014; Rommel et al., 1999) and in addition we would also expect high pAKT in the presence of LOH PTEN. Since we showed that NAC has a positive effect in the patients with activation of the PI3K–AKT–mTOR signaling pathway, high pAKT could be, in fact, a positive marker for
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the application of NAC in NSCLC. Also, all NAC protocols for our patients were platinum-based and it has been shown that its antitumor activity was promoted through AKT and ERK1/2 pathways (Zhang et al., 2014a,b). Our results are very important from the standpoint that it could imply to the possibility of more detailed stratification of the patients before the application of the therapy towards personalized treatment. The discovery of the so-called ‘driver mutations’ such as the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) has led to the improvement in personalized therapy for lung ADC (Giaccone, 2005; Kwak et al., 2010). However, no such available molecular targets exist for SCC. For this reason, the identification of novel druggable molecular targets in SCC would be a research priority. In conclusion, since there is a high rate of extrapulmonary relapses, possibly due to the presence of distant micro-metastases at the time of diagnosis of NSCLC, NAC is an accepted treatment modality for these patients. In this study we examined whether some of the key molecules associated with the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways could have predictive and prognostic value for the NAC application. LOH PTEN and low pERK expression is shown to be correlated with the longest survival of patients with SCC and ADC, respectively, who received NAC. In accordance with other studies these results imply that the application of NAC is beneficial in the treatment of NSCLC with the additional therapeutic targets for ADC and the new ones for SCC which would lead to the personalized treatment of these patients. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This study was supported by Grant III41031 from the Ministry of Education, Science and Technological Development of the Republic of Serbia. References Al-Haddad, S., et al., 1999. Psoriasin (S100A7) expression and invasive breast cancer. Am. J. Pathol. 155, 2057–2066. Al-Saad, S., et al., 2009. Diverse prognostic roles of AKT isoforms, PTEN and PI3K in tumor epithelial cells and stromal compartment in non-small cell lung cancer. Anticancer Res. 29, 4175–4183. Barbareschi, M., et al., 2012. PI3KCA mutations and/or PTEN loss in Her2-positive breast carcinomas treated with trastuzumab are not related to resistance to anti-Her2 therapy. Virchows Arch. 461, 129–139. Boudaya, M.S., et al., 2013. What outcome after the prescription of neoadjuvant chemotherapy in lung cancer? Asian Cardiovasc. Thorac. Ann. 21, 432–436. Cantrell, D.A., 2001. Phosphoinositide 3-kinase signaling pathways. J. Cell Sci. 114, 1439–1445.
Fig. 3. Kaplan–Meier survival curves. Patients who received NAC and had low pERK expression lived significantly longer (panel A, P = 0.05). Patients with SCC who carried LOH PTEN and received NAC lived significantly longer than those without this treatment (panel B, P = 0.006). Patients who had ADC, low pERK expression and received NAC had the longest survival, followed by patients with low pERK expression without chemotherapy, patients with high pERK and chemotherapy and finally patient with high pERK and no therapy (panel C, P = 0.01). The survival among analyzed groups of patients was considered significantly different if P ≤ 0.05.
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Contents lists available at ScienceDirect
Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
Prolonged survival after neoadjuvant chemotherapy related with specific molecular alterations in the patients with nonsmall-cell lung carcinoma Jelena Stojsic a, Tijana Stankovic b, Sonja Stojkovic b, Vedrana Milinkovic b, Jelena Dinic b, Zorica Milosevic b, Zorka Milovanovic c, Nikola Tanic b, Jasna Bankovic b,⁎ a b c
Department of Thoracopulmonary Pathology Service of Pathology, Clinical Centre of Serbia, Koste Todorovica 20/26, 11000 Belgrade, Serbia Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia Institute for Oncology and Radiology of Serbia, University of Belgrade, Pasterova 14, 11000 Belgrade, Serbia
a r t i c l e
i n f o
Article history: Received 13 November 2014 Accepted 18 November 2014 Available online 20 November 2014 Keywords: Non-small cell lung carcinoma Neodjuvant chemotherapy PTEN pERK pAKT
a b s t r a c t Lung cancer is the most common cause of neoplasia-related death worldwide. Accounting for approximately 80% of all lung carcinomas, the non-small cell lung carcinoma (NSCLC) is the most common clinical form with its two predominant histological types, adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Although surgical resection is the most favorable treatment for patients with NSCLC, relapse is still high, so neoadjuvant chemotherapy (NAC) is an accepted treatment modality. In this study we examined whether some of the key molecules associated with the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways could have predictive and prognostic value for the NAC application. To that end we examined the expression status of PTEN, pAKT, pERK and loss of heterozygosity (LOH) of PTEN in two groups of NSCLC patients, those who received and those who did not receive NAC. LOH PTEN and low pERK expression is shown to be correlated with the longest survival of patients with SCC and ADC, respectively, who received NAC. These results point that the application of NAC is beneficial in the NSCLC patients with specific molecular alterations which could further help to improve constant search for the druggable molecular targets used in personalized therapy. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Lung cancer is the most common cause of neoplasia-related death worldwide with a variety of histological types. The non-small cell lung carcinoma (NSCLC), which predominantly comprises of adenocarcinoma (ADC) and squamous cell carcinoma (SCC), is the most common histological subgroup of lung cancer, accounting for approximately 80% of all lung carcinomas. Surgical resection is the most favorable treatment for early-stage NSCLC, although relapse is still high, especially in stages II and III (Win et al., 2008). Management of locally advanced NSCLC represents a challenge, particularly because the role of surgery in this group is controversial in view of the fact that most patients relapse within 3 years from diagnosis, causing 5-year overall survival b10%. Considering the high rate of extrapulmonary relapses, possibly due to the presence of distant micro-metastases at the time of diagnosis, neoadjuvant chemotherapy (NAC) is an accepted treatment modality for patients with NSCLC. Potential disadvantages include the development of medical illness and treatment-related toxicity (Boudaya et al., 2013). Therefore, there is a need for the identification of appropriate molecular markers
⁎ Corresponding author at: Despot Stefan Boulevard 142, 11060 Belgrade, Serbia. E-mail address: [email protected] (J. Bankovic).
http://dx.doi.org/10.1016/j.yexmp.2014.11.010 0014-4800/© 2014 Elsevier Inc. All rights reserved.
which would identify patients who would benefit from neoadjuvant chemotherapy. The PI3K/AKT/mammalian target of rapamycin (mTOR) (PI3K) and the RAS/RAF/MAP kinase–ERK kinase (MEK)/extracellular-signal-regulated kinase (ERK) (MAPK) pathways are frequently deregulated in human cancers, as well as lung cancer, mostly as a result of genetic alterations in their components (De Luca et al., 2012). PI3K/AKT/mTOR signaling pathway has been shown to be involved in the regulation of cell proliferation and apoptosis, and is a key to the initiation and progression of malignancies, enhancing cell survival by the stimulation of cell proliferation and the inhibition of apoptosis (Cantrell, 2001). The main regulator of this pathway, PTEN (phosphatase and tensin homolog deleted on chromosome 10), frequently altered in lung cancer, reduces the downstream activity of AKT, thereby inducing cell-cycle arrest and apoptosis (Hosoya et al., 1999). Another signaling pathway very important in carcinogenesis is RAS/RAF/MEK/ERK and it promotes cell proliferation, angiogenesis, cell differentiation and migration (McKay and Morrison, 2007). In the current study we investigated the expression status of PTEN, pAKT, pERK and loss of heterozygosity (LOH) of PTEN in two groups of NSCLC patients, those who received and those who did not receive NAC. The aim was to evaluate whether these key components of RAS/ RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways could have the predictive and prognostic value for the rational application of NAC.
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2. Materials and methods
were assessed spectrophotometrically. The quality of DNA was verified by electrophoresis.
2.1. Tissue samples 2.3. LOH analysis Paired samples from cancer and adjacent normal lung tissue from 70 patients with NSCLC who underwent surgery, lobectomy or pneumonectomy with regional lymphadenectomy, at Clinic of Thoracic Surgery, Clinical Centre of Serbia, Belgrade, Serbia, were analyzed. Patients were divided into two groups. One group of 35 patients received NAC, while the other did not. All chemotherapy protocols were platinum-based ranging from I to IV cycles. The samples were collected and used after obtaining an informed consent and approval from the Ethics Committee, in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. The tumors were histologically classified according to the latest World Health Organization classification of the lung cancer (Travis et al., 2004) while the pathological stage of the tumors diagnosed before 2010 was reclassified according to the recommendation from 2009 (Travis, 2009; Vallières et al., 2009). Diagnosis of NSCLC, the histological grade and regional lymph node involvement were established by histological examinations of the surgical specimens. The patients' distribution by clinicopathologic parameters is presented in Table 1.
2.2. DNA extraction Specimens obtained from the patients without NAC were frozen in liquid nitrogen, where they were kept until DNA extraction. This DNA was extracted using the phenol/chloroform/isoamyl alcohol method (Sambrook et al., 1989). DNA from paraffin-embedded tumor material from patients with NAC was extracted using a Kappa Express Extract DNA extraction kit (KapaBiosystems, USA) according to the manufacturer's procedure. The concentrations of isolated nucleic acids
Table 1 Clinicopathological parameters of patients with and without NAC. Parameter
Patients with NAC
Patients without NAC
Total Age ≤50 N50 Gender Male Female NSCLC subtype Adenocarcinoma Squamous cell carcinoma Necrosisa n1 n2 n3 Inflammation Level 1 Level 2 Level 3 Histological gradeb g1 g2 g3 Stage I II III Lymph node invasion Positive Negative
35
35
6 29
11 24
26 9
23 12
16 19
15 20
27 5 3
25 6 4
9 20 6
15 17 3
10 15 10
6 24 5
5 15 15
2 12 21
25 9
29 6
NAC — neoadjuvant chemotherapy. a n1, obscure or no necrosis; n2, necrosis in 50% of tumor mass; n3, necrosis in more than 50% of tumor mass. b g1, well differentiated; g2, moderately differentiated; g3, poorly differentiated.
The DNA obtained from the malignant and normal lung tissue from all 70 patients was used to study the LOH of PTEN, tumor suppressor gene. LOH analyses were performed using highly polymorphic microsatellite markers. Five polymorphic microsatellite markers spanning the PTEN gene (D10S579, D10S1765, D10S215, AFM086wg9, and D10S541) were selected to cover deletions at the whole PTEN locus on chromosome 10q23. All forward primers were 5′-labeled with Fam, Vic, Ned, Pet, and Fam fluorescent dyes, respectively. The choice of the microsatellite markers and locus-specific PCR conditions were determined from published sources (Feilotter et al., 1998; Hahn et al., 1999). The PCR products were separated by capillary electrophoresis on an ABI Prism 3130 automated sequencer and sized using GeneScan-500 LIZ size standard (Applied Biosystems). The obtained data were analyzed with the GeneMapper software (Applied Biosystems). The DNA from normal lung tissue adjacent to tumors from the same patient was used as reference. On the one hand, a marker was defined as noninformative (homozygote) when only 1 allelic peak was detected in the DNA sample of the normal lung tissue. On the other hand, a marker was considered informative (heterozygote) when 2 major allelic peaks occurred in a normal specimen. The LOH score for the informative cases was calculated automatically by GeneMapper software according to the following equation: (peak height of normal allele 2)/(peak height of normal allele 1) divided by (peak height of tumor allele 2)/(peak height of tumor allele 1). A sample was considered to be an LOH candidate for particular locus if the ratio values were less than 0.66 and higher than 1.5. 2.4. Immunohistochemistry Tumor samples were fixed in buffered 10% formalin, embedded in paraffin blocks and cut in 3 μm for routine analysis. The following antibodies were used according to manufacturer's instructions: PTEN (1:50, clone: PN37, Invitrogen, USA), pERK (1:100, clone: P44/42MAPL-ERK1/2(137 FS), Cell Signaling, USA) and pAKT (1:40, clone: HCL-l AKT-Phos, Novocastra, Leica Biosystems, USA). Immunostaining was performed by incubating tissue sections with appropriate serum for 30 min at room temperature in humidity chamber, using the streptavidinbiotin technique (LSAB + Kit, Peroxidase Labeling, K0690, DAKO Cytomation, Denmark). Antigen–antibody complexes were visualized with diaminobenzidinehydrochloride (DAB, No. K3468, DAKO Cytomation, Denmark) substrate solution. The cell nuclei were contra-stained with Mayer's hematoxylin. At the same time, tissue samples with appropriate positive immunostaining were used as indicators of the quality of the target retrieval procedure. Positive immunoreactivity of pAKT in epidermis of human skin and PTEN and pERK in regular breast ductal epithelia was used as the internal positive control. Immunohistochemical (IHC) results were independently evaluated by two pathologists (J.S. and Z.M.) on microscope Leica DM2500 (Leica Microsystems, Germany). The immunoreactivity of PTEN was assessed using the semiquantitative method based on the score of percentage of stained cells—cytoplasm/nuclei (P) (0, no immunoreactivity; 1,1–10%; 2, 11–50%; 3, 51–100%) and intensity of staining (SI) (0, no immunoreactivity; 1, reduced staining intensity relative to the corresponding normal cells; 2, same as normal cells staining; 3, increased staining). Since cutoff levels for reduced PTEN expression by immunohistochemical methods have not been defined so far, we used the mean PTEN score as a cutoff point to designate reduced expression (Shoman et al., 2005). Accordingly, PTEN status was defined as follows: low expression if score was ≤ 4; and high expression if score was N4. The pAKT staining was evaluated by an H-score, which was calculated
J. Stojsic et al. / Experimental and Molecular Pathology 98 (2015) 27–32
by multiplying the P by the corresponding SI (1 = weak, 2 = moderate, and 3 = strong), giving a maximum score of 300 (100% × 3). A positive control for pAKT (human skin) was included in each analysis. H-scores N 50 were considered positive (Esteva et al., 2010). The pERK immunoreactivity levels of each case were assessed semi-quantitatively under a light microscope by assessing the average signal intensity (on scale of 0 to 3) and the proportion of cells showing a positive cytoplasmatic stain (0, none; 0.1, less than one tenth; 0.5, less than one half; and 1, greater than one half). The intensity and proportion scores were then multiplied to give the H-score (Al-Haddad et al., 1999). H-score more than 1 was considered as elevated pERK immunoreactivity (Handra-Luca et al., 2003).
2.5. Statistical analysis A statistical analysis was performed using STATISTICA 6.0 software (StatSoft, Inc., Tulsa, USA). The following clinicopathological and genetic parameters were evaluated: histological type, grade, tumor clinicopathologic stage of tumor, lymph node invasion, reactive inflammation, degree of tumor necrosis, PTEN alterations, PTEN, pERK and pAKT expression and patients' outcome. To test correlations between different parameters Fisher exact test and Mann–Whitney U test were used. Survival analyses were performed using the Kaplan and Meier product-limit method; to test the difference in overall survival, the log-rank test was used. The overall survival was calculated from day one after surgery to the last follow-
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up examination or death of the patient. Statistical differences were considered significant when P ≤ 0.05. 3. Results 3.1. LOH analysis of PTEN tumor suppressor gene Loss of heterozygosity of PTEN gene was evaluated by fragment analysis (Fig. 1). In the group of patients without neoadjuvant chemotherapy 19 tumor samples were noninformative for all 5 tested markers. All the rest of the 16 informative cases showed the loss of an allele (45.7%, Table 2). In the group of patients who received the neoadjuvant chemotherapy 32 samples were available for LOH analysis and 16 of them were noninformative for PTEN markers. Among 16 informative cases, which were heterozygous for at least 1 examined locus, 13 showed the loss of an allele (40.6%, Table 2). 3.2. pAKT, pERK and PTEN protein expression According to the established IHC scoring immunohistochemical studies of the patients' samples in the group without NAC were detected: the high level of PTEN expression in 13 (37.1%), of pAKT in 18 (51.4%) and of pERK in 11 (31.4%) out of 35 NSCLC patients (Table 2). The results obtained for the group of patients with NAC were similar with high pAKT in 19 (54.3%) and pERK in 9 (25.7%) out of 35 patients, while PTEN expression was slightly different being high in 7 patients
Fig. 1. Loss of heterozygosity (LOH) analysis of PTEN gene. A) LOH of PTEN detected using D10S579 microsatellite marker in the sample from patient 9 receiving NAC, B) LOH PTEN detected using D10S1765 microsatellite marker in the sample from patient 33 not receiving NAC, C) LOH of PTEN detected using D10S215 microsatellite marker in the sample from patient 21 receiving NAC. Arrows indicate allelic loss in tumor samples.
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Table 2 Gene alterations and immunophenotype in NSCLC tissue samples. Patients
LOH PTEN
High PTEN expression
High pAKT expression
High pERK expression
With NACa Without NAC
13/32 (40.6%) 16/35 (45.7%)
7/35 (20%) 13/35 (37.1%)
19/35 (54.3%) 18/35 (51.4%)
9/35 (25.7%) 11/35 (25.7%)
a
Neoadjuvant chemotherapy.
(20%) out of 35 patients (Table 2). Representative examples of staining intensities for PTEN, pAKT and pERK are shown in Fig. 2. 3.3. LOH PTEN, pAKT, pERK and PTEN expression in relation to clinicopathological parameters The correlation between LOH PTEN, pAKT, pERK and PTEN expression and clinicopathological parameters was assessed regarding two groups of patients, those with and those without NAC. Samples were grouped according to the following clinicopathological parameters: histological type, histological grade, tumor stage, lymph node invasion, degree of tumor necrosis, and reactive inflammation. The number of patients without lymph node invasion who received NAC and had low pAKT expression was significantly higher than the number of those who had high expression of this protein (Table 3, P = 0.01). 3.4. LOH PTEN, pAKT, pERK and PTEN expression and survival rate The overall survival of patients with and without NAC was evaluated in relation to LOH PTEN, pAKT, pERK and PTEN expression in their NSCLC samples (Fig. 3). pERK expression was significant for the survival of the patients with NAC. Namely, patients in this group who had low pERK expression lived significantly longer (Fig. 3A, P = 0.05). Further, all patients were stratified according to the tumor subtype and in those who had SCC and carried LOH PTEN, NAC had favorable effect on survival, i.e. those who received it lived significantly longer than those who did not (Fig. 3B, P = 0.006). Patients who had ADC, low pERK expression and received NAC had the longest survival, followed by patients with low
pERK expression without NAC, patients with high pERK and NAC and finally patient with high pERK and no therapy (Fig. 3C, P = 0.01). 4. Discussion The aim of this study was to evaluate whether key molecules of RAS/ RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways could have the predictive and prognostic value for the rational application of NAC in the treatment of NSCLC. The expression status of PTEN, pAKT, pERK and LOH PTEN in two groups of NSCLC patients, those who received and those who did not receive NAC was evaluated. The expression levels of pAKT, pERK as well as LOH PTEN were similar in both groups of patients, in those who received and in those who did not receive NAC. Only PTEN expression was slightly higher in the group without NAC (37.1% vs. 20%) but with no statistical significance. Similarly, studies that included breast or rectal cancer samples demonstrated the same modality, i.e. low or lost PTEN expression did not correlate with chemotherapy (Yonemori et al., 2009; Barbareschi et al., 2012; Erben et al., 2011). Further investigation included assessment of the correlations of LOH PTEN, pAKT, pERK and PTEN expression and clinicopathological parameters among patients with and without NAC. The obtained results showed that in the group of patients who received NAC and were without lymph node invasion the number of those with low pAKT expression was significantly higher than the number of those with high pAKT expression. Hence, it could be said that chemotherapy lowers the expression of this protein suggesting its beneficial usage in the tumors with activation of PI3K/AKT/mTOR pathway and clear lymph nodes which is usually expected in the early-stage tumors. This is supported by the fact that PI3K and pAKT expression has already
Fig. 2. Immunohistochemical analysis of pAKT, PTEN and pERK expression in non-small cell lung carcinoma. Representative examples of: A) positive antibody controls: pAKT skin (10× objective); PTEN breast tissue (10× objective); pERK breast tissue (10× objective); B) weak nuclear pAKT immunoreactivity (20× objective), weak cytoplasmatic PTEN immunoreactivity (20× objective) and weak cytoplasmatic pERK immunoreactivity (20× objective); C) moderate nuclear pAKT immunoreactivity (20× objective), moderate cytoplasmatic PTEN immunoreactivity (20× objective) and moderate cytoplasmatic pERK immunoreactivity (20× objective); D) strong nuclear pAKT immunoreactivity (20× objective), strong cytoplasmatic PTEN immunoreactivity (20× objective) and strong cytoplasmatic pERK immunoreactivity (20× objective).
J. Stojsic et al. / Experimental and Molecular Pathology 98 (2015) 27–32 Table 3 pAKT expression in patients without lymph node invasion. Variables
NACb Without With a b c
pAKT expression Low
High
NPa (%)
NP (%)
1 (16.7) 8 (88.9)
5 (83.3) 1 (11.1)
P value
0.01c
NP, number of patients per group. Neoadjuvant chemotherapy. Bold indicates statistically significant values, P ≤ 0.05.
been detected in the early stage NSCLC (Al-Saad et al., 2009; David et al., 2004). In addition, Yip P. et al. reported that patients with early-stage NSCLC who expressed higher levels of nuclear pAKT had a poorer prognosis than those with lower levels of expression (Yip et al., 2014). Also, it was shown that down-regulation of pAKT could suppress invasion and migration of osteosarcoma cells (Long et al., 2013). When analyzing patients' survival in the relation to the expression status of PTEN, pAKT, pERK and LOH PTEN, patients in the group with NAC who had low pERK expression lived significantly longer than those with high pERK expression. The investigation on lung ADC cell line A549 also showed that their migration and invasion could be suppressed via PKC-α-ERK1/2-NF-κB pathway (Cheng et al., 2014). Similar results were obtained on osteosarcoma cells, whose growth and invasion were inhibited possibly through inhibition of the MAPK/ERK pathway (Miao et al., 2014). In addition, neoadjuvant therapies administered to skin carcinoma and colorectal adenocarcinoma induced a synergistic cytotoxicity after a prolonged ERK inhibition (Weyergang et al., 2013). This all suggests that low pERK could be an important factor for the prolonged survival of our patients with NSCLC who received NAC regardless of tumor histological type. However, the most interesting results were obtained when the NSCLC samples were grouped according to the histological type. Namely, patients with SCC, LOH PTEN and NAC lived significantly longer than those who did not have this type of treatment. Among the patients with ADC, those who had low pERK expression and received NAC had the longest survival, followed by patients with low pERK expression without chemotherapy, patients with high pERK and chemotherapy and finally patient with high pERK and no therapy. Previous studies reported that the activation of the PI3K– AKT–mTOR signaling pathway inhibits ERK1/2 activation (Milosevic et al., 2014; Rommel et al., 1999) and in addition we would also expect high pAKT in the presence of LOH PTEN. Since we showed that NAC has a positive effect in the patients with activation of the PI3K–AKT–mTOR signaling pathway, high pAKT could be, in fact, a positive marker for
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the application of NAC in NSCLC. Also, all NAC protocols for our patients were platinum-based and it has been shown that its antitumor activity was promoted through AKT and ERK1/2 pathways (Zhang et al., 2014a,b). Our results are very important from the standpoint that it could imply to the possibility of more detailed stratification of the patients before the application of the therapy towards personalized treatment. The discovery of the so-called ‘driver mutations’ such as the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) has led to the improvement in personalized therapy for lung ADC (Giaccone, 2005; Kwak et al., 2010). However, no such available molecular targets exist for SCC. For this reason, the identification of novel druggable molecular targets in SCC would be a research priority. In conclusion, since there is a high rate of extrapulmonary relapses, possibly due to the presence of distant micro-metastases at the time of diagnosis of NSCLC, NAC is an accepted treatment modality for these patients. In this study we examined whether some of the key molecules associated with the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways could have predictive and prognostic value for the NAC application. LOH PTEN and low pERK expression is shown to be correlated with the longest survival of patients with SCC and ADC, respectively, who received NAC. In accordance with other studies these results imply that the application of NAC is beneficial in the treatment of NSCLC with the additional therapeutic targets for ADC and the new ones for SCC which would lead to the personalized treatment of these patients. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This study was supported by Grant III41031 from the Ministry of Education, Science and Technological Development of the Republic of Serbia. References Al-Haddad, S., et al., 1999. Psoriasin (S100A7) expression and invasive breast cancer. Am. J. Pathol. 155, 2057–2066. Al-Saad, S., et al., 2009. Diverse prognostic roles of AKT isoforms, PTEN and PI3K in tumor epithelial cells and stromal compartment in non-small cell lung cancer. Anticancer Res. 29, 4175–4183. Barbareschi, M., et al., 2012. PI3KCA mutations and/or PTEN loss in Her2-positive breast carcinomas treated with trastuzumab are not related to resistance to anti-Her2 therapy. Virchows Arch. 461, 129–139. Boudaya, M.S., et al., 2013. What outcome after the prescription of neoadjuvant chemotherapy in lung cancer? Asian Cardiovasc. Thorac. Ann. 21, 432–436. Cantrell, D.A., 2001. Phosphoinositide 3-kinase signaling pathways. J. Cell Sci. 114, 1439–1445.
Fig. 3. Kaplan–Meier survival curves. Patients who received NAC and had low pERK expression lived significantly longer (panel A, P = 0.05). Patients with SCC who carried LOH PTEN and received NAC lived significantly longer than those without this treatment (panel B, P = 0.006). Patients who had ADC, low pERK expression and received NAC had the longest survival, followed by patients with low pERK expression without chemotherapy, patients with high pERK and chemotherapy and finally patient with high pERK and no therapy (panel C, P = 0.01). The survival among analyzed groups of patients was considered significantly different if P ≤ 0.05.
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