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 Table of Contents  
ORIGINAL ARTICLE
Year : 2012  |  Volume : 37  |  Issue : 3  |  Page : 172-177

Detection of tyrosine hydroxylase m-RNA in the peripheral blood of neuroblastoma patients and its relation to treatment response


1 Department of Clinical Pathology, South Egypt Cancer Institute, Assiut University, Assiut, Egypt
2 Department of Pediatric Oncology, South Egypt Cancer Institute, Assiut University, Assiut, Egypt
3 Department of Clinical Pathology, Faculty of Medicine, South Valley University, Qena, Egypt

Date of Submission10-Feb-2012
Date of Acceptance30-Mar-2012
Date of Web Publication21-Jun-2014

Correspondence Address:
Hosny B. Hamed
Department of Clinical Pathology, South Egypt Cancer Institute, Assiut University, 7111 Assiut
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.7123/01.EJH.0000416548.07291.ab

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  Abstract 

Background

Neuroblastoma (NB) is the most common malignant solid tumor in childhood and, among all childhood malignancies, is second only to leukemia. NB originates before birth in the neural crest. Tyrosine hydroxylase (TH) is the first enzyme in the pathway of catecholamine synthesis and the detection of TH m-RNA is very useful as a tumor marker for NB.

Aim of the study

The aim of the present study is to detect the TH m-RNA in the peripheral blood of patients with NB by real-time reverse transcriptase-PCR (RT-PCR) at diagnosis and during therapy to determine the role of TH m-RNA in the diagnosis of NB and its relation to the treatment response.

Patients and methods

Blood samples were collected from 32 children with advanced stages NB (stages III and IV), either newly diagnosed (n=11) or receiving treatment (n=21). Another 12 blood samples were also obtained from age-matched children with other pediatric cancers as a control group. We used real-time RT-PCR for the detection of TH m-RNA.

Results

TH m-RNA expression was detected in peripheral blood samples obtained from five of 11 (45.4%) newly diagnosed cases and in five of 19 (26.3%) children receiving treatment. A significant difference was found between stages (III and VI) at presentation (P=0.04), LDH level (P=0.04), and bone marrow disease (P=0.025) with the detection of TH m-RNA in peripheral blood. Although no significant effect on survival outcomes had been reported in our patients, there were significant differences (P=0.04) in the response to treatment and TH m-RNA expression.

Conclusion

Detection of TH m-RNA by real-time RT-PCR is a reliable, quick, and easy way to detect the expression of NB cell in blood at diagnosis. Also, TH expression can be used as a tumor marker for the accurate diagnosis of NB and a predictor of treatment response.

Keywords: neuroblastoma, real-time polymerase chain reaction, tyrosine hydroxylase


How to cite this article:
Hamed HB, Sayed HA, Ali SS. Detection of tyrosine hydroxylase m-RNA in the peripheral blood of neuroblastoma patients and its relation to treatment response. Egypt J Haematol 2012;37:172-7

How to cite this URL:
Hamed HB, Sayed HA, Ali SS. Detection of tyrosine hydroxylase m-RNA in the peripheral blood of neuroblastoma patients and its relation to treatment response. Egypt J Haematol [serial online] 2012 [cited 2019 Dec 9];37:172-7. Available from: http://www.ehj.eg.net/text.asp?2012/37/3/172/135055


  Introduction Top


Neuroblastoma (NB) is the most common solid malignant extra cranial tumor in childhood; it develops from the sympathetic precursor cells of neural crest origin. The disease is predominantly found in young children and infants and is characterized by its clinical heterogeneity; it ranges from slow-growing resectable tumors to disseminated forms with widespread metastases at diagnosis and poor survival despite intensive multimodal therapy 1. As with many cancers, metastases in children with NB are routinely identified by clinical evidence of spread using imaging studies and detection of tumor cells in bone marrow by histologic examination. The tumor stage ranges from localized tumors (stage I) with good outcome to tumors with bone or bone marrow metastases (stage IV) with a poor prognosis 2. NB belongs to the small round cell tumors, which include other solid tumors such as Ewing’s sarcoma, rhabdomyosarcoma, and malignant lymphoma 3. Small round cell tumors are histologically ambiguous; thus, it is necessary to analyze adequate tumor markers for an accurate diagnosis 4.

A large majority of NBs produce catecholamines, thus expressing tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. Because catecholamines are produced by 98% of all NB, TH m-RNA was used as a target for the detection of NB cells by a reverse transcriptase-PCR (RT-PCR) 5. Other targets for the detection of NB cells by RT-PCR including PGP 9.5 6, NFM and SYN 7, and MAGE and GAGE 8 have been evaluated, but TH m-RNA appears to be the single most useful target 9. TH m-RNA has not been detected in normal peripheral blood 10.

Although RT-PCR increases the sensitivity of NB cell detection 10, 11, its clinical value is difficult to assess from the current literature, with reported frequencies in peripheral blood from children with stage 4 disease at diagnosis varying from 25 to 100% 11. Recent reports have concluded that the concentrations of TH m-RNA at diagnosis predict the outcome for high-risk NB patients, suggesting a possible novel way of stratification for different treatment strategies in these children 12.

The aim of the present study is to detect TH m-RNA in peripheral blood of patients with NB at diagnosis and during therapy by real-time RT-PCR, to confirm the diagnosis of NB, and to study TH m-RNA as a prognostic factor.


  Patients and methods Top


Blood samples were collected from 32 children with advanced stages NB (stages III and IV), as all the attending children in Pediatric Oncology Department were in advanced stages, both newly diagnosed (n=11) and those receiving treatment (n=21), attending the South Egypt Cancer Institute, Assiut University. Twelve blood samples were also obtained from age-matched children as a control group (four acute lymphoblastic leukemia, two acute myeloid leukemia, one germ cell tumor, two rhabdomyosarcomas, one hepatoblastoma, one Wilms’ tumor, and one Ewing’s sarcoma).

Institutional ethical approval was obtained and parental consent was taken for all children from whom blood was sampled. For each patient, evaluation was carried out by assessment of history and examination, routine laboratory investigations, and imaging studies for local and metastatic disease.

NB staging was defined according to the international NB staging system 13.

All patients (with stage III and IV disease) received the same treatment, which included six courses of OPEC alternating with OJEC chemotherapy. After the third and sixth courses, patients were evaluated for response to chemotherapy by clinical examination, laboratory investigation, and imaging studies according to the initial presentation.

RNA extraction

RNA was extracted from the peripheral blood using QIAamp RNA (QIAgen, Germany) blood mini kit for total RNA purification from human whole blood.

cDNA

cDNA was obtained from the extracted RNA using the Transcriptor First Strand cDNA synthesis kit (Roche Diagnostics GmbH, Mannheim Germany).

Real-time polymerase chain reaction

Light Cycler TaqMan Master was used [ready-to-use hot start reaction mix for PCR on the Light Cycler Carousel-based system with Hydrolysis TaqMan Probes (Roche Diagnostics GmbH)].

Principles

Hydrolysis probes, also called the TaqMan assay, use a single probe containing two labels: a fluorescent reporter dye and a fluorescent quencher. While the probe is intact, the quencher is close to the reporter dye and suppresses reporter fluorescence by fluorescence resonance energy transfer. When the probe is hybridized to the target sequence, the 5′ nuclease activity of the polymerase can cleave the hydrolysis probe, separating the reporter and quencher. With an increasing amount of target sequence during PCR, more probes are cleaved and the fluorescence signal of the unquenched reporter dye increases.

Interpretation of results

We used the relative quantification method in which the result is expressed as a relative ratio of the target of interest to a reference genes. Here, we used glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a reference gene (each sample run in two tests: one for the TH and the other for GAPDH). For each test, the crossing point (CP), the time at which the fluorescence intensity is greater than the background and the curve begins to rise, of the target gene and reference genes were determined. Then, the soft ware of the Light Cycler calculates the relative ratio between the target and the reference genes [Figure 1].
Figure 1: Amplification curve. This graph shows the fluorescence signal against the cycles that show the CP, the cycle at which the curve rises, for each target and its reference. In this curve, sample number 1 is negative as the curve of the reference had CP (Cycle number 28), but the target had no CP. The sample number 2 is positive as the target and the reference had CP (Cycle numbers 34 and 23, respectively). Number 5 is a negative control.

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Reagents

The primers and TaqMan probe sequences of TH and GAPDH are presented in [Table 1].
Table 1: Primers and probes sequences used in the real-time polymerase chain reaction

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The Light Cycler TaqMan Master protocol is presented in [Table 2].
Table 2: Protocol on Light Cycler TaqMan Master

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Statistical analysis

Data were collected and analyzed using the computer program SPSS version 17 (SPSS Inc., Illinois, Chicago, USA). Data were expressed as mean±SD, number, and percentage. The t-test was used to determine significance for numeric variables and the χ2 to determine significance for nonparametric variables. Person’s correlation was also used for numeric variables in the same group.


  Results Top


Blood samples were collected from 32 patients, both newly diagnosed (n=11) and those receiving treatment (n=21). There were 20 males and 12 females, M : F=1.6 : 1. All had stage IV disease; six patients had stage III disease.

Only 30 patients were evaluable for the study after the exclusion of two patients: the first one refused treatment after diagnosis and the other patient was lost to follow-up shortly after treatment. The median age at the time of diagnosis was 3 years (range 4 months to 11 years). The mean level of lactate dehydrogenase (LDH) was 1176.5±123.8 IU/l. Most of our patients (80%) had metastatic disease; of these, 87.5% had bone barrow disease and 53.8% had bone disease.

[Table 3] shows the clinical characteristics of all the patients included in the study. A positive correlation was found between age at diagnosis and LDH level (P<0.009) among our patients.
Table 3: Characteristics of all patients included in the study

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Ten (33.3%) out of 30 collected blood samples were expressing TH m-RNA. It was detected in samples obtained from five of 11 (45.4%) newly diagnosed cases and five of 19 (26.3%) cases receiving treatment. The level ranged between 0.003 and 0.0006 relative ratio (this ratio, which is the relative expression of TH m-RNA to the expression of the housekeeping gene GAPDH, which is normally expressed, is 0.003–0.0006). In the control group, other round cell tumors, TH m-RNA, were not detected.

None of the patients with stage III disease expressed TH m-RNA except one; this patient did not achieve complete remission after treatment and was subjected to second-line treatment, whereas all patients with positive TH m-RNA expression had stage IV disease, except one. In analytic studies, these results showed a significant difference between stages III and IV with TH m-RNA expression (P=0.04; [Table 4].
Table 4: Relation between tyrosine hydroxylase m-RNA and other variables

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Also, we found a significant difference between the detection of TH m-RNA in peripheral blood and both LDH level (P=0.04) and bone marrow disease (P=0.025), whereas no significant difference was found in the level of TH m-RNA in peripheral blood samples obtained from newly diagnosed cases versus those receiving chemotherapy.

Among 10 children in whom TH m-RNA was expressed in their peripheral blood, progressive and residual disease were reported in 50 and 20%, respectively; also, relapse was reported in one of the three remaining patients achieving remission. However, 70% of children with a negative expression of TH m-RNA achieved complete remission, whereas only 20% had progressive disease. This finding implies a significant difference (P=0.04) in the response to treatment and TH m-RNA expression; accordingly, TH m-RNA may be used as a predictive factor for response to treatment. [Table 4] shows the relation between TH m-RNA expression and other variables.

The 3-year median overall survival for our patients was 1.8 and the median disease-free survival was 1.6±0.515 [Figure 2] and [Figure 3]. TH m-RNA expression had no effect on survival outcome in this study, whereas stage, age, and LDH level at the time of diagnosis affected the overall survival significantly (P=0.04, 0.02, and 0.001, respectively; [Table 5] and [Figure 4], [Figure 5], [Figure 6].
Table 5: Prognostic significance of tyrosine hydroxylase m-RNA detected in peripheral blood and other common variables at diagnosis for overall survival

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Figure 2: Kaplan–Meier graph showing overall survival of all patients with neuroblastoma included in the study.

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Figure 3: Kaplan–Meier graph showing disease-free survival of all patients with neuroblastoma included in the study.

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Figure 4: Kaplan–Meier graph showing overall survival of all patients with neuroblastoma according to stage.

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Figure 5: Kaplan–Meier graph showing overall survival of all patients with neuroblastoma according to age.

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Figure 6: Kaplan–Meier graph showing overall survival of all patients with neuroblastoma according to lactate dehydrogenase.

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  Discussion Top


A majority of children with NB have widespread disease at diagnosis 14. Despite intensive multimodal therapy, only a minority of the high-risk patients achieve long-term survival. Therapy for children with NB is increasingly being tailored for an individual on the basis of the risk factors assessed at the time of diagnosis 15. Advanced stage disease at diagnosis was one of three significant poor prognostic factors, in addition to age 16 and elevated serum LDH levels 17. Several studies have also shown that the presence of metastasis determined by imaging and histologic examination in NB differs with age and correlates with event-free survival 18, MYCN amplification 2, and other prognostic biologic tumor features also shown to have prognostic significance.

The detection of tumor cells in peripheral blood from clinically disease-free children is a powerful tool to predict those most likely to relapse. TH m-RNA in peripheral blood was found before catecholamine levels increased and clinical relapse occurred as Burchill et al. 19 reported that the presence of TH m-RNA in peripheral blood of children 1 year old at the end of therapy was rare, but associated with rapidly progressing disease and a high relapse rate; in most children, TH m-RNA was not detected 8–10 weeks after the start of therapy, which compares well with the clearance of tumor cells from peripheral blood reported using immunocytology. The detection of TH m-RNA in peripheral blood from children on treatment was not of prognostic significance.

Other reports confirm the use of the detection of minimal residual disease as a prognostic marker or have reported an association of molecular detection of TH and disease outcome 20.

The aim of this prospective study was to determine the diagnostic and prognostic significance of disease detected in peripheral blood by RT-PCR in patients with advanced NB, focusing on the association between TH m-RNA expression and response to treatment.

Detection of NB cells by RT-PCR for TH has been reported by several groups 21–25. These methods involve post-PCR manipulations, which are time consuming and laborious, and they introduce the risk of carryover contamination. To overcome these limitations of post-PCR sample handling, we used real-time PCR for the detection of TH in peripheral blood from patients with NB the real time PCR overcome all these problems.

Our results are in agreement with the results of Rie et al. 4, who found that TH m-RNA was expressed in all NB cell lines (100%) and in 23 of 25 (92%) clinical NB tumor samples, but not expressed in any of the other cell lines and their clinical tumor samples. We found that TH m-RNA was expressed specifically in NB, and this specific expression can be used to distinguish NB from other small round cell tumors.

TH m-RNA expression used in our study correlated to stage at diagnosis, bone marrow disease, LDH, and response to chemotherapeutic drugs, where TH m-RNA expression was detected in peripheral blood samples from 10 of 30 (33.3%) patients, and a significant difference (P=0.04) between stages III and IV. These results are in agreement with the results of Burchill et al. 19, who reported that RT-PCR of peripheral blood from children with NB for TH m-RNA detection identifies those with an unfavorable prognosis by association with the disease stage at diagnosis and within the advanced age group. Similar results were also obtained by Trager et al. 12.

Also, we found a significant difference between the detection of TH m-RNA in peripheral blood and both LDH level (P=0.04) and bone marrow disease (P=0.025), whereas no significant difference was found in the presence of TH m-RNA in peripheral blood samples obtained from newly diagnosed cases versus those on treatment. However, the response to chemotherapy is better in children who are negative for TH m-RNA than those who are positive for TH m-RNA. These results are in agreement with the results of Burchill et al. 19, who reported that in a multivariate analysis, the presence of TH m-RNA in peripheral blood and a serum LDH>1500 IU/l were the most powerful poor prognostic factors.

Burchill et al. 19 reported that the overall and event-free survival remain significantly better in children without TH m-RNA in peripheral blood. In the present study, we found that TH m-RNA expression had no significant effect on survival outcomes, but it can be an important predictor of tumor response to treatment, which suggests a possible novel way of stratification of different treatment methods in these children. This was implied by the significant difference (P=0.04) in the response to treatment and TH m-RNA expression.

Progressive and residual disease (50 and 20%, respectively) were reported in 10 children with TH m-RNA expressed in their peripheral blood also reporting relapse in out of three remaining cases achieving remission. However, 70% of children with a negative expression of TH m-RNA achieved complete remission, whereas only 20% had progressive disease.

In the present study, we did not detect TH m-RNA in any samples of the round cell tumors (four acute lymphoblastic leukemia, two acute myeloid leukemia, one germ cell tumor, two rhabdomyosarcomas, one hepatoblastoma, one Wilms’ tumor, and one Ewing’s sarcoma). These results are in agreement with the result of Rie et al. 4, who found that TH m-RNA was not detected in the osteosarcoma, osteochondroma, Wilms tumor, small round cell tumor samples, and rhabdomyosarcoma. Therefore, TH m-RNA can be used to confirm the diagnosis of NB.


  Conclusion Top


In conclusion, taking into consideration that our patient group is relatively small, we found that the detection of TH m-RNA by real-time PCR is reliable, quick, and easy to perform to study NB cells in peripheral blood at diagnosis. Also, TH expression can be used as a tumor marker for the accurate diagnosis and staging of NB and as a predictor of treatment response.[25]

 
  References Top

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2.Brodeur GM, Seeger RC, Schwab M. Amplification of MYCN in untreated human neuroblastomas correlates with advanced disease stage. Science. 1984;224:1121–1124  Back to cited text no. 2
    
3.Horowitz ME, Malawer MM, Delaney TF, Tsokos MG Principles and practice of pediatric oncology. 2002 Philadelphia Lippincott Williams & Wilkins:795–821  Back to cited text no. 3
    
4.Rie I, Satoru A, Shigeki K, Shigeyasu M, Hiroyuki S, Motoaki C, et al. Usefulness of tyrosine hydroxylase mRNA for diagnosis and detection of minimal residual disease in neuroblastoma. Biol Pharm Bull. 2004;27:315–318  Back to cited text no. 4
    
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21.Miyajima Y, Horibe K, Fukuda M, Matsumoto K, Numata S, Mori H, Kato K. Sequential detection of tumor cells in the peripheral blood and bone marrow of patients with stage IV neuroblastoma by the reverse transcription polymerase chain reaction for tyrosine hydroxylase mRNA. Cancer. 1996;77:1214–1219  Back to cited text no. 21
    
22.Kuroda T, Saeki M, Nakano M, Mitzutani S. Clinical application of minimal residual neuroblastoma cell detection by reverse transcriptase-polymerase chain reaction. J Pediatr Surg. 1997;32:69–77  Back to cited text no. 22
    
23.Lode HN, Handgretinger R, Schuermann U, Seitz G, Klingebiel T, Niethammer D, Beck J. Detection of neuroblastoma cells in CD34-selected peripheral stem cells using a combination of tyrosine hydroxylase nested RT-PCR and anti-ganglioside GD2 immunocytochemistry. Eur J Cancer. 1997;33:2024–2030  Back to cited text no. 23
    
24.Burchill SA, Lewis IJ, Selby P. Improved methods using the reverse transcriptase polymerase chain reaction to detect tumour cells. B J Cancer. 1999;79:971–977  Back to cited text no. 24
    
25.Gilbert J, Habe M, Bordow SB, Marshall GM, Norris MD. Use of tumor-specific gene expression for the differential diagnosis of neuroblastoma from other pediatric small round-cell malignancies. Am J Patho. 1999;155:17–21  Back to cited text no. 25
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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