DOI: 10.31038/CST.2017215

Abstract

Laryngeal squamous carcinoma (LSC) represents one of the refractory malignant tumors of head and neck squamous cell carcinoma (HNSCC), leading to high patient mortality and unsatisfactory overall 5-year survival. New therapeutic targets and modalities for effective chemoprevention and treatment are required to improve the survival of patients with LSC. Evidence from previous studies has suggested that proteasome inhibitors (PIs) possess effective antitumor activity and have been approved by the United States Food and Drug Administration (FDA) to treat myeloma. However, resistance of myeloma to PIs has been reported, which prevents PIs from their clinical uses in cancer treatment. So far, the underlying mechanisms of tumor cells anti-PIs effects remain unclear. In the present study, we found that constitutive activation of signal transducer and activator of transcription 3 (STAT3) was associated with clinicopathlologic factors in LSC patients, and persistent activation of STAT3 was found in Hep-2 laryngeal carcinoma cells. Moreover, we demonstrated that proteasome inhibitor MG-132 induces apoptosis and cell cycle arrest in Hep-2 cells, accompanied by increased activation of STAT3; knockdown of STAT3 expression by shRNA or application of STAT3 inhibitor AG490 potentiates the antitumor effects of MG-132 in vitro, which is attributed to the down-regulation of cyclin D1 and Bcl-2. Taken together, we identified that STAT3 activation plays a great role in therapeutic resistance to PIs in LSC; blocking STAT3 activation could potentiate the tumor killing effects of PIs in LSC or other malignancies involving STAT3 activation. Combined application of PIs and STAT3 inhibitor may implicate a promising strategy for managing LSC or even other HNSCC.

Keywords

Signal transducer and activator of transcription 3, STAT3; proteasome inhibitor; STAT3 inhibitor; laryngeal carcinoma; apoptosis

Introduction

Laryngeal squamous carcinoma (LSC) is a kind of head and neck squamous cell carcinoma (HNSCC), the latter of which ranks the sixth most common malignant cancer, and accounts for 35,00000 newly diagnosed cases annually worldwide [1]. Despite of successive advancement in diagnosis and treatment, the overall survival of HNSCC has just been slightly improved in recent years. In addition to distant metastasis and relapse, resistance to traditional therapeutic modalities has been the dominant cause of treatment failure, especially in advanced cases. For combating the therapeutic resistance of cancers, some novel concepts and strategies for targeted therapies have been introduced, most of which mainly focused on some key modulators of cell proliferation and apoptosis pathways. Although some preliminary results have been achieved, they are not as effective as expected.

Signal transducer and activator of transcription 3 (STAT3), a member of STAT family, is a focus of many oncogenic receptor tyrosine kinase pathways, such as EGFR, IL-6/JAK and Src [2]. It has been demonstrated that STAT3 can induce up-regulation of prosurvival proteins including Bcl-2, Bcl-xL, survivin and Mcl-1, and promote expression of angiogenesis factors, such as VEGF [3]. In fact, STAT3 is constitutively activated in many tumors [4-7] and plays a great role in proliferation, cell cycle progression and apoptosis of tumor cells [8]. In addition, activation of STAT3 is also associated with chemoresistance in many malignancies [9, 10]. Results from our previous study demonstrate that knockdown of STAT3 with specific shRNA can enhance radiosensitivity in LSC Hep-2 cells both in vitro and in vivo [11, 12]. Therefore, targeting STAT3 is a potential therapeutic strategy for LSC.

As is known, due to rapid proliferation and insufficient blood supplies, the microenvironments of most solid cancers are accompanied by hypoxia, glucose starvation and low PH, leading to endoplasmic reticulum (ER) stress due to causal accumulations of incorrect proteins [13]. Sequently, cancer cells must adapt to these unfavorable conditions for their survival via inhibiting synthesis of proteins and degrading unfolded proteins accumulated in ER by proteasome [14]. Therefore, targeting proteasome becomes a promising strategy for cancer therapy.

Early studies demonstrated that tumor-inhibiting effects of proteasome inhibitors (PIs) are attributed to repression of nuclear factor-κB (NF-κB) signaling and regulation of proteins associated with both cell cycle and apoptosis, such as p27^Kip1 [15], p21^cip/WAF1 [16], p53 [17], Bcl-2 [18, 19], Bax [17], Bim and Bik [20]. It is now believed that PIs induce ER stress-related cell death through unfolded protein response (UPR) [21] and confer preferential cytotoxicity towards hypoxic tumor cells [22]. However, cases of resistance to PIs are emerging, whereas the related mechanisms are not systematically elucidated [23]. Interestingly, a recent study has demonstrated that a proteasome inhibitor Bortezomib promotes STAT3 activation in HNSCC, suggestive of potential roles of STAT3 in the resistance of HNSCC to the proteasome inhibitor [24]. Therefore, it would be of great interest to elucidate whether the STAT3 plays a role in resistance LSC to PIs.

In the present study, we first assessed the associations between constitutive activation of STAT3 and clinicopathlological factors in LSC. We next investigated the role of STAT3 activation by protesome inhibitor and its impacts on proteasome-induced cell apoptosis in LSC.

Materials and Methods

Patients’ data

50 LSC patients (36 males and 14 females) who received surgical resection at Bethune International Peace Hospital were recruited, and their complete detailed clinicopathologic data were collected. Of the 50 cases, there were 17 cases of supraglottic and 34 cases of glottic cancers. The age of the present series ranged from 36 to 86 years, with a mean age of 58 years. All patients had undergone neither chemotherapy nor radiotherapy prior to surgery. According to UICC cancer staging system (2002), there were 7 cases of stage I, 6 cases of stage II, 17 cases of stage III and 20 cases of stage IV tumors. With regard to the histological grading, 6 cases were well differentiated, 13 cases were moderately differentiated and 31 cases were poorly differentiated squamous cell carcinomas (SCC). Twenty-two cases had neck node metastasis as proven by postoperative pathologic examinations.

Tumor tissue collection

With the informed consent of all patients, tissue specimens were collected immediately after surgical removal. Paired tumor-free paracancerous tissues (PCT) were taken at least 0.5 cm beyond the tumor margins. Each sample was cut in halves. One half was promptly frozen at -70 ℃ for subsequent Western blot, while the other was fixed immediately with 10% neutrally buffered formalin for histopathologic evaluation and immunohistochemistry. The acquisition of paraffin tissues and frozen specimens were approved by the Institutional Review Boards at Bethune International Peace Hospital. Neck dissection specimens were evaluated by two pathologists to confirm the status of neck node metastasis in these patients.

Immunohistochemistry and immunocytochemistry

Immunohistochemical and immunohistocytochemical studies were performed as described previously [25] using SP-9002 kit (including 0.3% hydrogen peroxide, 10% goat serum, biotinated secondary antibodies and horseradish peroxidase marked strepto- antibiotin, Zhong Shan Jin Qiao, Beijing) with some modifications. Briefly, for immunohistochemistry, consecutive sections were deparaffinized and hydrated, and subjected to 0.3% hydrogen peroxide for elimination of endogenous peroxidase at room temperature for 10 min. Slides were then heated for the retrieval of antigens in an autoclave sterilizer for 2 min. Goat serum (10%) was added to block nonspecific protein staining for 10 min. The sections were incubated with anti-STAT3 and anti-p-STAT3 (Santa Cruz Biotechnology Inc.) at 4 ℃overnight. Afterwards, they were rinsed by PBS and incubated at room temperature with biotinated secondary antibodies for 15 min under a humid condition followed by 15-min incubation with horseradish peroxidase-marked strepto- antibiotin at room temperature. Finally, they were detected with DAB kit (Zhong Shan Jin Qiao, Beijing). The immunoreactivity of STAT3 or p-STAT3 was evaluated as described previously [31]. Brown coloration was identified as positive staining. The distribution of positive staining cells was classified in 4 categories: weakly positive (+), positive (++), strongly positive (+++), and negative. For immunocytochemical study, Hep-2 cells were seeded in 6-well plates with a small cover slide in each well. When confluence reached 70% to 80%, cells grown on the cover slide were fixed with cold acetone at room temperature for 15 min. Cover slides were then subjected to 0.3% hydrogen peroxide for elimination of endogenous peroxidase at room temperature for 30 min, washed with PBS and permeabilized with 0.1% TritonX-100 on ice for 2min. The immunostaining of Hep-2 cells was performed as in immunohistocehmistry.

Cell line and culture

Hep-2 human LSC cell line (National Cancer Institute, USA) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) under the condition of 37℃, 20% O₂, 5%CO₂, 95% humidity in a CO₂ incubator. Cells were allowed for attachment overnight and those grown in logarithmic phase were used for subsequent experiments. All experimental groups came from the same parental cells. Cells were passaged every 2 to 3 days to maintain exponential growth.

Transfection of plasmids

The construction of STAT3-targeting shRNA expression vector and the transfection of plasmids were performed as our previous publications [12]. Briefly, the double strands of shRNA targeting gene of STAT3 (sense: 5′-CACCGCAGCAGCTGAACAACATGTTCAAGAGACATGTTGTTCAGCTGCTGCTTTTTTG-3′, the corresponding mRNA coding region is underlined) and the double strands of negative control gene (sense: 5′-CACCGTTCTCCGAACGTGTCACGTCAAGAGATTACGTGACACGTTCGGAGAATTTTTTG-3′, underline shows the target sequence) were inserted into pGPU6/GFP/Neo vector (Gene Pharma Co., Ltd), and named pGPU6/GFP/Neo-shSTAT3 and pGPU6/GFP/Neo-shNC, respectively. The procedure of transient transfection was performed according to Lipofectamine^TM2000 (Invitrogen, Corporation) manufacturer’s instructions in 96-well plates or/and 6-well plates. The thansfection efficiency was monitored by observation under a fluorescent microscope and by flow cytometry. Sequent assays were performed 24 hours after transfection.

MTT assay

The inhibition of hep-2 cells by chemicals was determined by MTT assay as described previously [26]. Transfected and untransfected hep-2 cells were subjected to MTT assay. After treatments with PIs (MG-132) (MERCK Corporation, Germany) and STAT3 inhibitor (AG940) (Sigma, USA) in different combinations and cisplatin (DDP, Shan Dong Qi Lu Pharmaceutics, China), 20μl 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, 5mg/ml, Sigma, USA) were added into each well of 96-well plates. After incubation at 37 ℃ for 4h, culture medium was replaced by DMSO to dissolve formazan for coloration. Absorbance was measured at 490 nm using Enzyme-linked Immunosorbent Detector (Model 550, Bio-Rad, USA).

Flow cytometry

The transfection efficiency, apoptosis rate and cell cycle distributions were determined by a FACScan analyzer (Becton Dickinson FAC Sort, USA). Hep-2 cells, with or without treatments, were collected after trypsinization. Fresh cells were used for detection of the efficacy of transfection by flow cytometry. To determine apoptosis rate and cell cycle distribution, cells were washed by PBS for 3 times, and then were fixed with 75% ethanol overnight at 4 ℃. Prior to analysis, ethanol was discarded and 80μl RNase (final concentration: 50mg/L) and 150μl propidium iodide (final concentration: 50mg/L, Sigmar, USA) were used for staining of DNA for 30 min at 4℃. Apoptotic cells appeared as a magnitude of the sub-G1 peak due to nuclear fragmentation and loss of DNA.

Western blot

Western blot was carried out as described previously [25] with minor modifications. For tissue specimens, lysates were obtained from 100mg sample of each frozen tissue by a homogenizer. Subjected Hep-2 cells were harvested following trypsinization and centrifugalization. The cell pellets were resuspended in cell lysis buffer containing 500mM HEPES 25ml (0.1mol/L)，100mM NaCl 5ml (1mol/L)，2mM EDTA 0.2 ml (0.5mol/ L)，sodium deoxycholate 0.25g (0.5%)，NP-400 5ml (1%)，10%SDS 0.5ml (0.1%)，2×10^-3 mol/L PMSF and 2×10^-6 g/L Lupeptin to collect lysates. Subsequently, lysates originating from tissues or hep-2 cells were acquired in the same way. After determination of protein concentrations in the lysates, equivalent protein (100mg from tissues or 30mg from cells) were separated by electrophoresis on 4% to 10% SDS-PAGE gels. The subjected gels were then transferred onto nitrocellulose filters, and blocked in confining liquid containing 1% nonfat dry milk, 0.02% Tween-20 (Sigmar, USA), 10mmol/LTris-Cl，0.15mmol/L NaCl at 4℃ overnight. The blocked filters were incubated with primary antibodies (Santa Cruz Biotechnology int and Cell Signaling Technology, USA) at 4℃ overnight followed by rinse in TBST. The filters were incubated with secondary antibodies marked by HRE (Zhong Shan Jin Qiao, BeiJing) at room temperature for 1 hour followed by rinse in TBST two times. Finally, the blots were visualized by ECL kit (Pierce, USA).

Statistical analysis

Results were analyzed for statistical significance with the use of SPSS 13.0. Data were presented as mean values ± SD. The method of statistical analysis included student’s t-test for comparision of two groups, one-way ANOVA for comparison of more than two groups combined with or without least significant difference (LSD) followed for comparison of each two groups, and Pearson correlation analysis for correlation evaluation of different factors. P < 0.05 was considered significant.

Results

Constitutive activation of STAT3 is associated with clinicopathlological factors in LSC, including clinical stage, pathological grading and cervical lymph node metastasis. It has been reported that STAT3 is aberrantly activated in many tumors in comparison with its transient activation in normal tissues [27-30]. To determine activation status and expression distribution pattern of p-STAT3 in LSC, we compared levels of p-STAT3 protein of primary LSC tissues with corresponding tumor-free PCT by immunohistochemistry. As can be seen in Fig 1A-D, p-STAT3 was constitutively expressed in LSC compared with PCT. In positive samples, weakly positive staining dominates PCTs, whereas strong positive coloration occupies most of cancer tissues; it was also noted that the staining of p-STAT3 in poorly differentiated LSC is stronger than that in well differentiated LSC.

Figure 1. The expression of phosphorylated STAT3 (p-STAT3) in human laryngeal squamous cell carcinoma (SP×400). Tissue specimens were collected and p-STAT3 protein expression levels were assessed by immunohistochemistry and western blot. A. Expression of STAT3 in well-differentiated laryngeal squamous carcinoma. B. The expression of p-STAT3 protein in moderately-defferentiated laryngeal squamous cell carcinoma (SP×400). C. The expression of p-STAT3 protein in poorly differentiated laryngeal squamous cell carcinoma (SP×400). D. The expression of p-STAT3 protein in tumor-free paracancerous laryngeal epithelial tissues (SP×200). Yellow arrow indicates representative staining cells. E. Expression of Stat3, p-STAT3, and cyclin Dl in human laryngeal carcinoma by western blot. Elevated levels of p-STAT3 and cyclin Dl in tumor tissues (T) compared to adjacent normal laryngeal mucosa (N) were further confirmed.

These findings suggest that STAT3 is activated constitutively and persistently in LSC rather than in PCT, and its activated form, p-STAT3, is likely to function in nucleus as in other malignancies. Moreover, expression of p-STAT3 is closely related to the expression of cyclin D1. Results from western blot analysis showed the same expression pattern for each protein (Fig 1E) in that there is a correlation between p-STAT3 and the expression of cyclin D1, suggesting that cyclin D1 is regulated by p-STAT3 in LSC tissues.

It has been reported in many studies that activation of STAT3 is related to poor prognosis of various cancers [31-33]. To test whether STAT3 activation is pertinent to clinicopathologic factors of LSC, we analyzed the relationship between p-STAT3 and critical prognostic factors. Results in Table 1 indicates that there is a significant correlation between p-STAT3 expression and clinical stage, pathologic grade and neck node metastasis rather than age, sex, T stage and even the site of primary tumors.

Table 1. The relationship between the expression of Stat3 and phosphorylated Stat3 protein and clinicophalogical features in laryngeal squamous cell carcinoma

n			Stat3		p-Stat3
			M number (rate%)	P value	M number (rate%)	P value
Age	≥60	28	23(82.14)	0.0715	17(60.71)	0.6609
	＜60	22	13(59.09)		12(54.54)
Sex	M	36	27(75.00)	0.6841	23(63.89)	0.1761
	F	14	9 (64.29)		6 (42.86)
Tumor position	Supra-glottis	17	10(58.82)	0.2473	10(58.82)	0.9325
	glottis	33	26(78.78)		19(57.58)
Patho- grade	Well to moderately differentiated	19	10(52.63)	0.0169	5 (26.32)	0.0004
	Poorly differentiated	31	26(83.87)		24(77.42)
T stage	T1-T2	17	12(70.58)	0.0685	7 (41.18)	0.0836
	T3-T4	33	27(81.81)		22(66.67)
Clinical stage	Ⅰ-Ⅱ	13	5 (38.46)	0.0056	4 (30.77)	0.0208
	Ⅲ-Ⅳ	37	31(83.78)		25(67.57)
Lymph node metastasis	Yes	22	20(90.90)	0.0083	17(77.27)	0.0144
	No	28	16(57.14)		12(42.86)

Activation of STAT3 is in constant existence in Hep-2 laryngeal carcinoma cells (Hep-2 cells). To test whether STATA3 is also constitutively activated in Hep-2 cells, we examined the expression of STAT3 and its related proteins by immunocytochemistry and western blot. By immunocytochemistry, STAT3 was distributed in cytoplasm of cancer cells, while p-STAT3 was observed mostly within nucleus of Hep-2 cells, which resembles the expression pattern of STAT3 and p-STAT3 in LSC tissues (Fig 2A, B, C). To examine if these proteins were constantly and pervasively expressed, we detected expression of the two proteins using western blot at different time points after the setting of cell culture. The sequent 24 h, 48 h, and 72 h were chosen as time spots of analysis. The results of western blot showed that STAT3 and p-STAT3 (Fig 2D) can be detected at different time points, suggesting that constitutive activation of STAT3 is also present in Hep-2 cells as in primary LSC.

Figure 2. TExpression of STAT3 and p-STAT3 in human laryngeal carcinoma Hep-2 cells. Immunocytochemistry was used to demonstrate the intracellular distribution of STAT3 and p-STAT. PBS was used as a negative control. Besides, western blot was used to show the expression patterns of STAT3 and p-STAT3 overtimes (0h, 24h, 48h, 72h). β-actin represents the internal protein control. Constitutive expression and activation of STAT3 in human laryngeal carcinoma Hep-2 cells was noted. A. Cytoplasmic staining of STAT3 in Hep-2 cells (SP × 400). B. Nuclear staining of p-STAT3 in Hep-2 cells (SP × 400). C. Negative control (SP × 400). D. Expression of Stat3 and p-STAT3 in Hep-2 cells as demonstrated by western blot.

Proteasome inhibitor MG-132 induces apoptosis in Hep-2 cells, which is accompanied by additional activation of STAT3 and changes in the expression of several proteins related to cell cycle and apoptosis regulation. PIs have been approved by the United States Food and Drug Administration (FDA) for the treatments of multiple myeloma. In addition, PIs have been investigated for the treatments of many cancers. Herein, we aimed to test the therapeutic effects of PIs on Hep-2 cells. First, we tested the inhibition of proliferation in Hep-2 cells by a PI, MG-132, using MTT assay. Medium containing different concentrations of MG-132 was add into each well of treatment groups and maintained for different times. As seen in Fig 3A, the inhibition of Hep-2 cells by MG-132 displayed a concentration-dependent manner (24h：r=0.925，p<0.01；48h：r=0.944，p<0.01). Second, we attempted to confirm if MG-132 inhibits proliferation of Hep-2 cells in a time-dependent manner. For doing this, we selected the 2.5μM (IC50) of MG-132 to stimulate Hep-2 cells for 48 h. The results of Fig 3B show that the effects of MG-132 in proliferation inhibition is enhanced with prolongation of treatment time (r=0.945，p<0.01). Third, using flow cytometry, we determined apoptosis rate and cycle distribution of Hep-2 cells after treatment with MG-132. Similar with the inhibition of proliferation, MG-132 induces apoptosis in time- (r=0.888，p<0.01) and dose-dependent (r=0.842，p<0.01) manners (Fig 3C, 3D ).

Figure 3. Effects of a proteasome inhibitor, MG-132, on Hep-2 cells. Hep-2 cells were treated with different concentrations of MG-132 for 24 hours. A concentration of 2.5μM was chosen for stimulation of Hep-2 cells for different times. Alterations in cell apoptosis, cell cycle and protein expression of STAT3 and p-STAT3 were assessed simultaneously. A. The dosage and effect curve of MG-132 (n = 3). B. The time and effect curve of MG-132 (n = 3). C. Apoptosis rate of Hep-2 cells treated with MG-132 for different time (n = 3). D. Apoptosis rate of Hep-2 cells treated with MG-132 at different concentrations (*P < 0.05, versus untreated groups (0μM) (n = 3). E. The correlated expression of cell cycle regulation proteins in Hep-2 cells treated with MG-132 for different time. F. Variation of cell cycle of Hep-2 cells treated with MG-132 for different time (n = 3). G. MG-132-induced STAT3 activation and Bcl-2 expression in Hep-2 cells.

To confirm the mechanisms underlying the cell cycle changes induced by MG-132, we used western blot to check the expression levels of some cell cycle regulators. As seen in Fig 3E, the expression of P21 is elevated significantly compared with the decreased levels of the expression of cyclinD1, suggesting that the changes of cell cycle induced by MG-132 were associated with upregulation of P21 and downregulation of cyclinD1. Simultaneously, cell cycle arrest was noted at G0/G1 phase and/or G2/M phase overtimes after treatment with MG-132 (Fig 3F), the former of which is more prominent than the latter.

From the results of western blot in Fig 3G, we can confirm that STAT3 and its activated form, p-STAT3, are expressed at all times in cultured Hep-2 cells, even in the absence of MG-132 treatment, and the level of its expression was relatively constant and stable. In the presence of MG-132, the expression of p-STAT3 was elevated within short period of time and remained at a stable level; however, the increased expression of p-STAT3 was not related to the duration of MG-132 treatment. Bcl-2 was identified as a downstream of STAT3, and facilitates anti-apoptosis activity in cancer cells [34]. Expression of Bcl-2 protein increased overtime after MG-132 treatment, which may contributed to the MG-132-induced STAT3 activation and resistance of Hep-2 cells to MG-132-induced apoptosis.

Knockdown of STAT3 by shRNA, abrogates the expression and thus activation of STAT3, and potentiates the apoptosis-inducing effects of MG-132. To further confirm the role of the STAT3 activation in inducing resistance of Hep-2 cells to MG-132, we used RNAi to knockout the expression of STAT3, and thus the “constitutive” (inherent) and “PI-induced” (acquired) activation of STAT3. The transfection efficiency was 85.76% by flow cytometry (data not shown). The results of Fig 4A showed that combined group has a similar expression level of p-STAT3 with pshSTAT3 group, with a higher inhibition rate than any other groups (all p<0.01). Results from apoptosis detection Fig 4B showed a more striking apoptosis rate 55.80±3.12% in combined group than in any other group (all p<0.01). In pshSTAT3 group, moderate apoptosis and proliferation inhibition rates were induced. Therefore, blockade of STAT3 by shRNA, abrogates the expression and thus both “inherent” and “acquired” activation of STAT3, and potentiates the MG-132-induced apoptosis in Hep-2 cells.

Figure 4. Effects of MG-132 combined with STAT3 RNAi on proliferation inhibition and apoptosis in Hep-2 cells, as assessed by MTT assay and flow cytometry. Hep-2 cells were seeded into 96-well plates for MTT assay, or 6-well plates for flow cytometry. Subjected Hep-2 cells were categorized as five groups: normal control group (untreated Hep-2 cells), negative control group (Hep-2 cells transfected with negative plasmid, pshNeg group), MG-132 group (Hep-2 cells treated with 2.5μM MG-132 only), combined group (Hep-2 cells transfected with positive plasmid combined with 2.5μM MG-132 treatment), pshSTAT3 group (Hep-2 cells transfected with positive plasmid without any other treatment). A. The proliferation inhibition rate of Hep-2 cells in different groups (n=3) (versus pshNeg group, all *P<0.01; pshSTAT3 group versus MG-132 group, #P<0.01). B. The apoptosis rate of Hep-2 cells in different groups (n=3) (control group versus pshNeg group, *P<0.01; pshSTAT3 group versus MG-132 group, #P<0.01). C. Expression of p-STAT3, cyclin D1 and Bcl-2 in Hep-2 cells in different groups was analyzed by Western blotting.. Lane 1: control group; lane 2: pshNeg group; lane 3: MG-132 group; lane 4: combined group; lane 5: pshSTAT3 group.

With regard to the activation of STAT3 at different situations, as seen in Fig 4C, p-STAT3 was expressed at the highest level in MG-132 group. By contrast, p-STAT3, cyclin D1, and Bcl-2 were expressed at lower levels in combined group, compared with each of the normal control group, negative control group and MG-132 group. The similar expression and activation patterns of STAT3 noticed in combined group and pshSTAT3 group indicated that p-STAT3 may participate in the formation of resistance of Hep-2 cells to MG-132 at a transcriptional level. Taken together, activation of STAT3 is responsible for the resistance of Hep-2 cells to MG-132 at least in part by regulating the anti-apoptosis factor Bcl-2 expression.

Combined treatment with STAT3 inhibitor AG490 and proteasome inhibitor MG-132 induces apoptosis more effectively in Hep-2 cells. In view of the fact that activation of STAT3 played a role in the therapeutic resistance to MG-132 in Hep-2 cells, which can be sensitized by knockdown of STAT3, we studied the effects of combined using STAT3 inhibitor AG490 and proteasome inhibitor MG-132 on proliferation and apoptosis. As seen in Fig 5 A, B, combination treatment by AG490 and MG-132 can induce significant increase in cell proliferation inhibition and apoptosis, indicative of effective synergism.

Figure 5. Effects of AG-490 combined with MG-132 on Hep-2 cells. Hep-2 cells were seeded into 96-well or 6-well plates followed by treatment with 1μM of MG-132 for 48h with or without presence of different concentrations (10, 20, 40, 80μM) of AG490. A concentration of 10μmol/L was for analysis of AG490-induced apoptosis. A. The proliferation inhibition rate of Hep-2 cells treated with the different concentrations AG490 alone and in combination with MG-132 for 48h (n=3). B. The apoptosis rate of Hep-2 cells treated with AG490 alone and in combination with MG-132(1μM) for 48h (n=3) (compared with control group,*P<0.01; compared with AG490 group and MG-132 group, all #P<0.01).

Discussion

Like other malignancies in HNSCC, LSC often exhibits resistance to various therapeutic strategies including chemoradiation and targeted therapies, especially in advanced cases. This difficult situation is often attributed to aberrant expression and activation of some specific genes, such as STAT3, which are utilized by cancer cells to maintain their biological behaviors. In this regard, cancer cells may become vulnerable to the assigned treatment modalities secondary to abrogating the functions of major responsible oncogenes, the so-called “Achilles heel”, which constitutes the fundamental rationale for targeted therapy [35].

Previous studies have demonstrated that activation of STAT3 is associated with chemo- and/or radioresistance in Hep-2 cells, and knockdown of STAT3 by siRNA or blockade of STAT3 activation by its inhibitors sensitizes chemo- and/or radiotherapy both in vitro or in vivo [11, 12, 36, 37]. Importantly, we also demonstrated in the present investigation that constitutive activation of STAT3 was associated with clinicopathlologic factors in LSC, which include clinical stage, pathological grading and cervical lymph node metastasis. This is consistent with the findings in the study of Rosen et al, in which they demonstrated that p-STAT3 was associated with poor prognosis of ovarian cancer [38]. Moreover, results of Kusaba’s study [33] also demonstrated that p-STAT3 is correlated with invasion of vein, nodal metastasis and Dukes grades instead of pathological grading in ovarian cancer. In the present study, we found expression of cyclin D1, one of the important factors downstream of STAT3, was also elevated. Taken together, correlations between activation of STAT3 and clinicopathlological factors of different cancers may depend on cell types from which they originate.

In addition to intrinsic elements, cancer cells in solid tumors also depends on extrinsic factors that form surrounding microenvironment (also called “niche”) for their proliferation and survival. The feature of the niche is characterized by low PH, hypoxia and glucose-deprived starvation as a result of rapid proliferation by tumor cells and insufficient delivery by intra-tumor capillary [39]. Under the stressful conditions, a mass of misfolded or unfolded proteins accumulate in endocytoplasmic reticulum (ER). Tumor cells must activate UPR to inhibit synthesis of protein and mRNA and degrade excessive protein by proteasome to accommodate ER-stress, which is rarely experienced by normal tissue cells [40]. It has been demonstrated that severe ER-stress caused by accumulated misfolded proteins can activate programmed death in tumor cells [39]. Therefore, proteasome can serve as a potential target for cancer therapy.

Recently, PIs was introduced into clinical treatment of cancers, either as a single agent or a combined therapy to sensitize traditional chemo- and/or radiotherapy. However, the resistance to PIs remains a problem to solve, of which underlying mechanisms have not been explained pertinently [23]. The prerequisite for us to select PIs as an anti-LSC agent resides on the fact that PIs is effective against cyclin D1 highly-expressed cancers [41]. However, it remains to be elucidated whether other HNSCC cells were dependent or independent on cyclin D1 expression for their proliferation, survival and progression.

Given the fact that activation of STAT3 is associated with clinicopathologic behaviors and chemoradiation resistance in LSC, STAT3 is definitely a potential target for therapeutic purposes. The pro-survival and –proliferation effects of p-STAT3 let us to postulate that the constitutive (inherent) along with PI-induced (acquired) activation of STAT3 may constitute a major cause of resistance to PIs in LSC. Abrogation of both existed and acquired STAT3 activation may sensitize the LSC cells to PI treatment. In present investigation, we found for the first time that activation of STAT3 contributed to resistance of LSC to MG-132 at least in part by upregulating expression of antiapoptotic protein Bcl-2, which is partly consistent with results of a recent study [24]. Our results showed that PIs plus AG490 displayed a more powerful killing effect to LSC cells in a synergetic manner. However, such a combination may not be applied to all cancers, because overexpression of Bcl-2 is associated with a good prognosis in some other malignancies [42].

In the present study, we also demonstrated that MG-132 can induce G₀/G₁ cell cycle arrest, via downregulation of cyclin D1 in LSC, which is also observed in some other cancers [43-45]. It can be inferred that an ideal local control is to be achieved by PIs through inducing quiescence in LSC cells. However, a fact that cannot be ignored is that G₀/G₁ cycle arrest also constitutes a cause of therapeutic resistance in some tumors.

Although inhibiting STAT3 is capable of inducing quiescence and potentiating PI-induced apoptosis in LSC cells, there has been no effective STAT3 inhibitor that can be safely used for humans in clinic for treatment of HNSCC. We recently defined dihydroartemisinin as putative STAT3 inhibitor, which might be safely used in clinic for cancer treatment [46]. Much work has to be done for developing new STAT3-inhibiting agents and exploring new treatment strategies and modalities based on the synergized apoptosis-inducing effects of PIs with STAT3 inhibitors.

Acknowledgements: The research work in the present investigation is supported by Key Basic Research Project Fund of Hebei Province, China (Fund No. 14967721D) and in part by Scientific Research Fund of Bethune International Peace Hospital, China.

Conflict of Interest: None

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DOI: 10.31038/CST.2017214

Abstract

Purpose: To assess the relationship between IFN-γR expression and clinical radiotherapy outcome in nasopharyngeal carcinoma.

Methods: IFN-γR expression was examined by immunohistochemistry from 70 nasopharyngeal carcinoma patients. Then, statistical analysis was conducted to explore the correlation between IFN-γR expression and tumor clinicopathological charateristics. Finally, CNE-1 cells were employed to detect the effects of IFN-γR expression on radiotherapy outcomes by in vitro assays.

Results: IFN-γR was upregulated dramatically in nasopharyngeal carcinoma tissues. Elevated expression of IFN-γR correlated significantly with tumor stage, positive lymph node metastasis and poor prognosis in patients with nasopharyngeal carcinoma. In vitro assays demonstrated that overexpression of IFN-γR promoted the radio-resistance ability in CNE-1 cells. Knockdown of IFN-γR facilitated CNE-1 cells more sensitive to radiation, and a wobble mutant of IFN-γR could restore this effect.

Conclusions: Overexpression of IFN-γR may correlate positively with radiotherapy resistance of nasopharyngeal carcinoma.

Keywords

IFN-γR; overexpression; prognosis; radiation resistance; NPC

Introduction

Nasopharyngeal carcinoma is one of the most common malignancies and occupies the seventh leading cause of cancer-related deaths in China [1]. Particularly in South China, nasopharyngeal carcinoma is the most prevalent. Locally, advanced nasopharyngeal carcinoma patients are generally treated with radiotherapy, and the prognosis is related with radiotherapy [2]. Both local-regional failure and early systemic dissemination of the disease always contribute to treatment failure, which makes radio-sensitization promising in improving the outcome of therapeutic irradiation [3].

IFN-γ plays a pleiotropic role in modulating immunity and antiviral activity. It stimulates macrophages and NK cells to produce MHC class I and II molecules, while IFN-γR is not required for the development of the immune system [4]. Researches showed that IFN-γR was overexpressed in breast cancer and its amplification was closely related with lymph node metastasis and poor prognosis [5-7]. Until now, there are still no reports about whether IFN-γR has an effect on the radiotherapy for NPC. In the present study, we found for the first time that IFN-γR expression correlated with the prognosis of NPC patients who received radiotherapy. High IFN-γR levels improved the radiation resistance ability of NPC cells.

Patients and methods

Tissue samples

Seventy tissues of nasopharyngeal carcinoma were freshly collected by nasopharyngoscope and procured by the Department of Pathology, Cancer Center of Wuhan Union Hospital, China. The collection of tumor samples was approved by the institutional ethics committee. Informed consent forms had been signed for sample collection before. Tumor regions were technically separated by experienced pathologists and promptly stored at liquid nitrogen until needed. All patients included underwent concurrent chemotherapy (Cisplatin, 40mg/m2, weekly) with radiation therapy for the first time. They received irradiation with daily fraction doses of 2.12Gy for a total dose 70 Gy, which was administered with linear accelerators. The clinicopathologic features of patients were showed in Table 1.

Immunohistochemical staining

Immunohistochemical staining was conducted as previously described. After rehydration, the paraffin-embedded nasopharyngeal carcinoma tissues were immersed in 3% hydrogen peroxide solution for 10 min, and then heated in EDTA buffer (pH 8.0) for 25 min. The slides were blocked with 10% normal rabbit serum at 37°C for 30 min, and then incubated with rabbit polyclonal antibody against primary antibody (IFN-γR, 1:200, ABCAM) overnight at 37°C. The slides were washed with PBS, and then incubated with biotinylated second antibody (diluted 1:200) for another 30 min at 37°C. Finally, streptavidin-peroxidase reactivity was detected with DAB solution. Cell nucleus was counterstained with hematoxylin. The immunohistochemical slides were graded according to the ratio of tumor cells with nuclear labeling. The level of IFN-γR- positive staining was scored into three grades: 0 for < 10%, positive for 10–30%, strong positive for > 30%.

Plasmid constructs and small interfering RNA synthesis

Double-stranded oligonucleotide with the following sequence was synthesized into the pSilencer 3.1- H1 neo small interfering RNA (siRNA) expression vector, and the scrambled siRNA was applied as control. Mutant DNA-IFN-γR (BOSTER, Wuhan, China) was used to resist the effect of IFN-γR-RNAi.

Cell culture and transfection

The CNE-1 cells (one of the esophageal squamous carcinoma cell lines) were cultured in RPMI 1640 (Invitrogen) with 10% fetal bovine serum. Cell transfection was carried out using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instruction. Cells were harvested after transient transfection for 48 h. For stable transfection, cells were selected with 200 μg/ml G418, and passaged by serial dilution. During the next two weeks, colonies were screened by selective medium. The positive colonies with IFN-γR overexpression and IFN-γR knockdown were chosen for experiments.

Western blot analysis

Total proteins were isolated from patient tissues and cultured cells with lysis buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP40] containing protease inhibitors. After centrifugation (15,000 RCF, 30 min, 4°C), supernatants were recovered for immunoblot analysis. The proteins were subjected to SDS-PAGE and then transferred onto polyvinylidenedifluoride (PVDF) membranes (Millipore). Next, the membranes were blocked and then incubated with primary antibody against IFN-γR (1:500, ABCAM) at 4°C overnight, then incubated with horseradish peroxidase-conjugated accordingly secondary antibody for 1 h at room temperature and developed using ECL detection reagent (BOSTER).

Ionizing radiation

CNE-1 cell line was exposed to IR in a JL Shepherd Model 143 137Cesium gamma and irradiated at a rate of 2.4 Gy/min.

Colony formation assay

A total of 400 cells were aliquoted into 6- well plate and exposed to γ rays in a dose of 5 Gy in triplicate. After incubation for 10 days, visible colonies were fixed in methanol and stained with Giemsa. Colonies were counted in each well. Each experiment was performed three times independently.

Results

Overexpression of IFN-γR in esophageal squamous cell carcinoma

IFN-γR expression was examined in 70 NPC samples and adjacent normal tissue by immunohistochemistry. Results showed that IFN-γR was hardly expressed or revealed weak expression in normal epithelial cells. While, in 36 cases of 70 NPC samples, IFN-γR expression exhibited significantly enhanced trend compared to the normal epithelium [Table 1]. Statistical analysis indicated that elevated expression of IFN-γR was significantly related to tumor stage, clinical stage and lymph node metastasis (p < 0.05) [Table 2].

Next, the effect of IFN-γR expression on patient’s survival was analyzed. IFN-γR expression is positive in some patients and negative positive in others [Figure 1A]. After the included patients underwent chemotherapy combined with radiation therapy (55 out of 70), compared to NPC patients with low IFN-γR expression in cancerous tissue was significantly higher, patients with cancerous tissue of high IFN-γR expression showed significantly lower survival rate (p = 0.001) [Figure 1B].

Effects of IFN-γR on the radio-sensitivity of CNE-1 cells

To confirm the clinical data, CNE-1 cells were radiated with a dose of 5 Gy for 0, 2, 4, 8 and 12 h to know IFN-γR expression change. IFN-γR expression was increased after radiation in a time-dependent manner, which implied that IFN-γR may be associated with the radiation response of CNE-1 cells [Figure 2].

Colony formation assay was conducted to detect the effects of IFN-γR on the radio-sensitivity of CNE-1 cells [8]. IFN-γR knock-down cells showed a significantly lower survival fraction than control groups [Figures 3A and B]. Conversely, the survival fraction of cells with forced IFN-γR expression was higher [Figures 3C and D]. Therefore, results indicated a positive relationship between IFN-γR expression and radio-sensitivity of CNE-1 cells.

Discussion

Overexpression of IFN-γR has been observed in various types of human tumors, and elevated expression of IFN-γR indicated poor prognoses for patients, such as gastric carcinoma, prostate cancer, oral squamous cell carcinoma and non-small cell lung cancer. Particularly, IFN-γR hyperexpression was found to be associated with poor prognosis and reduced p27 expression in gastric carcinoma. Some studies have revealed that IFN-γR was amplified and upregulated in breast cancer. Moreover, its overexpression indicated worse tumor stage and lymphatic metastasis. However, there have been no reports on IFN-γR expression in NPC until now. Our results showed for the first time that IFN-γR expression increased in NPC, which significantly related with tumor stage and positive lymphatic metastasis. Coincidently, other studies have demonstrated that elevated IFN-γR level was correlated with poor prognosis in colorectal carcinoma [9,10]. However, no significance was shown between IFN-γR level and overall survival of NPC patients in the present study. We could not help putting forward the speculation whether IFN-γR expression affects radiotherapy outcome. Survival analysis unveiled that the overall survival rate of patients received radiation therapy was indeed affected by IFN-γR expression. Histological detection also showed a strong association betweeen IFN-γR hyperexpression and T-stage and lymphatic metastasis. Besides, in vitro assay further confirmed this point. This was the first report about the correlation between IFN-γR expression and radiation resistant of carcinoma.

Some studies reported that downregulation of IFN-γR suppressed prostate cancer cells proliferation, anchorage-independent growth and migration 11,12], while knockdown of Cks2 induced apoptosis [13,14]. Decreased IFN-γR promoted G2/M phase arrest and programmed cell death in human lung cancer cells [15]. Some study also found that overexpressin of IFN-γR in breast cancer cells repressed cell apoptosis through the MEK-Erk pathway [16]. However, no reports have described the apoptotic effects of IFN-γR expression in radiation-treated cells. In our next study, we may find out that overexpression of IFN-γR could induce apoptosis in CNE-1 cells after radiation.

To conclude, this study identified that overexpression of IFN-γR correlated with the poor prognosis of NPC patients receiving radiotherapy and elevated IFN-γR expression promoted the radiation resistance ability of NPC cells.

Conflict of interest: None declared.

Source of funding: None.

Consent: Written informed consent was obtained from the patients.

Acknowledgments: The Authors thank all the people involved in the project.

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DOI: 10.31038/CST.2017213

Abstract

Purpose: Several studies provided evidence for the efficacy of dose-escalation on biochemical control (BC) of prostate cancer and High-dose-rate brachytherapy (HDR) is one method for it.

Materials and Methods: Patients with histological diagnosis Gleason scored (GS), clinical stage T1 to T3a, no evidence of metastatic disease, prostate volume

Results: From 1997 to 2005 there were 273 patients treated with this treatment combination at AC Camargo Cancer Center, Sao Paulo, Brazil. The median age and FU time were 64.7 and 10.3 years, respectively. Two hundred thirteen (78.0%) patients had FU longer than 5 years. Actuarial 10-year overall survival (OS), Clinical Specific Survival (CSS) and BC were 89.8%, 63.6% and 71.8%, respectively. On univariate analysis GS<7, clinical stage<T2b, low risk group (LR), absence adjuvant androgen deprivation (ADT), age>65, PSAi<10, localized EBRT and 3D-HDR plan were associated with improved CSS and BC, excluding PSAi, age for the last one. Multivariate Cox regression analysis confirmed LR, GS<7, PSAi<10, absence of ADT, age

Conclusion: The present data represents a unique uni-institutional study at long FU for the given technique. A comparison with the current literature confirms the excellent results achieved with this treatment modality. HDR has also the advantage of treatment time reduction and increasing in the capability of work load of the linear accelerators, especially in developing countries, where waiting lists and lack of radiation oncology facilities are a reality.

Keywords

prostate cancer, radiotherapy, high-dose rate brachythrerapy, biochemical control, PSA

Abbreviations and Acronyms

ADT: Adjuvant Androgen Deprivation
AJCC: American Joint Committee on Cancer
BC: Biochemical Control
CSS: Clinical Specific Survival
EBRT: External Beam Radiotherapy
FU: Fallow Up
GS: Gleason Score
HDR: High-Dose-Rate Brachytherapy
LR: Low Risk Group
NAAD: Neoadjuvant Hormonal Therapy
OS: Overall Survival
PSAi: Initial Prostate-Specific Antigen
TRUS: Trans Rectal Ultrasound

Introduction

More than 62% of Prostate Cancers (PCa) are diagnosed in men over 65 years. It has become a public health and socioeconomic problem with increasing incidence, in special due to a rapidly aging population worldwide [1]. In Brazil it was expected the diagnosis of 61,200 new cases of PCa in 2016, and the crude mortality for 2013 was around 14,000 deaths [2].

Management options for localized and locally advanced PCa are controversial and include active surveillance, radical prostatectomy, external beam radiotherapy (EBRT) and brachytherapy with low or high dose rate sources. [3]

Several studies provided evidence for the efficacy of dose-escalation on biochemical control (BC) of PCa. Mature results from randomized trials show a direct relation between increasing the radiation dose given to the prostate and/or seminal vesicles and BC [4-7].

High-dose-rate after loading brachytherapy (HDR) is one method that can deliver a high localized radiation dose to the tumor with excellent BC when combined to EBRT [8]. One prospective randomized trial with up to 10 years follow up has proved that HDR plus EBRT is more efficient than EBRT alone in terms of BC with less acute rectal toxicity and improved quality of life [9].

The aim of this retrospective study is to evaluate the mature results of patients with local and locally advanced PCa treated with combination of HDR and EBRT.

Materials and Methods

Patients with confirmed histological diagnosis Gleason scored (GS) of PCa, AJCC clinical stage T1 to T3a, with no evidence of metastatic disease and initial PSA <60mg/ml, prostate volume

This single-centre institutional protocol of treatment was performed in compliance with the Declaration of Helsinki and approved by the local research Ethics Committee. Written informed consent was mandatory.

External Beam Radiotherapy

The EBRT target volume was defined using diagnostics CT images on conventional two dimensional or 3D planning. The targets were the prostate gland and the proximal seminal vesicles with a 1 to 1.5 cm margin except to the posterior region, which margins were reduced to 0.5 to 1.0cm. The EBRT dose ranged from 45 to 54 Gy prescribed to the intersection point. Further details of the radiotherapy schedules have been published previously [10].

High Dose Rate Brachytherapy

HDR was done under spinal anesthesia. Using TRUS with a perineal template affixed to perineum the exact needles positions were determined intraoperatively. In a first moment treatments were planned based on semi-orthogonal X-rays – two dimensional planning (2D) – and after that we moved to three dimensional (3D) planning, based on CT images. The prostate gland, the rectum, and the urethral trajectory and length were countered and identified in both situations. Implant dosimetry geometric optimization was initially utilized, followed later by use of inverse planning. Treatment parameters and dose constraints changed minimally throughout the years. The patients considered low risk had 16 Gy given in 4 fractions BID, one single implant. Intermediate and high risk patients had 20 Gy given in the same treatment schedule. The dose-volume histogram constraints were as follows: the TRUS or CT-based prostate´s volume receiving 100% of the dose (V100) should be >95%, the uniformity index should be more than 50%, and the V150 less than 30%. The urethra maximum punctual and the maximal dose to 1cc of anterior rectal wall should not exceed 135% and 75% of prescribed doses, respectively.

Definition of end points and statistical analysis

BC was measured using PSA tests and assessed according to the Phoenix definitions [11]. Clinical Specific Survival (CSS) was calculated from the start of treatment to the lost of BC, diagnose of metastatic disease or death from PCa. The BC was evaluated from the date of start the treatment until date of first biochemical failure. The follow up (FU) program also included clinical investigation, digital rectal examination and image studies.

The statistical program SPSS (statistical package for the social sciences) Inc., released 2008, Statistics for Windows, version 20.0 (SPSS Inc., Chicago, IL) was used for all statistical analysis. The analysis of OS, CSS and BC was made using the Kaplan–Meier method. The log-rank test was used to test the significance when comparing different subgroups. Univariate and multivariate Cox regression analysis were also performed. The alpha level considered for statistically significant differences was 0.05.

Results

Between March, 1997 and March, 2005 there were 305 patients treated with combination of HDR and EBRT at the Department of Radiation Oncology, AC Camargo Cancer Center, Sao Paulo, Brazil. Thirty two patients were lost of FU and the data of 273 patients was available for analysis. Sixty four (27.1%) patients had pelvic EBRT and the remaining 209 (76.5%) localized EBRT. Clinical and treatments characteristics are depicted in Tables 1 and 2.

Table 1. Patients Characteristics

		Median	Range	Variable	n	%
	Age (years)	64.7	42-82
	Prostate Vol (cc)	36.3	19-72	<35	121	44.3
				>35	152	55.7
	PSAi (ng/ml)	10.3	1-52	<10	173	16.8
				10-20	54	19.8
				>20	46	63.4
	Gleason Score			<7	190	69.6
				=7	58	21.2
				>7	25	21.2
				Yes	47	17.2
				No	226	82.8
	Clinical Stage			<T2b	192	70.3
				T2b-c	47	17.2
				>T2c	34	12.5
	Risk Group			Low	133	48.7
				Interm	76	27.8
				High	64	23.4
ADT	NAAD			Yes	93	34.1
				No	180	65.9
	ADJ				91	33.3
	Salvage				37	13.6
	WO				145	53.2
EBRT	Pelvic				64	23.4
	Localized				209	76.6
	Comorbidities			No	146	53.5
				SAH	42	15.4
				Diabetes	19	7.0
				Other	39	14.3
TOTAL					273	100.0

Legend: ADJ (adjuvant hormonal therapy), ADT (Androgen deprivation therapy), BF (Bichemical failure, EBRT (External beam radiotherapy), NAAD (neoadjuvant hormonal therapy), SAH (Systemic arterial hypertension), Salvage (Salvage hormonal therapy)

Table 2. Treatment  Characteristics

		Median	Range	Variable	n	%
Dose	EBRT	50	40-54	< 50	149	54.6
				>50	124	45.4
	HDR			16	133	48.7
		18.3	16-20	20	140	51.3
	HDR plan			2D	167	61.2
				3D	106	38.8
	Interval	18.5	9-61	<18	173	63.4
				>18	100	36.6

Legend: EBRT (External beam radiotherapy), HDR (High-dose-rate brachytherapy), HDR plan 2D/3D (two or three dimensional planning)

The median age and FU time were 64.7 (range, 42-82) and 10.3 (range, 1-15) years, respectively. Two hundred thirteen (78.0%) patients had FU longer than 5 years, and of these 153 (56.1%) longer than 10 years.

Androgen deprivation therapy

Androgen deprivation therapy (ADT) in a short course neo-adjuvant ADT, was prescribed for less than 6 months. Neoadjuvant hormonal therapy (NAAD) was administered to 34.1% of the patients. Adjuvant hormonal therapy (ADJ) was observed, mostly, for intermediate and high risk patients (33.4%), generally for no more than 6 months for intermediate risk and up to 3-years in high risk patients. Salvage ADT was observed in 37 (90.2%) of 41 patients dead due PCa. The profile of hormonal therapy is shown in Tables 3 and 4.

Table 3. Neoadjuvant Hormonal therapy according to Risk Group – Risk NAAD Cross tabulation

		NAAD
Risk Group		WO %		YES  %		Total %
	Low	74	27.1	8	2.9	82	30.0
	Interm	65	23.8	35	12.8	100	36.6
	High	41	15.0	50	18.3	91	33.3
Total		180	65.9	93	34.1	273	100

Legend: ADJ (adjuvant hormonal therapy), Interm (intermediate), NAAD (neoadjuvant hormonal therapy), WO (without hormonal therapy)

Table 4. Adjuvant and Salvage Hormonal therapy according to Risk Group

	WO   %		ADJ   %		Salv    %		Total    %
Low	72	26.4	7	2.6	3	1.1	82	30.0
Interm	57	20.9	34	12.5	9	3.3	100	36.6
High	16	5.9	50	18.3	25	9.2	91	33.3
Total	145	53.2	91	33.4	37	13.6	273	100

Legend: ADJ (adjuvant hormonal therapy), Interm (intermediate), NAAD (neoadjuvant hormonal therapy), Salv (Salvage hormonal therapy), WO (without hormonal therapy)

The crude 10-year overall survival (OS) rate at was 52.7%. Actuarial 5- and 10-year OS, CSS and BC were 80.1%, 89.8%, 83.7%, 63.6%, 85.5% and 71.8%, respectively. (Figures 1-3)

Univariate and multivariate analysis

On univariate analysis GS <7, clinical stage <T2b, low risk group, absence adjuvant ADT, older age (>65-years), PSAi Univariate analysis failed to identify neoadjuvant androgen deprivation therapy (NAAD) as a predictor for BC in all group risks (p=ns). When we pooled the intermediate and high risk group into a unique denominated unfavorable risk group, NAAD also failed to predict improved CSS and BC.

Multivariate Cox regression analysis confirmed low risk group (HR 0.03, 95% CI 0.006-0.116, p<0.001) and intermediate risk (HR 0.09, 95% CI 0.042-0.216, p<0.001) compared to high risk, presence of ADT (HR 0.39, 95% CI 0.179-0.868, p=0.021) as favorable predictors for CSS. GS >7 (HR 3.24, 95% CI 1.279-8.220, p<0.001), PSA >10 (HR 6.19, 95% CI 2.015-19.041, p=0.001), age >65 years (HR 2.87, 95% CI 1.264-6.504, p=0.012) and EBRT dose >50 Gy (HR 14.50, 95% CI 1.874-112.164, p=0.010) were confirmed as adverse predictors for CSS. Tables 6-8, Figures 4-9.

Low risk group compared to intermediate, (HR 0.70, 95% CI 0.023-0.213, p<0.001) and high risk (HR 0.99, 95% CI 0.052-0.190, p<0.001) groups, was a favorable predictive factor for BC. GS >7 (HR 3.09, 95% CI 1.473-6.473, p=0.003) and PSAi >10 (HR 6.18, 95% CI 2.331-16.393, p<0.001) were negative predictive factor for BC.

Low risk group was confirmed as the only predictive factor for OS when compared to intermediate (HR 0.28, 95% CI 0.133-0.608, p=0.001) and high (HR 0.38, 95% CI 0.223-0.665, p=0.001) risk groups.

Table 6. Cox regression for CSS

	B	SE	Wald	df	Sig.	Exp(B)	95.0% CI for Exp(B)
							Lower	Upper
LR			44.294	2	.000
IR	-3.641	.761	22.910	1	.000	.026	.006	.116
HR	-2.351	.418	31.579	1	.000	.095	.042	.216
EBRT Pelvic x Local	-.110	.312	.124	1	.724	.896	.486	1.651
2D x 3D	-1.861	1.134	2.693	1	.101	.156	.017	1.436
ADT	-.931	.403	5.336	1	.021	.394	.179	.868
PSAi <10			10.858	2	.004
PSAi (>10<20)	1.925	.661	8.481	1	.004	6.856	1.877	25.048
PSAi (>20)	1.824	.573	10.133	1	.001	6.195	2.015	19.041
GS <7			7.328	2	.026
GS = 7	.335	.709	.223	1	.636	1.398	.349	5.608
GS >7	1.176	.475	6.145	1	.013	3.243	1.279	8.220
CS <T2b			3.350	2	.187
CS T2b/T2c	-.662	.551	1.443	1	.230	.516	.175	1.519
CS >T2c	.464	.818	.321	1	.571	1.590	.320	7.895
Age <65y	1.053	.418	6.352	1	.012	2.867	1.264	6.504
EBRT <50Gy	2.674	1.044	6.562	1	.010	14.498	1.874	112.164
HDR dose <20Gy	1.763	1.023	2.973	1	.085	5.830	.786	43.261
Interval EBRT to HDR	-.187	.303	.381	1	.537	.829	.458	1.502

Legend: 2D (two dimensional plan), 3D (tridimensional plan), ADT (Androgen deprivation therapy), BC (biochemical control), Comorb (Comorbidites), CS (Clinical stage), CSS (Clinical Specific Survival), EBRT (External beam radiotherapy), GS (Gleason score), IR (Intermediate risk group), HDR (High-dose-rate brachytherapy), HR (High risk group), LR (Low risk group), NAAD (neoadjuvant hormonal therapy), OS (overall survival), P vol. (Prostate volume cc)

Table 7. Cox regression for BC

	B	SE	Wald	df	Sig.	Exp(B)	95.0% CI for Exp(B)
							Lower	Upper
LR			62.053	2	.000
IR	-2.660	.568	21.948	1	.000	.070	.023	.213
HR	-2.309	.330	48.920	1	.000	.099	.052	.190
2D x 3D	-.789	.656	1.448	1	.229	.454	.126	1.642
ADT	-.455	.307	2.192	1	.139	.634	.347	1.159
PSAi <10			13.634	2	.001
PSAi (>10<20)	1.822	.498	13.403	1	.000	6.182	2.331	16.393
PSAi >20	1.346	.462	8.494	1	.004	3.843	1.554	9.502
GS <7			8.944	2	.011
GS =7	.957	.465	4.240	1	.039	2.603	1.047	6.469
GS >7	1.127	.378	8.915	1	.003	3.088	1.473	6.473
CS <T2b			2.825	2	.244
CS T2b/T2c	-.647	.410	2.493	1	.114	.523	.234	1.169
CS >T2c	-.306	.580	.278	1	.598	.737	.236	2.294
EBRT 50Gy	1.411	.550	6.590	1	.010	4.101	1.396	12.044
HDR dose <20Gy	-.470	.680	.477	1	.490	.625	.165	2.370
Interval EBRT to HDR	-.064	.230	.078	1	.780	.938	.597	1.473

Legend: 2D (two dimensional plan), 3D (tridimensional plan), ADT (Androgen deprivation therapy), BC (biochemical control, Comorb (Comorbidites), CS (Clinical stage), CSS (Clinical Specific Survival), EBRT (External beam radiotherapy), GS (Gleason score), IR (Intermediate risk group), HDR (High-dose-rate brachytherapy), HR (High risk group), LR (Low risk group), NAAD (neoadjuvant hormonal therapy), OS (overall survival), P vol. (Prostate volume)

Table 8. Cox regression for OS

	B	SE	Wald	df	Sig.	Exp(B)	95.0% CI for Exp(B)
							Lower	Upper
ADJ	.149	0.3	0.3	1	.580	1.161	.684	1.970
Age <65y	.223	0.2	1.0	1	.322	1.250	.804	1.942
Comorbidity	-.310	0.2	2.1	1	.149	.734	.482	1.117
EBRT Pelvic x Local	-.089	0.2	0.1	1	.722	.915	.560	1.494
LR			14.3	2	.001
IR	-1.259	0.4	10.5	1	.001	.284	.133	.608
HR	-.954	0.3	11.7	1	.001	.385	.223	.665

Legend: ADJ (adjuvant hormonal therapy, EBRT (External beam radiotherapy), IR (Intermediate risk group), HDR, HR (High risk group), LR (Low risk group), OS (overall survival)

Discussion

Conventional EBRT to treat PCa is securely limited to doses of 64–70 Gy in 1.8–2.0 Gy fractions. These levels of doses are determined by the risk of long-term toxic effects to the bladder and rectum. The clinical and biochemical relapse rates associated with these dose levels are around 33% within 5 years. Peeters et al 12 published the results of a randomized trial comparing total doses of 68 Gy and 78 Gy using EBRT alone. The 5-year OS in the higher dose arm of that study was 83% using ASTRO definition and the BC was significantly better in the 78-Gy arm compared to the 68-Gy arm, with an adjusted hazard ratio of 0.74 (p=0.02). Other published reports using dose-escalated photon beam EBRT alone also point in the same direction with respect to their long-term BC results [13-14].

HDR can escalate the dose given to the prostate by the combination with EBRT, and further more, in locally advanced disease has also the possibility of including the seminal vesicles when they needed to be encompassed. HDR has also a potential biological advantage through the delivery of high doses per fraction [10]. It is important to note that the comparisons between series published are difficult due differences in the techniques and planning for both, EBRT and HDR.

The combination of values of PSAi, GS and CS to identify a more or less aggressive disease has being extensively discussed in the literature, and was confirmed by this study. The most frequent challenge is to identify, for example the indication of prostate versus pelvic EBRT, varying risk categories, absence or use of NAAD, ADT and their length. Despite this, the combination of HDR and EBRT provides an optimal modulation of dose delivery. Results of this combination, in terms of BC and with more than 5 years of FU, range from 57% to 100% according to the risk group for biochemical failure (Table 9).

Table 9. Results of biochemical control by risk groups in series of HDR plus EBRT with more than 5-year follow up

Reference	n	Median FU  (months)	Biochemical control by risk groups (%) Low       Intermediate       High			Dose in Gy (HDR(n.fx)/EBRT)
13	344	61		84	74	19.5(3) / 46
14	121	63		91		10(1)/50
15	313	68	100	88	79	23(2)/46
16	229	61	95	90	57	21(3)/50.4
17	64	105		84	80	18(3)/45
18	90	95			80	16.5(3)/45
19	264	75	97			18(3)/45
20	100	62		84	82	10(1)/60
21	64	61		100	91	21(3)/50
22	196	66		86		18(3)/46
23	131	63		87	71	30(4)/45

The search for factors predicting BC and CSS is important on defining what patients should be treated more aggressively. We, as other authors [26-28] observed that PSAi <10 ng/ml was confirmed as a favorable predictive factor related to BC and CSS.

Age<65 years was found to be an adverse prognostic factor in our analysis for CSS, with a marginally statically significance impact on OS, not confirmed on multivariate analysis. Smolska-Ciszewska et al, conversely to our results, noted that younger age at time of treatment impacted only on OS, not explaining if there was any association between treatment and side effects or worsening of associated comorbidities. As in our analysis, they found that low risk group and association of HDR to EBRT correlated with improved BC [29].

As in our results, Kamrava et al found that T stage, GS, and use of ADT were significantly associated with CSS on univariate analysis, but on multivariate analysis only GS and use of ADT were significantly associated CSS [30]

It is expected that the grouping of the patients in risk groups for biochemical failure based on PSAi, GS and CS, to aggregate patients with adverse features in the intermediate and high risk, leading to worse CSS and BC for the two last one, what was confirmed in our analysis. This was also observed by Morris et al [31].

In our series, as in others, patients who received adjuvant ADT had significantly higher risk features suggesting patient selection bias for CSS in this group of patients, instead of a negative interaction between HDR and EBRT [31-32].

Mature data in the literature evaluated the 10-year outcomes of intermediate- and high-risk patients noting a clear dose response by increasing the dose escalation through HDR doses [27]. More recently the use of Intensity modulated radiation therapy combined to HDR has being investigated. Chen et al. published the results of 148 patients treated with HDR – 22 Gy in 4 fractions followed IMRT up to 50.4Gy. All patients with Gleason score of 8 or higher had ADT for 1 year. They noted a 4-year actuarial CSS of 96.8% and of 100%, 100% and 94% for low, intermediate and high risk, respectively [32].

The results of the first randomized prospective trial, which has addressed dose escalation using an HDR and EBRT is a trial with a relatively slow accrual rate. There are some critics that must be addressed as the changes in EBRT technique during the time of the study, and, by current standards, the control arm is a relatively low-dose treatment. Despite that, the study reported the results of 218 patients treated between 1997 and 2005. There were 108 patients assigned to EBRT alone and 110 patients treated by EBRT followed by HDR. They noted that CSS was significantly higher in patients treated with combined modality (p = 0.04). In multivariate analysis the category treatment modality and ADT were significant covariates for BC, but with no differences in OS, as observed in our study. After a median follow-up time of 10.5 year follow-up, an 18% increase in CSS was obtained relative to EBRT alone, reflecting a 31% reduction in the risk of recurrence (p = 0.01) and no evidence of an increase in long-term severe morbidity [9].

Surgically induced gland deformation is inevitable during brachytherapy procedures. We observed that the use more of advanced methods of images (3D plan based on CT, MR or TRUS) images to identify the target, organs at risk and needles is important and have already been reported to impact on BC and CSS [33]. In our analysis Multivariate Cox regression analysis confirmed EBRT dose >50 Gy as predictor for CSS and BC, but with no impact on OS. This may be explained by the relative low dose per fraction schedule used in both groups. Of importance is to note that the dose given by HDR was relative constant thorough the risk groups, leading to higher biological effective dose to intermediate and high risk patients, and even though, these patients had a worse outcome, showing that there is space for further studies of dose escalation or treatment combination. After 2005 with the introduction of real time TRUS image acquisition and planning we have changed our protocol and moved forward for a more intense dose escalation, increasing dose and reducing the number of fractions.

Other information of this study is that presence of NAAD had no impact on DSS, BC or OS in any risk group, showing that this treatment strategy should be reserved for downsizing the prostate prior to treatment, in special for low risk patients.

In conclusion, this report demonstrates that HDR combined with EBRT is an important and effective method in achieving dose escalation in the radical radiotherapy of PCa. This combination has also the advantage of treatment time reduction and in increasing in the capability of work load of the linear accelerators, especially in developing countries, where waiting lists and lack of radiation oncology facilities are a reality. The present data represents a unique uni-institutional study at long FU for the given technique, and a comparison with the current literature confirms the excellent results achieved with this treatment modality. The satisfactory BC, CSS and OS are probably result of improved LC achieved with dose escalation, showing that HDR is an optimal alternative method of local dose escalation when combined to EBRT.

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DOI: 10.31038/CST.2017212

Abstract

Purpose: Platinum-based doublet chemotherapy had been the standard first-line treatment for advanced NSCLC, regardless of histologic subtypes. We report overall survival (OS) and time to treatment failure (TTF) in patients with squamous cell lung cancer (SCC) receiving doublet of platinum.

Patients and Methods: Patients (N = 82) with advanced NSCLC received doublet of platinum. Sixty five (79,2%) patients were treated with a combination of platinum plus gemcitabine and 17 (20,7%) received microtubules inhibitor (3 patients were treated with vinorelbine, 3 patients with docetaxel and 11 patients with paclitaxel).

Results: Median TTF was 2,53 months CI95% [2,21 – 2,84] and median OS was 8,246 months CI95% [5,8 –2,6]. Regarding doublet of chemotherapy, in patients in which gemcitabine was used there was an improvement in TTF of 1,2 months (p= 0,107; log rank) and 4,75 months in OS (p= 0,018; log rank).

Conclusion: Gemcitabine plus platinum must be the chemotherapy of ghoice in advanced SCC. Randomized clinical trials with gemcitabine in advanced SCC are needed.

Keywords

squamous cell lung cancer, chemotherapy, gemcitabine

Introduction

Primary lung cancer is the most common malignancy and the first death related causes from cancer in the worldwide. Nowadays, it is the most important cause of cancer mortality in men and women. Lung cancer is still increasing both in incidence and mortality worldwide. In Spain, more than 21,000 men were diagnosed of lung cancer in 2012, while over 17,000 died. Lung cancer is the leading cause of dead among Spanish men. Figures in women were near 5,000 and more than 3,500 respectively. Women got into the habit of smoking some decades later than men in Spain [1]. Non-small-cell lung cancers (NSCLC) account for 85%–90% of lung cancers. NCSLC includes several histologic subtypes such as adenocarcinoma, squamous cell carcinoma (SCC), and large cell carcinoma. Platinum-based doublet chemotherapy had been the standard first-line treatment for advanced NSCLC, regardless of histologic subtypes [2].

New agents has been developed recently (anti folate, anti VEGF, antiEGFR, etc) [3]. These agents play a crucial role in first-line systemic therapy for nonsquamous histology while they do not have activity in SCC.

On the other hand, several regimens of platinum-based doublet chemotherapy are currently the standard first-line therapy for advanced lung SCC, including platinum combined with gemcitabine, docetaxel, paclitaxel, or vinorelbine and these scheludes have similar effectiveness [4].

There are not any phase III studies focused on determining what is the most active platinum-based chemotherapy regimen to treat advanced lung SCC. This study examined the comparative effectiveness of various platinum-based regimens as first-line therapy for advanced lung SCC.

Material and Methods

Patients

This is a retrospective study. The study population included patients with newly diagnosed lung SCC from 2012 to 2014 in the University General Hospital of Ciudad Real (Spain). The following inclusion criteria were used to identify eligible patients: (1) pathologically proven initial diagnosis of lung SCC as the single primary cancer; (2) age ≥ 18 years; and (3) advanced disease stage at diagnosis, which was defined as stage IIIB or stage IV disease according to the American Joint Committee on Cancer, 7th edition.5 Patients who underwent surgery during the first course of treatment and those who underwent radiotherapy with curative intent (which was defined as a cumulative dose > 50 Gy) were excluded.

All patients received chemotherapy for advanced lung SCC. Main regimens considered in our study including cisplatin (P), carboplatin (CP), gemcitabine (G), docetaxel (D), paclitaxel (T), or vinorelbine (V). Platinum agent was considered and patients were classified into patients who received cisplatin or carboplatin.

Objective

The main objective was overall survival (OS). OS was determined according to the date of diagnosis of advanced lung SCC to the date of death.

Statistical analysis

Baseline demographic and clinical variables were summarized with descriptive statistics. Regard chemotherapy, G were recoded as antimetabolite and D, T, V as microtubules inhihitor (MI).

The OS was estimated using the Kaplan-Meier method, and the differences between the study groups were compared using the log-rank test. The Cox proportional hazard model was used to estimate the univariate or adjusted hazard ratios and associated 95% confidence intervals for detecting differences in the effects of treatments on overall mortality. The sex, age, brain metastases and type of platinum were adjusted in the Cox proportional hazard model. Subgroup analyses defined according to sex, age (< 70 or ≥ 70 years) and platinum were performed as sensitivity analyses to determine whether the differences in effects on mortality of platinum + antimetabolite compared with those of P + MI. Two-sided P values of ≤ .05 were considered statistically significant. All analyses were performed by SPSS for windows v. 18.

Results

Baseline characteristics

Eighty two patients were included in this study. Among them, 79 (96.3%) were men, 41 (50%) were aged ≥ 70 years, and 2 (2,7%) had brain metastases (Table 1). Sixty five (79,2%) patients were treated with a combination of platinum plus gemcitabine and 17 (20,7%) received microtubules inhibitor (3 patients were treated with vinorelbine, 3 patients with docetaxel and 11 patients with paclitaxel). Patients aged < 70 years were more likely to receive chemotherapy with microtubules inhibitors than older patients (70,6% vs. 44,6%, P = 0.057). Up to December the 31th, 2014, 66 patients (80,5%) were with progression disease, 68 (82,2%) had died and 45 patients (54,9%) were controlled by the Palliative Care Unit. The median follow-up time was 6 months (table 1).

Table 1. Baseline characteristics of patients with advanced lung squamous cell carcinoma in our series

Baseline Characteristics	Gemcitabine	Microtubule inhibitor	All	p
N (%)	65 (79,2%)	17 (20,7%)	82 (100%)
Age (median, range)	71 (44-88)	64 (40-81)	70 (40-84)	0,118
Years (n, %) <70 años >70 años	29 (44,6) 36 (55,4)	12 (70,6) 5 (29,4)	41 (50,0) 41 (50,0)	0,057
Gender (n, %) Male Female	63 (96,9) 2 (3,1)	16 (94,1) 1 (5,9)	79 (96,3) 3 (3,7)	0,583
Brain Metastases (n, %) No Yes	63 (96,9) 2 (3,1)	17 (100,0) 0 (0,0)	80 (97,6) 2 (2,4)	0,464
Platinium (n,%) Cisplatin Carboplatin	21 (32,3) 44 (67,7)	3 (17,6) 14 (82,4)	24 (29,3) 58 (70,7)	0,237
Progression first line (n, %) No Yes	11 (16,9) 54 (83,1)	5 (29,4) 12 (70,6)	16 (19,5) 66 (80,5)	0,247
Second line  (n, %) Taxanes Gemcitabine TKI	27 (41,5) 21 (77,7) 0 (0,0) 6 (22,2)	4 (23,5) 2 (50,0) 1 (25,0) 1 (25,0)	31 (37,8) 23 (74,19) 1 (3,3) 7 (22,5)	0,173
Control by Palliative Care Unit (n, %) No Yes	29 (44,6) 36 (55,4)	8 (47,1) 9 (52,9)	37 (45,1) 45 (54,9)	0,857
Estatus Alive Exitus	12 (18,5) 53 (81,5)	2 (11,8) 12 (88,2)	14 (17,1) 68 (82,9)	0,514

In patients, the main causes of death were progression disease in 59 patients (86,7%), pulmonary embolism in 2 patients (2,9%), stroke in 1 patient (1,47%), chronic obstructive pulmonary disease in 3 patients (4,4%), myocardial infarction in 1 patient (1,47%), hemoptysis in 1 patient (1,47%) and chemotherapy related toxicity in 1 patient (1,47%) (Figure 1).

Time to treatment failure

Median TTF was 2,53 months CI95% [2,21 – 2,84] (Figure 2). Considering age, sex, brain metastases, platinum compound and doublet of chemotherapy, we did not find differences (Table 2). Regarding doublet of chemotherapy, in patients in which gemcitabine was used there was an improvement in TTF of 1.2 months (p= 0.107, log rank) (Figure 3).

Table 2. Medians and hazard ratios of time to treatment failure regarding age, sex, brain metastases, platinum compound and doublet of chemotherapy

	Median months, IC95%	P, log rank	Hazard ratio, IC95%	P, cox
Age <70 años >70 años	2,9 [2,07-3,9] 2,3 [2,1-2,5]	0,264	0,757 [0,46-1,24]	0,269
Sex Male Female	2,5 [2,2-2,8] 1,9 [0,0-5,04]	0,714	0,806 [0,25-2,6]	0,718
Brain Metastases (n, %) No Yes	2,5 [2,1-2,8] 2,3 [n.r.]	0,771	0,747 [0,1-5,5]	0,774
Platinum (n,%) Cisplatin Carboplatin	2,3 [2,01-2,7] 2,6 [1,9- 3,2]	0,895	1,037 [0,6-1,7]	0,896
Doublet of CT (n, %) Microtubules inhibitor Gemcitabine	1,7 [0,0-4,3] 2,5 [2,2- 2,8]	0,107	1,677 [0,8-3,1]	0,115

Overall survival

Median OS was 8,246 months CI95% [5,8 –2,6] in all patients (Figure 4). Overall survival rate al 6, 12 and 18 months were 62,8%, 35,1% and 3,8% respectively. Considering age, sex, brain metastases, platinum compound and doublet of chemotherapy there were statistically significant differences regard sex and doublet of CT (Table 3).

Table 3. Medians and hazard ratios of time to treatment failure regarding age, sex, brain metastases, platinium compound and doublet of chemotherapy

	Median months, IC95%	P, log rank	Hazard ratio, IC95%	P, cox
Age <70 años >70 años	7,5 [4,6-10,5] 8,9 [6,4-11,3]	0,619	0,884 [0,54-1,43]	0,619
Sex Male Female	8,3 [6,2-10,5] 3,2 [2,4-4,1]	0,006	0,217 [0,06-0,719]	0,012
Brain Metastases (n, %) No Yes	8,2 [5,8-10,6] 4,3 [n.r.]	0,596	1,466 [0,35-6,1]	0,599
Platinium (n,%) Cisplatin Carboplatin	10,48 [3,9-17,02] 7,65 [4,9- 10,33]	0,260	0,764 [0,42-1,2]	0,263
Doublet of CT (n, %) Microtubules inhibitor Gemcitabine	5,7 [3,8-7,6] 9,45 [6,7- 12,1]	0,018	1,989 [1,1-3,5]	0,021

In multivariate analyses adjusted for sex, age (< 70 or ≥ 70 years) and platinium the first line chemotherapy regimen based in antimetabolites was a predictor of OS (p=0.018).

Second line

Only 31 (37,8%) patients received second line therapy of treatment. Most common agents were docetaxel (17 patients, 54,8%), paclitaxel (6 patients, 19,9%), erlotinib (6 patients, 19,4%), gefitinib (1 patient, 3,2%) and gemcitabine (1 patient, 3,2%).

Median OS posprogression (OSpp) was 6,637 months CI95% [5,4 –7,8]. With respect to type of treatment, patients treated with docetaxel had a median of 9,01 months CI95% [5,1 –12,9], patients treated with paclitaxel 6,6 months CI95% [0,5 –12,7] and patients treated with erlotinib 5,8 months CI95% [4,6 –7,1]. Patient treated with gefitinib and gemcitabina were alive while 5,4 months and 3,2 months respectively. There were statistically significant differences in median OSpp regarding type of treatment (p=0,026 log rank). Hazard ratio for use of taxanes was 0,695, CI95% [0,26 –1,83], p=0,4.

Discussion

Squamous cell NSCLC is a particularly aggressive type of lung cancer, and few treatments are effective [6]. The NCCN and ESMO frontline recommendations include chemotherapy doublets, which are considered the cornerstone of initial therapy for squamous NSCLC [7,8]. To our knowledge, no prospective clinical trial has specifically compared cytotoxic chemotherapies for advanced lung SCC so there is is uncertainty about the best option of treatment.

Respect gender, women is represented in 3,7% in our study. In most published studies, little is reported about women diagnosed of advanced squamous NCSLC treated with CT. Overall survival among women in our study is poor (3,2 months versus 8,3). Recently, it has been described that females patients with squamous NCSLC had a significantly higher rate of human papillomavirus (HPV) infection compared to males with SCC [9] and HPV infection appears to be involved in cancer progression in SCC by promoting the expression of p53. On the other hand, patterns of mutation in SCC are unknown being KRAS, FGFR1 and PIK3CA most frequently reported and women seem to have less PIK3CA mutation than men. Moreover, there may exist unknown endocrine mechanisms this particularly lack of response and bad prognosis in women. To our knowledge, there are no differences in smoking patters that could explain this fact [10].

Elderly and young patients, defined by >70 years or <70 years, have similar proportion in our series. Although doublets with microtubule inhibitors are less used in old patients overall survival is similar in both groups. The definition of elderly patients varies widely across trials and and an uniform definition is lacking. Particularly in the actual world setting, older patients are often untreated even even when free of comorbid illnesses. Most studies in advanced NSCLC define elderly patients as those older than 70 years for the treatment of advanced NSCLC [11] and the benefit of platinum-based doublet regimens in this population seems to be greater than single-agent chemotherapy. So avoiding the use of doublets in elderly  is not justified in absence of comorbid status. Studies in elderly patients show similar response rates than in younger ones even with aggressive regimens of chemotherapy.

Palliative Care Unit (PCU) has played a preeminent role in the management of patients. More than half of our patients were remitted to PCU (45 patients, 54,9%) This fact allowed better control of symptoms, especially at home, and ultimately improved patients’ quality of life [12]. Early integration of palliative care into the treatment strategy should be mandatory because it may improve quality of life, particularly end-of-life care and improve overall survival. Our institution is working to make success this target.

Median time to treatment failure was 2,53 months and overall survival 8,24 months. The use of cisplatin or carboplatin has no impact on TTF or on OS, although use of cisplatin has a tendency to improve overall survival in 3 months over the use of carboplatin. Our data are similar to the reported about Veterans Health Administration data [12]. The use of gemcitabine as platinum doublet has demonstrated increased survival in our patients over microtubule inhibitors (9,45 months versus 4,7 months). A phase III trial of cisplatin and pemetrexed compared with cisplatin and gemcitabine revealed a statistically significant improvement in OS with cisplatin and gemcitabine in patients with squamous cell histology (10.8 vs 9.4 months) [13,14]. A retrospective study in patients with advanced lung SCC, revealed the use of various regimens did not have a significant effect on survival outcomes [15].

Finally, 31 patients (37,8%) received second line of treatment being docetaxel the most common agent used in this setting followed by paclitaxel and erlotinib. Although the number of patients is small, there is an improvement in survival in the group treated with taxanes respect to the group treated with tyrosine kinase inhibitor TKI). In TITAN phase III study, [16] patients were randomized to receive TKI or CT (taxanes or pemetrexed) and both treatments were similar in efficacy with different toxicity profiles.

We conclude that gemcitabine must be the CT of choice in advanced SCC. On the other hand, female patients had worse prognosis, patients aged >70 years old must receive a double of platinum, cisplatin is similar to carboplatin and cisplatin must be used when there is no contraindication for its use. Aditional agents and strategies must be developed in this setting to improve quality of life and survival.

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DOI: 10.31038/CST.2017211

Letter to the Editor

A thoughtful editorial previously published in Cancer Studies and Therapeutics pondered the question of “What is the Main Cause of Cancer?’ [1]. Certainly there are no simple answers.

Perhaps a study by Poutahidis et al (2015) [2] provides some clues to this ‘What Causes Cancer?’ enigma. Their studies in animal models revealed multigenerational cancer phenomena that were transplantable using fecal microbiota alone. These findings raise the possibility that disrupted microbiota, arising from societal practices such as refined diets or antibiotics during earlier generations, may have carcinogenic consequences in subsequent generations. The authors postulated that detrimental microbial effects in utero and during infancy lead to a dysregulated host immune system featuring premature thymic involution, possibly via epigenetic mechanisms. Under these immune-suppressed conditions, future infant mucosal surfaces become more permeable to environmental threats including sepsis [3]. Extrapolating across generations, microbiota may function as part of a quorum sensing mechanism ultimately influencing host immune and hormonal homeostasis, thus altering cancer susceptibility of progeny animals [4]. In those studies, grandchildren of mice consuming ‘fast food’ diets were at high risk to develop cancer at a young age, even without other predisposing genetic or environmental risks.

These intriguing data are supported by other findings suggesting that bacteria should be on our ‘What Causes Cancer?’ radar screen [5-7]. Firstly, direct evidence exists in humans with Helicobacter pylori infection and inflammation-associated gastric cancer [8]. Likewise, in the lower bowel, infection with a related microbe H. hepaticus leads to inflammation-associated colon and mammary tumors in mice [9, 10]. Further, certain pathogenic Escherichia coli organisms are shown to cause DNA damage in gut epithelia [6], and even to invade the bloodstream and extra-intestinal tissues. Indeed, E. coli has been implicated in mastitis and breast cancer in women [11], whereas Lactobacillus sp apparently inhibits mammary cancer development [12]. This raises the possibility that certain microbiota serve as invisible mutagens or guardians that help to explain the enigma.

And there’s more. Many studies have now shown that cancer-fighting capacity of our immune system can be mobilized or inhibited by our gut bacteria [10, 13-15]. Animal model systems mimicking complex cancer processes in human subjects reveal that microbes indirectly modulate tissue injury repair capacity and risk for tumor development and progression [3, 7, 9, 13-17]. For example, Poutahidis et al (2013) found that microbe therapy in mice led to proficient wound repair occurring twice-as-fast as in untreated controls [16]. Another study by Varian et al (2016) showed that microbe monotherapy was sufficient to increase thymus gland size, inhibit intestinal polyp formation, and increase lifespan in mouse models [18]. The proposed immune mechanisms involved microbial up-regulation of transcription factor Forkhead box protein N1 (FoxN1), the protein that is entirely lacking in athymic nude mice rendering them without T lymphocytes and as a result highly permissive to cancer growth [18, 19].

This leads us back to the original question of ‘What causes Cancer?’. The original author posited that for a theory of to be widely accepted, the answer should explain the striking differences in cancer risk by age and among tissues [1]. We should at least consider the possibility that our modernized lifestyle practices using antibiotics, Caesarian births, and refined diets have depleted valuable diversity and beneficial organisms in our microbiome with carcinogenic consequences to future generations. After all, oral supplementation with a model organism Lactobacillus reuteri, once believed to be widespread in humans but now dwindling to <4% of people worldwide [4], was sufficient to rescue multigenerational health impairments in infant mice [2, 20].

Further research is needed to better understand the roles of microbiota among the many possibilities for “What is the Main Cause of Cancer?’. However, based on existing data, opportunities abound for engineering diets and microbe cocktails to reinforce host balance and extinguish cancer for generations to come.

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