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Clinical Cancer Research 14, 1059, February 15, 2008. doi: 10.1158/1078-0432.CCR-07-1513
© 2008 American Association for Cancer Research
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Imaging, Diagnosis, Prognosis

Thymidylate Synthase Expression in Gastroenteropancreatic and Pulmonary Neuroendocrine Tumors

Paolo Ceppi1, Marco Volante2, Anna Ferrero3, Luisella Righi2, Ida Rapa2, Rosj Rosas2, Alfredo Berruti3, Luigi Dogliotti3, Giorgio V. Scagliotti1 and Mauro Papotti2

Authors' Affiliations: 1 Thoracic Oncology Unit, 2 Pathology Division, and 3 Division of Medical Oncology, Department of Clinical and Biological Sciences, University of Torino, San Luigi Hospital, Torino, Italy

Requests for reprints: Paolo Ceppi, Department of Clinical and Biological Sciences, University of Turin, San Luigi Hospital Regione Gonzole 10, 10043 Orbassano, Torino, Italy. Phone: 39-011-9026644; Fax: 39-011-9026753; E-mail: paolo.ceppi{at}unito.it.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The predictive role of the quantification of thymidylate synthase (TS) in tumors treated with antifolate drugs, such as 5-fluorouracil (5-FU), has been extensively reported in a variety of human tumors. Neuroendocrine tumors (NET) represent potential targets of antifolate agents, but no data on TS expression level in these tumors are currently available.

Experimental Design: A series of 116 NETs were collected, including 58 gastroenteropancreatic (GEP) and 58 lung NETs. In 24 well-differentiated GEP neuroendocrine carcinomas (WD-NEC), a 5-FU–based treatment was given. Total RNA was extracted from microdissected paraffin blocks. TS mRNA quantification was done by real-time PCR, whereas protein expression was evaluated by immunohistochemistry.

Results: By means of both quantification by real-time PCR and immunohistochemistry, a higher TS expression in pulmonary small cell lung cancer and large cell NEC compared with typical and atypical carcinoids was observed (P < 0.01). Similarly, in GEP tumors, a higher TS expression in poorly differentiated carcinomas than both WD-NEC and benign tumors (P < 0.01) was found. In patients with WD-NEC treated with 5-FU, high TS mRNA levels were associated with shorter time to progression (P = 0.002) and overall survival (P = 0.04). This negative prognostic role was confirmed in multivariate analysis adjusting for major prognostic variables (P = 0.01). No association between TS mRNA and survival was observed in WD-NEC patients not receiving 5-FU.

Conclusions: This study, for the first time, (a) reports the differential TS expression in the spectrum of NETs and (b) indicates TS as a possible predictive marker of treatment efficacy in WD-NEC patients treated with 5-FU.

Thymidylate synthase (TS) is an enzyme that plays an important role in cellular proliferation and growth (1), catalyzing the methylation of fluorodUMP to dTMP, an essential precursor for DNA synthesis (2). 5-Fluorouracil (5-FU) is an anticancer agent active in several types of cancer through TS inhibition and the block of DNA synthesis (3). Previously, several studies investigated the level of TS expression, mostly testing transcript quantification by real-time PCR, and higher TS levels were associated with poor prognosis and/or an adverse response to 5-FU treatment in esophageal (4), breast (5), head and neck (6), bladder (7), and non–small cell lung cancer (8), although conflicting results have been reported in colorectal carcinoma (9, 10).

Neuroendocrine tumors (NET) are a rare and heterogeneous group of tumors with specific biological, histopathologic, and clinical features (11–13). NETs of the gastroenteropancreatic (GEP) system are classified according to WHO (14) as follows: well-differentiated (WD) NETs with benign behavior, WD neuroendocrine carcinomas (NEC) with low-grade malignancy, and poorly differentiated (PD) NECs (PD-NEC) with a high grade of aggressiveness and poor prognosis. The classification of pulmonary NETs differentiates typical carcinoid (TC) and atypical carcinoid (AC) and PD-NECs of the small–cell lung cancer (SCLC) and large–cell NEC (LCNEC) types (15–17). Both classifications identify a benign group of tumors (WD-NET and TC) and a highly malignant counterpart (SCLC, LCNEC, and PD-NEC), being low-grade malignant tumors (atypical carcinoid, WD-NEC) in an intermediate position. In WD-NEC, surgical resection is the preferred treatment, but in the case of a neoplastic spread outside the primary site or recurrence after surgery, no standard systemic treatment is currently recommended. Therapy with IFN-{alpha} or somatostatin analogues, such as octreotide, have been used as a first-line treatment, but they are generally associated to a low response rate (18–20). Treatment of NETs with 5-FU has been proposed with promising results (21–25), although no data are currently available on the expression of TS in NETs to support antifolate drug based strategies.

This study aimed at testing TS expression levels in a large series of GEP and pulmonary NETs by means of quantification by real-time PCR and immunohistochemistry on paraffin-embedded specimens. In addition, the potential prognostic or predictive role of TS expression in NETs was investigated.


   Materials and Methods
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Patients and samples. One hundred and sixteen paraffin-embedded surgical or biopsy specimens were collected (from the pathology files of the University of Turin Medical School) from patients affected by NETs of GEP (n = 58) or lung (n = 58) origin from 1992 to 2006. These included 50 WD (6 benign and 44 low grade malignant) and 8 PD GEP tumors, as well as 5 pulmonary TCs, 17 ACs, 16 LCNECs, and 20 SCLCs. Patients characteristics are summarized in Table 1 . Overall, 32 of 116 (28%) patient samples were obtained from metastatic sites. Tumors of the GEP tract were from pancreas (n = 18), intestine (n = 29), liver (n = 2), stomach (n = 2), and of occult origin (n = 7). Among the malignant cases (n = 105), 24 patients (20%) with advanced GEP WD-NEC were prospectively enrolled in a trial which investigated the administration of octreotide and 5-FU protracted continuous infusion (25). In this subset of patients, two had unresectable locally advanced disease and 22 had metastatic disease, of which 18 showed the presence of liver metastases (82%).


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  		Table 1. Characteristics of the 116 patients

 
In the remaining 81 patients, 11 (13%; four GEP WD-NECs, three ACs, and four LCNECs of the lung) had no available outcome information and were excluded from the survival analysis. The patients considered for survival analysis (n = 70) were grouped as WD-NEC (n = 30) and PD-NEC (n = 40) independently of tumor site and were treated with surgery, somatostatin analogues, or chemotherapy (not 5-FU based) when indicated according to clinical protocols. Clinicopathologic data included age, sex, tumor location, treatment, follow-up, and, when available, performance status, response to therapy and time to progression. The study was approved by the institutional review board of the hospital. All histologic samples were reviewed by two pathologists (M.V. and L.R.). Both tissues and clinical data were deidentified and coded by a staff member not taking part in the molecular analysis; none of the researchers conducting gene and protein expression analyses had access to disclosed clinicopathologic data.

Immunohistochemistry in paraffin-embedded tissues. From each paraffin block, 5-µm-thick sections were obtained and stained with H&E for conventional histologic examination. In addition, serial sections collected on charged slides were processed for immunohistochemical staining. Briefly, after deparaffinization and rehydration through graded alcohols and phosphate-buffer saline at pH 7.5, endogenous peroxidase activity was blocked by methanol and 0.3% hydrogen peroxide for 15 min. For antigen retrieval, the sections were treated in a microwave oven for 15 min in EDTA buffer (pH 8.0). The slides were then incubated for 40 min at room temperature with the primary mouse anti-TS antibody (clone TS106, dilution 1:100; Zymed). The immune reaction was revealed with a biotin-free detection system based on a dextran chain-linked to the secondary antibody and peroxidase (En Vision, Dako Cytomation), using 3,3'-diaminobenzidine (Dako) as chromogen. Slides were counterstained with hematoxylin, dehydrated, and mounted. A colorectal carcinoma specimen was included as a positive control, whereas negative controls were obtained omitting the primary antibody. For statistical analyses, the immune reaction was considered as positive when present in >5% of the tumor cell population.

Microdissection, RNA isolation, and cDNA synthesis. From each paraffin block of representative tumor areas, serial sections with a thickness of 10 µm were prepared and stained with nuclear Fast Red (Sigma-Aldrich). Malignant cells were selected under microscope magnification (50x to 100x) and dissected from the slide using a scalpel. RNA isolation was done as previously described (26). In brief, tissue samples were heated at 92°C for 30 min in 4 mol/L DTT-GITC/sarcosine [4 mol/L guanidinium isothiocyanate, 50 mmol/L Tris-HCl (pH 7.5), 25 mmol/L EDTA; Invitrogen]. Fifty microliters of 2 mol/L sodium acetate (pH 4.0) followed by 600 µL of freshly prepared phenol/chloroform/isoamyl alcohol (250:50:1) were added to the tissue suspensions. The suspension was centrifuged at 13,000 rpm for 8 min in a chilled (8°C) centrifuge. The upper aqueous phase was removed and combined with glycogen (10 µL) and 300 to 400 µL of isopropanol. The tubes were placed at –20°C for 30 to 45 min to precipitate the RNA. After centrifugation at 13,000 rpm for 7 min in a chilled (8°C) centrifuge, the supernatant was carefully poured off, the pellet was resuspended in 50 µL of 5 mmol/L Tris, and the cDNA synthesis was done as previously described (27).

Real-time PCR analysis. Relative cDNA quantification of TS and β-actin (internal reference gene) was done using a fluorescence-based real-time detection method, as previously described (28). Each measurement was done in duplicate, and the comparative Ct method was used. To further normalize across samples, the highest {Delta}Ct value was subtracted from each {Delta}Ct to give the {Delta}{Delta}Ct values. These values were converted to relative expression levels by the following formula: 2–{Delta}{Delta}Ct (29). The sequences of the primers and probe used were as follows (30): TS forward 5'-GGCCTCGGTGTGCCTTT-3', reverse 5'-GATGTGCGCAATCATGTACGT-3', probe (FAM)-5'-AACATCGCCAGCTACGCCCTGC-3'-(TAMRA); β-actin forward 5'-TGAGCGCGGCTACAGCTT-3', reverse 5'-TCCTTAATGTCACGCACGATTT-3', probe (FAM)-5'-ACCACCACGGCCGAGCGG-3'-(TAMRA). The PCR reaction mixture consisted of 1,200 nmol/L of each primer, 200 nmol/L probe, 200 nmol/L each of dATP, dCTP, dGTP, dTTP, 3.5 mmol/L MgCl2, and 1x Taqman Universal PCR Master Mix to a final volume of 20 µL (all reagents were from PE Applied Biosystems). Cycling conditions were 50°C for 2 min, 95°C for 10 min, followed by 46 cycles at 95°C for 15 s and 60°C for 1 min.

Data analysis. To test differential TS protein expression among different histopathologic groups, the {chi}2 for trend test was used. The Mann-Whitney U and Kruskall-Wallis tests were used to verify differential TS mRNA levels between groups. The Ki67 score of the 5-FU–treated patients was previously assessed in a clinical study recently presented (25). For the purpose of a correlation analysis with TS expression, a Spearmans' test was used. In the survival analysis, tumors were divided into groups according to tertiles of mRNA expression levels. First and second tertiles of TS distribution were grouped as low TS and compared with the third tertile (high TS). Survival analysis was done by Kaplan-Meier curves, and the significance was verified by log-rank test. Univariate and multivariate analysis of time to progression were carried out by means of the Cox proportional hazard model. All analyses were done using the statistical PC software package. The level of significance was set at P < 0.05.


   Results
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Discussion
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TS protein expression in NETs. TS immunohistochemistry staining in paraffin-embedded tumor sections was found positive in 50 of the 116 samples (43%), using 5% of positive cells as a cutoff value. According to different histopathologic categories, TS protein expression progressively increased from WD-NET to WD-NEC and PD-NEC (P < 0.0002). TS immunohistochemistry was present in only 5 of 44 WD-NEC (11%). In lung NETs, a significantly higher rate of TS expression in SCLC and LCNEC compared with TC and AC (P < 0.0001, all {chi}2 tests for trend) was observed. As a source of potential pitfall, no differences in the TS immunohistochemical distribution were observed comparing tumor samples obtained from small biopsies or surgical specimens. The results are summarized in Table 1. Figure 1 shows TS immunostaining in WD-NEC and PD-NEC. In the subgroup of 24 patients treated with 5-FU + long acting octreotide, we also investigated the correlation between Ki67 score, which was assessed previously to enroll patients in the clinical trial (25), and TS expression, and the results showed that the two markers were independently expressed (Rs = 0.04, P = 0.85).


Figure 1
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  		Fig. 1. Scattered TS immunoreactive cells are evident in a pancreatic WD-NEC (A), whereas an intense cytoplasmic and nuclear staining is present in a duodenal PD-NEC (B). Scattered positive lymphocytes (bottom right) serve as internal positive control (A and B immunoperoxidase, 100x).

 
Quantification of TS mRNA levels in NETs. TS expression level (ranging from 1 to 52.5, unit less ratios) was measured with the relative {Delta}Ct method from all the investigated samples. Table 1 shows the relative TS mRNA levels according to different histologic tumor types. Consistently with immunohistochemical data, TS mRNA transcripts were significantly higher among PD-NEC of the GEP system compared with WD-NEC and WD-NET (P = 0.002) and, among lung tumors, in SCLC and LCNEC compared with pulmonary TC and AC (P < 0.001, all Kruskall-Wallis tests). No significant differences were found comparing samples obtained from primary tumors (84 cases) and from metastases (32 cases), as well as among GEP tumors of different locations, in terms of TS expression. TS mRNA expression levels in GEP and pulmonary NETs are shown in Fig. 2A and B , respectively.


Figure 2
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  		Fig. 2. TS mRNA levels in GEP (A) and pulmonary (B) NETs. The line in the middle represents the median values. The boxes represent the distance between the 25th and 75th percentiles. The whiskers represent the upper and lower values. All P values are Mann-Withney U tests.

 
Prognostic and predictive significance of TS mRNA levels in NETs. Because of the lower rate of positive cases detected by immunohistochemistry in the group of WD tumors, TS transcript quantification was selected for the survival analysis. Patients were split by adopting cutoff values according to tertiles of distribution of TS mRNA expression, and patients in first and second tertile (low TS) were compared with those in the third one (high TS).

In the subset of malignant cases not treated with 5-FU (n = 70), survival analysis revealed that TS levels were not correlated with survival once the tumors were grouped by histologic subtypes (P > 0.05) or grade of differentiation. In the latter case, Kaplan Meier curves did not show significant statistical differences neither in WD (n = 30, P = 0.55; Fig. 3A ) nor in PD carcinomas (n = 40, P = 0.86; Fig. 3B) irrespective of their GEP or pulmonary origin.


Figure 3
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  		Fig. 3. Kaplan-Maier univariate analysis of survival of WD-GEP and AC (A) and PD-GEP, SCLC and LCNEC (B) NEC patients not treated with 5-FU according to TS mRNA expression levels. Patients with low TS are those in the first and second tertiles of distribution of mRNA expression levels, whereas those with high TS are in the third tertile. P values are log-rank tests.

 
In WD-NECs (n = 24) homogeneously treated with somatostatin analogue and continuous 5-FU infusion, the median time to progression was 20.4 months, whereas the median overall survival was not reached. Univariate analysis showed that 5-FU–treated patients with higher TS levels (third tertile, n = 8) had worse outcome in terms of time to progression (P = 0.002; Fig. 4A ) and overall survival (P = 0.04; Fig. 4B) than those with lower levels (first and second tertiles, n = 16). None of the other prognostic considered variables (age, gender, performance status, site of primary disease, and response to therapy) were significantly correlated with survival, except for the Ki67 score (5% cutoff value), as reported elsewhere (25). In multivariate analysis, both TS (hazard ratio, 4.81; 95% confidence interval, 1.45-7.67; P = 0.01) and Ki67 (hazard ratio, 4.80; 95% confidence interval, 1.14-7.97; P = 0.03) were found to be independent prognostic markers (Table 2 ).


Figure 4
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  		Fig. 4. Kaplan-Maier univariate analysis of survival (A) and time to progression (B) of patients affected by GEP WD-NEC treated with 5-FU compared with TS transcript levels. P values are log-rank tests.

 

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  		Table 2. Cox univariate and multivariate analysis of time to progression of 5-FU–treated patients

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
This is the first report in which TS expression level and its prognostic role were analyzed in a large series of NETs. Both quantification by real-time PCR and immunohistochemistry detected a significantly higher expression of TS in PD-NECs compared with WD-NETs or carcinomas both of GEP and pulmonary origin (all P < 0.01). According to these results, TS expression at mRNA and protein level seems to be strictly correlated with tumor differentiation, this being higher in PD tumors of each group. Because the efficacy of TS-inhibiting drugs, such as 5-FU, was shown to be inversely correlated with TS expression (31), the results of this study could provide further insights on the limited activity of 5-FU in PD-NEC (32, 33).

In addition, this study contributes to define the prognostic effect of TS in NETs by analyzing a potential relationship between expression levels and survival in TS-inhibiting drug naive patients. In untreated colorectal cancer patients, the association between TS levels and survival had already been shown (34, 35). However, in the malignant cases not treated with 5-FU (n = 70) of the present study, TS transcript levels in NECs grouped by histologic subtypes or grade of differentiation failed to correlate with survival (all P > 0.05). With regard to the predictive value of TS expression, 24 patients with metastatic GEP NECs, included in a trial testing the administration of octreotide and 5-FU, were separately considered for survival analysis. Interestingly, a strong correlation between TS levels and time to progression (P = 0.002), as well as overall survival (P = 0.04), was found. In an attempt to explain the predictive value of TS, its expression was compared with cell proliferation, as assessed by Ki67 immunostaining, which was previously shown to be correlated with prognosis (25); however, by means of Spearmans' correlation method, no significant association between Ki67 and TS was found (P = 0.85). Interestingly, both markers were correlated with time to progression in the univariate and retained in the multivariate analysis (TS, P = 0.01 and Ki67, P = 0.03) and showed that they were two independent factors affecting the outcome of 5-FU–treated patients. Conversely, response to therapy was not correlated to prognosis (Table 2), and this could be related to the general indolence of this type of tumors. These data indicate that TS may be a predictive, rather than a prognostic marker in NECs, information possibly useful in the pharmacogenomic selection of patients to be treated with 5-FU and with other antifolate agents. The patients in the third tertile of TS expression (the highest) are, in fact, those more likely to develop drug resistance, whereas no difference was consistently found among patients within the first and second tertiles in terms of clinical outcome. The PD highly malignant NETs are not usually treated with antifolate drugs, whereas 5-FU has been shown some efficacy in WD-NEC of the GEP tract and in pulmonary carcinoid tumors (21–25). Therefore, the result of this study could provide the rationale for the administration of 5-FU in the subset of WD tumors only.

In conclusion, this work for the first time (a) reports a differential TS expression in the spectrum of NETs and (b) indicates the possible predictive role of TS expression levels in NEC patients treated with 5-FU–based therapy. This study adds novel insights which might open the line of future prospective trials based on 5-FU administration in patients with WD-NETs.


   Footnotes
 
Grant support: Italian Ministry of University and Research (Rome; ex 60% to M. Papotti, M. Volante, and G.V. Scagliotti) and Regione Piemonte (Turin) research grant 2006 (G.V. Scagliotti).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/22/07; revised 9/18/07; accepted 10/19/07.


   References
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 References
 

  1. Navalgund LG, Rossana C, Muench AJ, Johnson LF. Cell cycle regulation of thymidylate synthetase gene expression in cultured mouse fibroblasts. J Biol Chem 1980;255:7386–90.[Abstract/Free Full Text]
  2. Friedkin M, Crawford EJ, Donovan E, Pastore EJ. The enzymatic synthesis of thymidylate: III. The further purification of thymidylate synthetase and its separation from natural fluorescent inhibitors. J Biol Chem 1962;237:3811–4.[Free Full Text]
  3. Spears CP, Gustavsson BG, Mitchell MS, et al. Thymidylate synthetase inhibition in malignant tumors and normal liver of patients given intravenous 5-fluorouracil. Cancer Res 1984;44:4144–50.[Abstract/Free Full Text]
  4. Harpole DH, Moore MB, Herndon JE, et al. The prognostic value of molecular marker analysis in patients treated with trimodality therapy for esophageal cancer. Clin Cancer Res 2001;7:562–9.[Abstract/Free Full Text]
  5. Pestalozzi BC, Peterson HF, Gelber RD, et al. Prognostic importance of thymidylate synthase expression in early breast cancer. J Clin Oncol 1997;15:1923–31.[Abstract/Free Full Text]
  6. Johnston PG, Mick R, Recant W, et al. Thymidylate synthase expression and response to neoadjuvant chemotherapy in patients with advanced head and neck cancer. J Natl Cancer Inst 1997;89:308–13.[Abstract/Free Full Text]
  7. Mizutani Y, Wada H, Ogawa O, et al. Prognostic significance of thymidylate synthase activity in bladder carcinoma. Cancer 2001;92:510–8.[CrossRef][Medline]
  8. Huang CL, Yokomise H, Kobayashi S, Fukushima M, Hitomi S, Wada H. Intratumoral expression of thymidylate synthase and dihydropyrimidine dehydrogenase in non-small cell lung cancer patients treated with 5-FU-based chemotherapy. Int J Oncol 2000;17:47–54.[Medline]
  9. Johnston PG, Lenz HJ, Leichman CG, et al. Thymidylate synthase gene and protein expression correlate and are associated with response to 5-fluorouracil in human colorectal and gastric tumors. Cancer Res 1995;55:1407–12.[Abstract/Free Full Text]
  10. Allegra CJ, Parr AL, Wold LE, et al. Investigation of the prognostic and predictive value of thymidylate synthase, p53, and Ki-67 in patients with locally advanced colon cancer. J Clin Oncol 2002;20:1735–43.[Abstract/Free Full Text]
  11. Livolsi VA, Asa SL. Endocrine pathology. New York: Churchill Livingstone; 2002.
  12. Wick MR. Neuroendocrine neoplasia. Current concepts. Am J Clin Pathol 2000;113:331–5.[Free Full Text]
  13. Couvelard A, O'Toole D, Turley H, et al. Microvascular density and hypoxia-inducible factor pathway in pancreatic endocrine tumours: negative correlation of microvascular density and VEGF expression with tumour progression. Br J Cancer 2005;92:94–101.[CrossRef][Medline]
  14. Solcia E, Kloppel G, Sobin LH, Williams ED; World Health Organization. Histological typing of endocrine tumors. 2nd ed. Springer; 2000.
  15. Travis WD, Gal AA, Colby TV, Klimstra DS, Falk R, Koss MN. Reproducibility of neuroendocrine lung tumor classification. Hum Pathol 1998;29:272–9.[CrossRef][Medline]
  16. Travis WD, Brambilla E, Muller-Hermelink HK, et al. Pathology and genetics of tumors of the lung, pleura, thymus and heart. Lyon: IARC Press; 2004.
  17. Brambilla E, Travis WD, Colby TV, Corrin B, Shimosato Y. The new World Health Organization classification of lung tumours. Eur Respir J 2001;18:1059–68.[Abstract/Free Full Text]
  18. Wymenga AN, Eriksson B, Salmela PI, et al. Efficacy and safety of prolonged-release lanreotide in patients with gastrointestinal neuroendocrine tumors and hormone-related symptoms. J Clin Oncol 1999;17:1111.[Abstract/Free Full Text]
  19. De Herder WW, Hofland LJ, Van der Lely AJ, Lamberts SW. Somatostatin receptors in gastroentero-pancreatic neuroendocrine tumours. Endocr Relat Cancer 2003;10:451–8.[Abstract]
  20. Fazio N, De Braud F, Delle Fave G, Oberg K. Interferon-{alpha} and somatostatin analog in patients with gastroenteropancreatic neuroendocrine carcinoma: single agent or combination? Ann Oncol 2007;18:13–9.[Abstract/Free Full Text]
  21. Bajetta E, Rimassa L, Carnaghi C, et al. 5-Fluorouracil, dacarbazine, and epirubicin in the treatment of patients with neuroendocrine tumors. Cancer 1998;83:372–8.[CrossRef][Medline]
  22. Kouvaraki MA, Ajani JA, Hoff P, et al. Fluorouracil, doxorubicin, and streptozocin in the treatment of patients with locally advanced and metastatic pancreatic endocrine carcinomas. J Clin Oncol 2004;22:4762–71.[Abstract/Free Full Text]
  23. Jensen RT, Niederle B, Mitry E, et al. Gastrinoma (duodenal and pancreatic). Neuroendocrinology 2006;84:173–82.[CrossRef][Medline]
  24. Wirth LJ, Carter MR, Janne PA, et al. Outcome of patients with pulmonary carcinoid tumors receiving chemotherapy or chemoradiotherapy. Lung Cancer 2004;44:213–20.[CrossRef][Medline]
  25. Dogliotti L, Brizzi MP, Ferrero A, et al Phase II trial of continuous/metronomic 5-fluorouracil infusion plus long acting Octreotide in advanced neuroendocrine carcinoma. J Clin Oncol 2007 ASCO Annu Meet Proc Part I. Vol 25, No. 18S (June 20 Supplement), 2007:15003.
  26. Lord RV, Salonga D, Danenberg KD, et al. Telomerase reverse transcriptase expression is increased early in the Barrett's metaplasia, dysplasia, adenocarcinoma sequence. J Gastrointest Surg 2000;4:135–42.[CrossRef][Medline]
  27. Kuramochi H, Hayashi K, Uchida K, et al. Vascular endothelial growth factor messenger RNA expression level is preserved in liver metastases compared with corresponding primary colorectal cancer. Clin Cancer Res 2006;12:29–33.[Abstract/Free Full Text]
  28. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996;6:986–94.[Abstract/Free Full Text]
  29. McGurk CJ, Cummings M, Koberle B, et al. Regulation of DNA repair gene expression in human cancer cell lines. J Cell Biochem 2006;97:1121–36.[CrossRef][Medline]
  30. Ceppi P, Volante M, Saviozzi S, et al. Squamous cell carcinoma of the lung compared with other histotypes shows higher messenger RNA and protein levels for thymidylate synthase. Cancer 2006;107:1589–96.[CrossRef][Medline]
  31. Leichman CG, Lenz HJ, Leichman L, et al. Quantitation of intratumoral thymidylate synthase expression predicts for disseminated colorectal cancer response and resistance to protracted-infusion fluorouracil and weekly leucovorin. J Clin Oncol 1997;15:3223–9.[Abstract]
  32. Havsteen H, Sorenson S, Rorth M, Dombernowsky P, Hansen HH. 5-FU in the treatment of small cell anaplastic carcinoma of the lung: a phase II trial. Cancer Treat Rep 1981;65:123–5.[Medline]
  33. Bajetta E, Catena L, Procopio G, et al. Are capecitabine and oxaliplatin (XELOX) suitable treatments for progressing low-grade and high-grade neuroendocrine tumours? Cancer Chemother Pharmacol 2007;59:637–42.[CrossRef][Medline]
  34. Van Triest B, Pinedo HM, Blaauwgeers JL, et al. Prognostic role of thymidylate synthase, thymidine phosphorylase/platelet-derived endothelial cell growth factor, and proliferation markers in colorectal cancer. Clin Cancer Res 2000;6:1063–72.[Abstract/Free Full Text]
  35. Lenz HJ, Danenberg KD, Leichman CG, et al. P53 and thymidylate synthase expression in untreated stage II colon cancer: associations with recurrence, survival, and site. Clin Cancer Res 1998;4:1227–34.[Abstract]



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Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online