Molecular Diagnostics | Open | Published:

# Quantitative expression analysis and prognostic significance of L-DOPA decarboxylase in colorectal adenocarcinoma

British Journal of Cancer volume 102, pages 13841390 (27 April 2010) | Download Citation

## Abstract

### Background:

L-DOPA decarboxylase (DDC) is an enzyme that catalyses, mainly, the decarboxylation of L-DOPA to dopamine and was found to be involved in many malignancies. The aim of this study was to investigate the mRNA expression levels of the DDC gene and to evaluate its clinical utility in tissues with colorectal adenocarcinoma.

### Methods:

Total RNA was isolated from colorectal adenocarcinoma tissues of 95 patients. After having tested RNA quality, we prepared cDNA by reverse transcription. Highly sensitive quantitative real-time PCR method for DDC mRNA quantification was developed using the SYBR Green chemistry. GAPDH served as a housekeeping gene. Relative quantification analysis was performed using the comparative CT method $(2-ΔΔCT)$.

### Results:

DDC mRNA expression varied remarkably among colorectal tumours examined in this study. High DDC mRNA expression levels were found in well-differentiated and Dukes’ stage A and B tumours. Kaplan–Meier survival curves showed that patients with DDC-positive tumours have significantly longer disease-free survival (P=0.009) and overall survival (P=0.027). In Cox regression analysis of the entire cohort of patients, negative DDC proved to be a significant predictor of reduced disease-free (P=0.021) and overall survival (P=0.047).

### Conclusions:

The results of the study suggest that DDC mRNA expression may be regarded as a novel potential tissue biomarker in colorectal adenocarcinoma.

## Main

Colorectal carcinoma (CRC) is the third most common malignant tumour and the fourth most common cause of cancer death in the world (Parkin et al, 2001a; Jemal et al, 2009). The prognosis of patients with CRC largely depends on the degree of penetration of the tumour through the bowel wall, the nodal status, and the presence or absence of distal metastases (Steinberg et al, 1986). These three characteristics are the key features of all clinical staging systems developed for this disease (Compton and Greene, 2004).

In spite of the fact that clinicopathological staging separates patients with CRC into groups with distinct outcomes, it provides little information about response to treatment in individual patients (Walther et al, 2009). In an attempt to refine prognostication and predict the benefit derived from systemic treatment, several protein and genetic markers have been evaluated in patients with CRC, including allelic loss of chromosome 18q (Jen et al, 1994; Martinez-Lopez et al, 1998; Ogunbiyi et al, 1998; Popat and Houlston, 2005), absence of the deleted in colorectal carcinoma (DCC) protein (Shibata et al, 1996; Reymond et al, 1998; Popat and Houlston, 2005), decreased SMAD4 mRNA expression (Boulay et al, 2002; Alazzouzi et al, 2005), expression and/or abnormalities of cytoplasmic oncoprotein p53 (TP53) (Sun et al, 1992; Munro et al, 2005; Russo et al, 2005), protein levels and/or gene haplotype of thymidylate synthetase (TYMS) (Popat et al, 2004; Suh et al, 2005; Tsourouflis et al, 2008), microsatellite instability (MSI) (Gryfe et al, 2000; Popat et al, 2005), and chromosomal instability (CIN) (Walther et al, 2008). Nevertheless, none of these biomarkers has been prospectively validated and established so far in clinical practice. Hence, the identification of new, reliable prognostic and predictive biomarkers that will contribute utmost to clinical decision-making, remains an important research topic (Locker et al, 2006; Walther et al, 2009).

L-DOPA decarboxylase (DDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyses the decarboxylation of 3,4-dihydroxy-L-phenylalanine (L-DOPA) to dopamine (DA) and 5-hydroxy-L-tryptophan (S-HTP) to serotonin (5-HT) (Christenson et al, 1972). DDC is expressed in the central nervous system as well as in peripheral organs, such as the liver, kidney, pancreas and placenta (Lindstrom and Sehlin, 1983; Maneckjee and Baylin, 1983; Ichinose et al, 1989; Mappouras et al, 1990; Siaterli et al, 2003). Moreover, enzymatically active DDC was recently found in human leukocytes as well as in the histiocytic lymphoma cell line U-937, thus suggesting a cross-talk between the nervous and the immune systems and raising new questions about the regulatory role of DDC in immune responses (Kokkinou et al, 2009a, 2009b). Recently, two endogenous inhibitors of the enzymatic activity of DDC have been identified and purified, one from human serum and another from the membrane fraction of human placental tissue, yet their biological significance remains unexplored (Vassiliou et al, 2005, 2009).

The structure of the human DDC gene has been fully determined. The single-copy gene encoding DDC maps to chromosome 7p12.2, close to the epidermal growth factor receptor (EGFR) gene, and is composed of 15 exons spanning a genomic region of more than 85 kb (Ichinose et al, 1989; Sumi-Ichinose et al, 1992). Furthermore, two other DDC mRNA transcripts encoding distinct DDC protein isoforms as well as alternative splicing in 5′-untranslated region have been identified and characterised (Krieger et al, 1991; Ichinose et al, 1992; O’Malley et al, 1995; Vassilacopoulou et al, 2004).

It is worth mentioning that DDC is regarded as a general biomarker for neuroendocrine tumours (Gazdar et al, 1988; Gilbert et al, 1995; Ippolito et al, 2005). High DDC mRNA expression has been noticed in small-cell lung carcinoma (SCLC), neuroblastoma, and pheochromocytoma (Jensen et al, 1990; Gilbert et al, 1999; Uccella et al, 2006). Moreover, it has been suggested that DDC mRNA levels could be a potential biomarker for the detection of minimal residual disease in patients with neuroblastoma and for the discrimination of neuroblastoma from other small round-cell tumours of childhood (Gilbert et al, 1999; Bozzi et al, 2004).

Recent studies revealed that DDC is also implicated in prostate cancer neuroendocrine differentiation, accounting for abnormal prostate cell proliferation and differentiation (Wafa et al, 2007), as it is an androgen receptor (AR) coregulator protein acting at the cytoplasmic level to enhance AR activity and to differentially modulate AR-regulated genes (Wafa et al, 2003; Margiotti et al, 2007). Not surprisingly, DDC gene expression at the mRNA level has been proposed as a novel tissue biomarker in prostate cancer (Avgeris et al, 2008). DDC is also overexpressed in peritoneal dissemination of gastric carcinoma, and its quantification has been shown to be reliable and effective for the selection of patients for adjuvant intraperitoneal chemotherapy, aiming at preventing peritoneal recurrence (Sakakura et al, 2004). Still, the role of DDC in CRC remains unclear.

The above data encouraged us to analyse the DDC mRNA expression in colorectal adenocarcinoma specimens, developing an ultra-sensitive and highly accurate quantitative real-time PCR methodology using the SYBR Green chemistry, and to examine its potential prognostic significance and clinical application as a novel molecular tissue biomarker for colorectal adenocarcinoma.

## Materials and methods

### Tissue samples and RNA isolation

Included in this study were tumour specimens from 95 patients having undergone surgical treatment for primary colorectal adenocarcinoma between 2000 and 2003. The selection criteria for the specimens included the availability of sufficient tissue mass for RNA extraction and assay. Tumour tissues had been frozen in liquid nitrogen immediately after their surgical resection.

Tissue specimens were pulverised and then dissolved in TRI Reagent (Ambion (Europe) Ltd., Huntingdon, UK). Following the manufacturer's instructions, we extracted and diluted total RNA in an RNA Storage Solution (Ambion Ltd), and stored it at −80°C until use.

Patient age ranged from 35 to 88 years with a mean±s.e. of 67.3±1.01. Other patients’ characteristics and stage of tumours are shown in Tables 1,2,3. Follow-up information was available for 72 patients and included survival status (alive or deceased) and disease status (disease-free or recurrence/metastasis) along with the dates of the events and cause of death.

The study was performed with respect to the ethical standards of the 1975 Declaration of Helsinki Principles, as revised in 1996, and has been approved by the ethics committee of the University General Hospital ‘Attikon’.

### cDNA synthesis

First-strand cDNA was produced from total RNA by using an RNA PCR Kit Version 3.0 (TaKaRa Bio Inc., Tokyo, Japan), according to the manufacturer's instructions. The reaction mixture contained 2 μg of total RNA diluted in 11 μl of diethylpyrocarbonate (DEPC)-treated water, 2.5 pmol Oligo dT-Adaptor primer, 2 μl RT Buffer (10 × , 100 mM Tris-HCl (pH=8.3), 500 mM KCl), 4 μl 5 mM MgCl2, 2 μl 10 mM dNTP mix, 20 U RNase inhibitor (Rnase Inhibitor; 40 U μl−1; TaKaRa Bio Inc.) and 1.25 U reverse transcriptase (AMV Reverse Transcriptase XL; 5 U μl−1; TaKaRa Bio Inc.). The final reaction volume was 20 μl. The reaction mixture was incubated at 30°C for 10 min, 60°C for 30 min, and the reaction was terminated by heating the mixture at 99°C for 5 min and cooling it at 5°C for 5 min.

### Real-time quantitative PCR

Quantitative real-time PCR was performed using the SYBR Green chemistry in a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) (Figure 1A). On the basis of the information of the DDC and GAPDH cDNA sequences, two pairs of gene-specific primers were designed. The reaction mixture contained 50 ng of cDNA diluted in 2.5 μl of DEPC-treated water, 5 μl Power SYBR Green PCR Master Mix (2 × ) (Applied Biosystems), and 2 μl of gene-specific primers (final concentration, 50 nM each), in a final reaction volume of 10 μl. The DDC real-time PCR primers were 5′-GAACAGACTTAACGGGAGCCTTT-3′ and 5′-AATGCCGGTAGTCAGTGATAAGC-3′, producing a 90-bp PCR amplicon, and the GAPDH real-time PCR primers were 5′-ATGGGGAAGGTGAAGGTCG-3′ and 5′-GGGTCATTGATGGCAACAATATC-3′, resulting in a 107-bp PCR amplicon. The cycling conditions were as follow: a denaturation step at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 60 s, and a final step for the generation of a dissociation curve to distinguish between the main PCR product and primer-dimers (Figure 1B).

Calculations were made with the use of the comparative CT $(2-ΔΔCT)$ method. GAPDH was used as an internal control gene to normalise the PCRs for the amount of RNA added to the reverse transcription reactions, whereas the breast adenocarcinoma epithelial cell line MCF7 was used as a calibrator for making PCRs from distinct runs comparable (Livak and Schmittgen, 2001). ΔΔCT represents the difference between the mean ΔCT value of a colon sample and the mean ΔCT of the calibrator, both calculated after the same PCR run, whereas ΔCT is the difference between the threshold cycle (CT) of the target gene (DDC) and the CT of the endogenous reference gene (GAPDH) of the same sample.

Normalised results were expressed as the ratio of DDC mRNA copies to GAPDH mRNA copies calculated for each colon tissue sample in relation to the same ratio calculated for MCF7 cells. The normalised $(2-ΔΔCT)$ amounts of tissue sample DDC mRNA levels were then multiplied with the average ratio of DDC mRNA copies to GAPDH mRNA copies of MCF7 cells (2−13.384), calculated from the intercept of the regression line shown in Figure 1C, thus resulting in comparable results that do not depend on the DDC mRNA expression levels of MCF7 cells. Finally, these results were multiplied by 1000, thus yielding c/Kc (DDC mRNA copies per 1000 GAPDH mRNA copies). Each real-time PCR reaction was performed in triplicate to evaluate the reproducibility of data.

### Statistical analysis

The X-tile algorithm was used to generate an optimal cut-off point for DDC (Camp et al, 2004), as there are no established cut-off points concerning its expression in colorectal adenocarcinoma. Having corrected for the use of minimum P-value statistics, the X-tile software yielded an optimal cut-off of 12.82 c/Kc, equal to the 75th percentile, with a calculated Monte Carlo P-value <0.05. According to this cut-off, DDC mRNA expression was classified as positive or negative, and associations between DDC status and other qualitative clinicopathological parameters were analysed using the χ2-test or the Fisher's exact test, where appropriate.

Cox proportional hazard regression model was developed to assess the association between the prognostic markers and the relative risks for relapse and death of patients (Cox, 1972). Cox analysis was conducted at both univariate and multivariate levels. Only patients for whom the status of all variables was known were included in the multivariate regression models, which incorporated DDC mRNA expression and all other variables for which the patients were characterised. The multivariate models were adjusted for nodal status, histologic tumour grade, and Dukes’ stage (Dukes, 1932; Hamilton and Aaltonen, 2000).

Survival analyses were also performed by constructing Kaplan–Meier disease-free survival (DFS) and overall survival (OS) curves (Kaplan and Meier, 1958). The differences between the curves were evaluated by the log-rank test.

## Results

### Validation of the comparative CT$(2-ΔΔCT)$ method for DDC mRNA quantification

For the ΔΔCT calculation to be valid, the amplification efficiencies of the target and reference genes must be approximately equal (Livak and Schmittgen, 2001). For this purpose, the CT values for DDC and GAPDH, corresponding to the number of cycles at which the fluorescence emission monitored in real time reached a threshold of 10 times the standard deviation of the mean baseline emission from cycles 3 to 15 (Figure 1A) (Giulietti et al, 2001), were measured in serial dilutions of a control cDNA over a 100-fold range, and the ΔCT (namely the difference CT,DDC–CT,GAPDH) was plotted vs the log cDNA dilution. The absolute value of the slope of the resulting plot is close to zero, which implies similar amplification efficiencies of both amplicons (Figure 1C).

### DDC expression status in colorectal adenocarcinoma tissues and its association with patients’ clinicopathological variables

mRNA expression of DDC in colorectal adenocarcinoma tissues varied from 0.04 to 91.95 c/Kc (DDC mRNA copies per 1000 GAPDH mRNA copies) with a mean±s.e. of 9.02±1.51 (Table 1). Table 2 shows the association of DDC mRNA expression status of the tumour with various clinicopathological variables. DDC values were classified into two categories (positive or negative), as described in the Materials and Methods section. Out of 95 colon adenocarcinomas examined, 24 (25.3%) were classified as positive for DDC expression and 71 (74.7%) as negative. DDC positivity was found more frequently in well-differentiated tumours, whereas high-grade colorectal tumours were found to be DDC-negative (P=0.011). Significant associations between DDC status and patient age, nodal status, or Dukes’ stage were not observed.

### DDC expression status and colorectal adenocarcinoma survival

Complete follow-up information was available for 72 patients, among whom 27 (38%) had relapsed and 22 (31%) had died. In Cox univariate analysis, histologic tumour grade, and disease Dukes’ stage were significant predictors of DFS and OS, as expected. In addition to these established prognostic factors, DDC mRNA expression was found to be an important predictor of DFS and OS (P=0.021 and P=0.047, respectively). DDC-positive patients were found to have a significant lower risk to relapse (HR=0.18) or die (HR=0.23) (Table 3). The Kaplan–Meier survival curves also show that patients with DDC-positive tumours have remarkably longer DFS (P=0.009) and OS (P=0.027), compared to those with DDC-negative malignancies (Figure 2). In multivariate analysis, when all parameters were included in the Cox regression model, only Dukes’ stage retained its prognostic significance for DFS and OS of patients with colorectal adenocarcinoma (P=0.004 and P=0.021, respectively) (Table 3).

## Discussion

Cancer of the colon and rectum is an important public health issue, constituting a major cause of worldwide morbidity and mortality (Pisani et al, 2002; Jemal et al, 2009). In Europe, CRC is the second most common malignancy among women after breast cancer and the third most frequent in men after prostate and lung cancer. Moreover, CRC is the second most common cause of cancer death for both sexes, accounting for 12% of all tumour-related deaths (Parkin et al, 2001b; Ferlay et al, 2010). Early diagnosis of CRC and early detection of recurrence after surgery are critical for effective treatment and/or positive clinical outcome. Endoscopic examination of the colon remains the most reliable screening method for this type of malignancy (Newcomb et al, 1992; Lieberman et al, 2000).

All existing classification systems for CRC distinguish between patients with early-stage CRC and those with very advanced-stage disease; nonetheless, they are less efficient in predicting the prognosis of patients with intermediate levels of tumour burden (McLeod and Murray, 1999). On the basis of studies published over the last few years, the American Society of Clinical Oncology Tumour Marker Panel and the European Group on Tumour Markers have recently suggested that preoperative carcinoembryonic antigen (CEA) levels may be used as an independent prognostic factor, assisting in staging and surgical treatment planning. It should also be noted that CEA is the marker of choice for monitoring the response of metastatic disease to systemic therapy (Duffy et al, 2003; Locker et al, 2006). However, neither CEA nor any other biomarkers that have been proposed in the past, such as CA19-9, have enough sensitivity for colon cancer detection (van der Schouw et al, 1992; Pokorny et al, 2000; Locker et al, 2006).

L-DOPA decarboxylase is a PLP-dependent enzyme participating in the catecholamine biosynthesis pathway, responsible principally for the synthesis of the key neurotransmitters DA and 5-HT (Christenson et al, 1972). Biogenic amines are generally considered to participate in various processes, such as angiogenesis, cell proliferation, differentiation and apoptosis (Berry et al, 1996; Medina et al, 1999; Lang et al, 2005), which implies a potentially significant role of DDC in cancer pathobiology and progression. Interestingly enough, it has recently been shown that catecholamines, including DA itself, inhibit erythrocyte apoptosis by preventing scramblase activation and subsequent phosphatidylserine exposure on the cell membrane (Lang et al, 2005), which in turn triggers clearance of apoptotic cells by macrophages.

DDC mRNA expression is used for the differential diagnosis of neuroblastoma from other pediatric small round-cell malignancies (Gilbert et al, 1999; Bozzi et al, 2004). Quantification of DDC mRNA expression using real-time RT-PCR has been proposed as a method useful for the prediction of peritoneal recurrence in patients with gastric carcinoma (Sakakura et al, 2004). In addition, DDC protein expression is a biomarker of neuroendocrine differentiation in SCLC cells and prostate carcinoma (Gilbert et al, 2000; Wafa et al, 2007). DDC mRNA levels were also found to be particularly elevated in cancerous prostate tissue, in comparison with benign prostate hyperplasia. High-expression levels of DDC were found to be associated with more aggressive prostate tumours (Avgeris et al, 2008).

Several peripheral cancers are characterised by an extremely high DDC activity, associated with the tumour. This is especially apparent with lung cancers of small-cell origin, although variants showing no protein expression have been observed as well (Berry et al, 1996). Remarkable increase in DDC activity, in comparison with normal tissue levels, is also seen in primary intestinal cancer and its related metastases in the spleen and liver (Gilbert et al, 1995). The significance of this increase in DDC activity and resultant monoamine synthesis by the cancer cells is still unknown (Berry et al, 1996), yet it is closely related to the implication of DDC in cancer.

In this study, we investigated the expression of the DDC gene in colorectal adenocarcinoma and its prognostic significance. Our study revealed a statistically significant, negative association between DDC mRNA expression levels and the histologic tumour grade (P=0.011). Colorectal tumours of low histologic grade (I) were more frequently DDC-positive, in contrast with malignancies of high grade (II/III). These data seem to indicate that higher DDC expression is associated with well-differentiated intestinal tumours.

In accordance with the above-mentioned results, the Kaplan–Meier analysis showed significantly higher DFS and OS time for patients having positive DDC mRNA expression (P=0.009 and P=0.027, respectively). Cox univariate regression analysis showed that DDC-positive patients had a significantly lower risk of relapse (5 times) and a higher probability of survival (4 times). In the Cox multivariate regression model, the levels of DDC mRNA were adjusted for patients’ nodal status, Dukes’ stage and histologic tumour grade. When all these non-molecular parameters were included in the multivariate analysis model, DDC mRNA expression did not show statistically significant independence as a prognostic factor for DFS or OS of patients with colorectal adenocarcinoma.

In conclusion, this study revealed that higher mRNA expression levels of DDC are related with less advanced and/or aggressive tumours. Our results imply that DDC mRNA overexpression is linked to favorable prognosis in patients with colorectal adenocarcinoma and may constitute a useful tissue biomarker. Involvement of DDC in apoptosis of colon cancer cells and/or response of DDC-positive tumours to chemotherapy are two potential explanations, but further investigation is required to clarify the role of DDC in CRC.

## References

1. , , , , , , , , , (2005) SMAD4 as a prognostic marker in colorectal cancer. Clin Cancer Res 11: 2606–2611

2. , , , (2008) Expression analysis and clinical utility of L-Dopa decarboxylase (DDC) in prostate cancer. Clin Biochem 41: 1140–1149

3. , , , (1996) Aromatic L-amino acid decarboxylase: a neglected and misunderstood enzyme. Neurochem Res 21: 1075–1087

4. , , , , , , , , (2002) SMAD4 is a predictive marker for 5-fluorouracil-based chemotherapy in patients with colorectal cancer. Br J Cancer 87: 630–634

5. , , , , , , , , (2004) Molecular detection of dopamine decarboxylase expression by means of reverse transcriptase and polymerase chain reaction in bone marrow and peripheral blood: utility as a tumor marker for neuroblastoma. Diagn Mol Pathol 13: 135–143

6. , , (2004) X-tile: a new bio-informatics tool for biomarker assessment and outcome-based cut-point optimization. Clin Cancer Res 10: 7252–7259

7. , , (1972) On the identity of DOPA decarboxylase and 5-hydroxytryptophan decarboxylase (immunological titration-aromatic L-amino acid decarboxylase-serotonin-dopamine-norepinephrine). Proc Natl Acad Sci USA 69: 343–347

8. , (2004) The staging of colorectal cancer: 2004 and beyond. CA Cancer J Clin 54: 295–308

9. (1972) Regression models and life tables. R Stat Soc B 34: 187–202

10. , , , , , , , , (2003) Clinical utility of biochemical markers in colorectal cancer: European Group on Tumour Markers (EGTM) guidelines. Eur J Cancer 39: 718–727

11. (1932) The classification of cancer of the rectum. J Pathol Bacteriol 35: 323

12. , , (2010) Estimates of cancer incidence and mortality in Europe in 2008. Eur J Cancer 46: 765–781

13. , , , , , , , (1988) Expression of neuroendocrine cell markers L-Dopa decarboxylase, chromogranin A, and dense core granules in human tumors of endocrine and nonendocrine origin. Cancer Res 48: 4078–4082

14. , , , , (1999) Use of tumor-specific gene expression for the differential diagnosis of neuroblastoma from other pediatric small round-cell malignancies. Am J Pathol 155: 17–21

15. , , (1995) Elevated aromatic-L-amino acid decarboxylase in human carcinoid tumors. Biochem Pharmacol 50: 845–850

16. , , (2000) The aromatic-L-amino acid decarboxylase inhibitor carbidopa is selectively cytotoxic to human pulmonary carcinoid and small cell lung carcinoma cells. Clin Cancer Res 6: 4365–4372

17. , , , , , (2001) An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25: 386–401

18. , , , , , , , (2000) Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer. N Engl J Med 342: 69–77

19. , (eds) (2000) Pathology and Genetics. In Tumours of the Digestive System. IARC Press: Lyon

20. , , , , (1989) Isolation and characterization of a cDNA clone encoding human aromatic L-amino acid decarboxylase. Biochem Biophys Res Commun 164: 1024–1030

21. , , , , , (1992) Tissue-specific alternative splicing of the first exon generates two types of mRNAs in human aromatic L-amino acid decarboxylase. Biochemistry 31: 11546–11550

22. , , , , , , , (2005) An integrated functional genomics and metabolomics approach for defining poor prognosis in human neuroendocrine cancers. Proc Natl Acad Sci USA 102: 9901–9906

23. , , , , , (2009) Cancer statistics, 2009. CA Cancer J Clin 59: 225–249

24. , , , , , , , , (1994) Allelic loss of chromosome 18q and prognosis in colorectal cancer. N Engl J Med 331: 213–221

25. , , , , (1990) A comparison of synaptophysin, chromogranin, and L-Dopa decarboxylase as markers for neuroendocrine differentiation in lung cancer cell lines. Cancer Res 50: 6068–6074

26. , (1958) Nonparametric observation from incomplete observations. J Am Stat Assoc 53: 457–481

27. , , (2009a) The U937 macrophage cell line expresses enzymatically active L-Dopa decarboxylase. J Neuroimmunol 216: 51–58

28. , , , , (2009b) Expression of enzymatically active L-DOPA decarboxylase in human peripheral leukocytes. Blood Cells Mol Dis 42: 92–98

29. , , , (1991) Different mRNAs code for Dopa decarboxylase in tissues of neuronal and nonneuronal origin. Proc Natl Acad Sci USA 88: 2161–2165

30. , , , , , , , , , , , , (2005) Inhibition of erythrocyte ‘apoptosis’ by catecholamines. Naunyn Schmiedebergs Arch Pharmacol 372: 228–235

31. , , , , , (2000) Use of colonoscopy to screen asymptomatic adults for colorectal cancer. Veterans Affairs Cooperative Study Group 380. N Engl J Med 343: 162–168

32. , (1983) Mechanisms underlying the effects of 5-hydroxytryptamine and 5-hydroxytryptophan in pancreatic islets. A proposed role for L-aromatic amino acid decarboxylase. Endocrinology 112: 1524–1529

33. , (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25: 402–408

34. , , , , , , , , (2006) ASCO 2006 update of recommendations for the use of tumor markers in gastrointestinal cancer. J Clin Oncol 24: 5313–5327

35. , (1983) Use of radiolabeled monofluoromethyl-Dopa to define the subunit structure of human L-Dopa decarboxylase. Biochemistry 22: 6058–6063

36. , , (1990) Purification and characterization of L-Dopa decarboxylase from human kidney. Mol Cell Biochem 94: 147–156

37. , , , , , (2007) Androgen-regulated genes differentially modulated by the androgen receptor coactivator L-Dopa decarboxylase in human prostate cancer cells. Mol Cancer 6: 38

38. , , , , , , , , (1998) Allelic loss on chromosome 18q as a prognostic marker in stage II colorectal cancer. Gastroenterology 114: 1180–1187

39. , (1999) Tumour markers of prognosis in colorectal cancer. Br J Cancer 79: 191–203

40. , , , (1999) Histamine, polyamines, and cancer. Biochem Pharmacol 57: 1341–1344

41. , , (2005) P53 abnormalities and outcomes in colorectal cancer: a systematic review. Br J Cancer 92: 434–444

42. , , , , (1992) Screening sigmoidoscopy and colorectal cancer mortality. J Natl Cancer Inst 84: 1572–1575

43. , , , , (1995) The human aromatic L-amino acid decarboxylase gene can be alternatively spliced to generate unique protein isoforms. J Neurochem 65: 2409–2416

44. , , , , , , , , , (1998) Confirmation that chromosome 18q allelic loss in colon cancer is a prognostic indicator. J Clin Oncol 16: 427–433

45. , , , (2001a) Estimating the world cancer burden: Globocan 2000. Int J Cancer 94: 153–156

46. , , (2001b) Cancer burden in the year 2000. The global picture. Eur J Cancer 37(Suppl 8): S4–S66

47. , , (2002) Estimates of the world-wide prevalence of cancer for 25 sites in the adult population. Int J Cancer 97: 72–81

48. , , (2000) What's new with tumor markers for colorectal cancer? Dig Surg 17: 209–215

49. , (2005) A systematic review and meta-analysis of the relationship between chromosome 18q genotype, DCC status and colorectal cancer prognosis. Eur J Cancer 41: 2060–2070

50. , , (2005) Systematic review of microsatellite instability and colorectal cancer prognosis. J Clin Oncol 23: 609–618

51. , , (2004) Thymidylate synthase expression and prognosis in colorectal cancer: a systematic review and meta-analysis. J Clin Oncol 22: 529–536

52. , , , , , (1998) DCC protein as a predictor of distant metastases after curative surgery for rectal cancer. Dis Colon Rectum 41: 755–760

53. , , , , , (2005) The TP53 colorectal cancer international collaborative study on the prognostic and predictive significance of p53 mutation: influence of tumor site, type of mutation, and adjuvant treatment. J Clin Oncol 23: 7518–7528

54. , , , , , , , , , , , , , (2004) Overexpression of Dopa decarboxylase in peritoneal dissemination of gastric cancer and its potential as a novel marker for the detection of peritoneal micrometastases with real-time RT-PCR. Br J Cancer 90: 665–671

55. , , , , , , , , (1996) The DCC protein and prognosis in colorectal cancer. N Engl J Med 335: 1727–1732

56. , , (2003) Cloning and expression of human placental L-Dopa decarboxylase. Neurochem Res 28: 797–803

57. , , , (1986) Prognostic indicators of colon tumors. The Gastrointestinal Tumor Study Group experience. Cancer 57: 1866–1870

58. , , , , (2005) Thymidylate synthase gene polymorphism as a prognostic factor for colon cancer. J Gastrointest Surg 9: 336–342

59. , , , , (1992) Molecular cloning of genomic DNA and chromosomal assignment of the gene for human aromatic L-amino acid decarboxylase, the enzyme for catecholamine and serotonin biosynthesis. Biochemistry 31: 2229–2238

60. , , , , , , (1992) Prognostic significance of cytoplasmic p53 oncoprotein in colorectal adenocarcinoma. Lancet 340: 1369–1373

61. , , , , , (2008) Prognostic and predictive value of thymidylate synthase expression in colon cancer. Dig Dis Sci 53: 1289–1296

62. , , , , , , , , , (2006) Histidine decarboxylase, DOPA decarboxylase, and vesicular monoamine transporter 2 expression in neuroendocrine tumors: immunohistochemical study and gene expression analysis. J Histochem Cytochem 54: 863–875

63. , , , , (1992) Comparison of four serum tumour markers in the diagnosis of colorectal carcinoma. Br J Cancer 66: 148–154

64. , , , (2004) Identification and characterization of a novel form of the human L-Dopa decarboxylase mRNA. Neurochem Res 29: 1817–1823

65. , , (2009) Detection, purification and identification of an endogenous inhibitor of L-Dopa decarboxylase activity from human placenta. Neurochem Res 34: 1089–1100

66. , , (2005) Purification of an endogenous inhibitor of L-Dopa decarboxylase activity from human serum. Neurochem Res 30: 641–649

67. , , , , , , , (2003) Isolation and identification of L-Dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system. Biochem J 375: 373–383

68. , , , , , , , , (2007) Comprehensive expression analysis of L-Dopa decarboxylase and established neuroendocrine markers in neoadjuvant hormone-treated versus varying Gleason grade prostate tumors. Hum Pathol 38: 161–170

69. , , (2008) Association between chromosomal instability and prognosis in colorectal cancer: a meta-analysis. Gut 57: 941–950

70. , , , , , (2009) Genetic prognostic and predictive markers in colorectal cancer. Nat Rev Cancer 9: 489–499

## Acknowledgements

The project was supported by a PENED grant co-funded by the European Union – European Social Funds (75%) and National Resources – Ministry of Development – General Secretariat for Research & Technology of Greece (25%) as well as ELPEN A.E. through EPAN.M.8.3 – 3rd Community Support Programme.

## Affiliations

1. ### Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Athens, Athens GR-15701, Greece

• C K Kontos
• , E G Fragoulis
•  & A Scorilas

## Corresponding author

Correspondence to A Scorilas.

### DOI

https://doi.org/10.1038/sj.bjc.6605654

• ### Expression of vimentin (VIM) and metastasis-associated 1 (MTA1) protein in laryngeal squamous cell carcinoma are associated with prognostic outcome of patients

• Sotirios Karamagkiolas
• , Ioannis Giotakis
• , Efthimios Kyrodimos
• , Evangelos I. Giotakis
• , Agapi Kataki
• , Fani Karagianni
•  & Andreas M. Lazaris

American Journal of Otolaryngology (2019)

• ### L-Dopa decarboxylase interaction with the major signaling regulator ΡΙ3Κ in tissues and cells of neural and peripheral origin

• Alice G. Vassiliou
• , Maria-Zacharenia Siaterli
• , Efseveia Frakolaki
• , Panayiota Gkogkosi
• , Ioannis Paspaltsis
• , Dido Vassilacopoulou
•  & Niki Vassilaki

Biochimie (2019)

• ### Basal level of autophagy and MAP 1 LC 3B‐ II as potential biomarkers for DHA ‐induced cytotoxicity in colorectal cancer cells

• Helle Samdal
• , Malin A. Sandmoe
• , Lene C. Olsen
• , Elaf A. H. Jarallah
• , Therese S. Høiem
• , Svanhild A. Schønberg
•  & Caroline H. H. Pettersen

The FEBS Journal (2018)

• ### Kallikrein-related peptidases and associated microRNAs as promising prognostic biomarkers in gastrointestinal malignancies

• , Panagiotis Tsiakanikas
•  & Andreas Scorilas

Biological Chemistry (2018)

• ### False-positive findings on 6-[18F]fluor-l-3,4-dihydroxyphenylalanine PET (18F-FDOPA-PET) performed for imaging of neuroendocrine tumors

• Annika M A Berends
• , Michiel N Kerstens
• , Janne W Bolt