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ORIGINAL RESEARCH Table of Contents   
Year : 2008  |  Volume : 19  |  Issue : 4  |  Page : 315-319
Reverse transcriptase polymerase chain reaction study to evaluate dissemination of cancer cells into circulation after incision biopsy in oral squamous cell carcinoma


1 Department of Oral Pathology and Microbiology, KLES's Institute of Dental Sciences, Belgaum, Karnataka, India
2 Department of Microbiology and Molecular Biology, Maratha Mandal Dental College and Hospital, Belgaum, Karnataka, India

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Date of Submission03-Jan-2007
Date of Decision21-Apr-2008
Date of Acceptance29-Apr-2008
 

   Abstract 

Background: Tissue manipulation by incisions, radiotherapy, and palpation may lead to dissemination of cancer cells into circulation. Circulating cancer cells in blood play a central role in metastatic process. Their numbers can be very small and for their detection,reverse transcriptase polymerase chain reaction (RT-PCR) has been successfully used in this study.
Materials and Methods: To examine whether cancer cell dissemination results from incision biopsy, we tried to detect oral squamous cell carcinoma (OSCC) cells in the peripheral blood sample before and after incision biopsy by CK19 RT-PCR. The study group consisted of 25 OSCC patients and the control group consisted of five patients with oral submucos fibrosis and five with leukoplakia. Five ml of blood collected before and twice (15 and 30 min) after incision were used for CK19 RT-PCR.
Results: Four (16%) of 25 cases of OSCC were positive for CK19 transcripts in their peripheral blood drained 15 min after incision. CK19 transcripts were not detected in the control group.
Conclusion : Surgical invasion, in the form of incisional biopsy, causes dissemination of cancer cells into circulation, resulting in increased risk of metastasis.

Keywords: Cytokeratin 19 (CK19), incisional biopsy, oral squamous cell carcinoma (OSCC), reverse transcriptase polymerase chain reaction (RT-PCR).

How to cite this article:
Dyavanagoudar S, Kale A, Bhat K, Hallikerimath S. Reverse transcriptase polymerase chain reaction study to evaluate dissemination of cancer cells into circulation after incision biopsy in oral squamous cell carcinoma. Indian J Dent Res 2008;19:315-9

How to cite this URL:
Dyavanagoudar S, Kale A, Bhat K, Hallikerimath S. Reverse transcriptase polymerase chain reaction study to evaluate dissemination of cancer cells into circulation after incision biopsy in oral squamous cell carcinoma. Indian J Dent Res [serial online] 2008 [cited 2019 Oct 20];19:315-9. Available from: http://www.ijdr.in/text.asp?2008/19/4/315/44534
Head and neck cancer represents a major health problem. The majority of malignant tumors of oral cavity is squamous cell carcinoma (OSCC) arising from mucosal surfaces. The clinical management of patients with malignant diseases should be based on utilizing reliable and detailed morphologic assessment of the tumor, the type, and grade of malignancy. In order to gain such information, tissue pieces or cells must be removed from the tumor for microscopic analysis. The established methods of obtaining diagnostic material from tumors include surgical incision biopsy, excision biopsy, and tissue biopsy with a large caliber needle and aspiration with a fine needle.

Incision biopsy of oral carcinomas has long been recognized as a very useful method for establishing a firm diagnosis and for directing management of specific lesions. However, a number of clinicians are concerned that this procedure may spread cancer cells beyond the borders of tumor into the surrounding normal tissue; thus, promoting local spread as well as increasing the potential for metastasis. Tissue metastasis is a complex process involving essential steps. [1] The critical steps are escape from the primary tumor, dissemination through the circulation, lodgment in small vessels at distant sites, penetration of vessel wall, and growth in the new site as a secondary tumor. It has been indicated that surgical trauma inflicted on malignant tumors may increase their metastatic potency. [2],[3] The major physical barriers to the migration of tumor cells during this process are connective tissue and basement membrane. The surgical intervention may destroy these barriers and facilitate invasion of vascular system at the site of the injury. There have been reports of increased frequency of neck metastasis from stage I and stage II OSCC after incision biopsy. [4],[5]

Cytokeratins (CK) are one of the main families of intermediate keratin filament. They make up the cytoskeleton of both normal and malignant cells of epithelial origin. Among them, CK19, a 40 kDa epithelial cytoskeletal protein, has been used as a marker for cancers of epithelial origin. [6],[7],[8] CK19 is not expressed in normal hematopoietic cells; detection of CK19 transcript in peripheral blood of a patient with known OSCC should indicate the presence of carcinoma cells. Expression of CK19 in peripheral blood is detected by utilizing the highly sensitive reverse transcriptase polymerase chain reaction (RT-PCR) technique. Thus, this study was conducted to determine whether dissemination of cancer cells results from incision biopsy and to evaluate the efficacy of RT PCR in detecting CK19 mRNA expression in peripheral blood samples.


   Materials and Methods Top


The study comprised of clinically confirmed cases of OSCC visiting the out-patient department of K.L.E.S's Institute of Dental Sciences, Belgaum, India. The control group consisted of five cases of oral submucous fibrosis and five cases of leukoplakia. Patients receiving chemotherapy, radiotherapy, having a history of previous biopsy, with recurrence of OSCC, and patients with metastasis were excluded from the study.

TNM staging was done for all the cases using n0 evilles grading system. Two ml of blood sample was obtained from the median forearm vein using 18-guage disposable syringe by following appropriate aseptic measures. Three peripheral blood samples were collected, immediately before incision biopsy (sample A), 15 min after incision biopsy (sample B), and 30 min after incision biopsy (sample C). these blood samples were stored in citrate. Incision biopsy of the lesion was performed under local anesthesia. Histopathological examination of the biopsy specimen was done. The blood samples were then subjected to RT-PCR.


   RNA Preparation/Extraction Protocol Top


One ml of peripheral blood sample was transferred to an Eppendorf tube and centrifuged for plasma separation. The plasma was discarded. 200 l of 0.2% NaCl (hypotonic saline) was added to the sample and mixed thoroughly for one minute to lyse the red blood cells. The sample was then centrifuged and the deposit was washed with phosphate buffer saline to remove traces of hemoglobin. Four types of buffer solutions were prepared (solution A, guanidium isothiocynate; solution B, tris HCl; solution C, phenol, chloroform, and isoamylalcohol; and solution D, chloroform and isoamylalcohol.)Four hundred microliters of solution A, 50 l of solution B, and 550 l of solution C were placed in a tube along with the deposit. After the solutions were thoroughly mixed, the tube was placed in an ice bath for 30 min and then centrifuged at 10,000 rpm for 15 min. The upper aqueous phase was transferred to a new tube into which one volume of solution D was previously added. The tube was kept again in an ice bath for 30 min followed by centrifugation. To the upper aqueous phase one volume of 100% isopropanol was added and kept in an ice bath for 30 min. The solutions were centrifuged and the supernatant was discarded. The pellet was air-dried and was dissolved in 20 l of RNAse-free water. Extracted RNA was stored at -20C.

RT-PCR was carried out using QIAGEN R one-step RT-PCR kit (Genetix C-88 Kitri Nagar, New Delhi 110015). The kit contained optimized components that allowed both RT and PCR amplification.


   RT-PCR Top


Master mix was prepared by adding 8 l of RNAse-free water, 2 l of buffer, 1 l of reverse transcriptase, 2 l of dNTP's mix, 1 l RNAse inhibitors, 1 l primer 86 (CK2: 5′-TTATTGGCAGGTCAGGAGAAGAGCC-3′), and 5 l of template RNA in a 20 l reaction tube. The ingredients were mixed thoroughly and kept in the thermal cycler for 1 h at 37C for conversion of RNA to cDNA. Hot start PCR was performed by adding 25 l of master mix, 8 l of distilled water, 1 l of primer 85 (CK1: 5′-AAGCTAACC ATGCAGAACCTCAACGACCGC-3′), 1 l of primer 86 (CK2: 5′-TTATTGGCAGG TCAGGAGAAGAGCC-3′), and 15 l sample (template DNA) in a 50 l reaction tube. Master mix contained all the ingredients required for amplification, except primers and template DNA.

Thirty-five cycles were performed consisting of 2 min denaturation at 94C, 30 seconds annealing at 60C, and 30 seconds annealing polymerase extension at 72C. t0 o confirm the integrity of RNA, 10 l of PCR products were run on 2% agarose gel.

In each batch of amplification, a known positive control (extracted DNA from biopsy specimen of OSCC patient) and a known negative control (extracted DNA from biopsy specimen of a healthy patient) were included. Association between the attributes was tested using Chi-square with Yate's correction.


   Results Top


To examine the dissemination of normal epithelial cells into the circulation by mucosal incision, CK19 RT-PCR was performed on blood samples obtained from five patients with leukoplakia and five with oral submucous fibrosis (nine male patients and a female patient [Table 1]. All the ten cases in control group were negative for CK19 RT-PCR before 15 min and 30 min after biopsy [Figure 1].

The study group consisted of twenty-five patients (20 men and 5 women) aged between 31 and 60 years. The oral cancers were classified into two cases of T 1 , six cases of T 2 and T 3 each, and eleven cases of T 4 . All the patients were negative for CK19 transcripts in the peripheral blood before biopsy. Three of eleven (27.27%) patients in T 4 tumors and one of six patients (16.66%) in T 3 were positive for CK19 transcripts in the peripheral blood taken 15 min after incision into lesional tissue. No statistical difference was observed in T 3 and T 4 tumors (P > 0.9). Two of eleven patients in stage III and two of eleven patients in stage IV showed positivity for CK19 transcripts in the peripheral blood taken 15 min after incision into lesional tissue. No statistical difference was observed in stage III and stage IV tumors (P = 1). Four (16%) cases of OSCC were positive for CK19 transcripts in peripheral blood samples collected 15 min after incision biopsy. CK19 transcripts were negative in all the peripheral blood samples (25 cases) drawn 30 min after incision biopsy. No statistical difference was observed between the study and control group with P > 0.05. The positivity for CK19 mRNA transcripts 15 min after incision biopsy of case number 11 and 17, and negative expression for CK19 transcripts 15 min after incision biopsy of case number 18 are shown in [Figure 2]. The details of all the study cases are shown in [Table 2].


   Discussion Top


The present study demonstrated four (16%) cases of OSCC to be positve for CK19 transcripts in the blood samples collected 15 min after incision biopsy, while . Ten patients in the control group were negative for CK19 transcripts in the blood samples collected 15 min after incision biopsy. Our study strongly supports the findings of Kusukawa et al, [10] who found 20% (2/10) positivity for CK19 transcripts in peripheral blood after 15 min of incision. They also reported negative CK19 transcripts in control group as well as in excision biopsy group. It has been suggested that connective tissue and basement membrane are major physical barriers to the migration of tumor cells into circulation.

Weiss [11] suggested the number of cancer cells in circulation at any point of time not only depends on detachment of cells from the primary tumors but also on their accessibility to vascular channels and the rates at which they are removed from the circulation.

RT-PCR for CK19 is a sensitive and specific technique to detect mammary carcinoma cells in peripheral blood and bone marrow of patients with breast cancer. Osborne MP and colleagues [12] demonstrated that RT-PCR assay is capable of detecting two or three mammary carcinoma cells in one million normal peripheral blood mononuclear cells which suggests that the technique is among the most sensitive assay available for detection of occult breast carcinoma.

Krismann et al, [8] pointed out that RT-PCR may detect nonspecific constitutive low-level expression of CK19 mRNA in peripheral blood.

Datta et al, [7] indicated that illegitimate transcription of CK19 mRNA or presence of CK19 expressing epithelial cells is rare in normal peripheral blood. Furthermore, Ruud et al, [13] identified CK19 pseudogene that might lead to false-positive result. In the present study, the blood samples obtained before incision biopsy from the control group were negative for CK19 transcripts. These results eliminate the possibility of the presence of pseudogene.

Contamination of blood samples with dermal cells can occur at needling. In addition, dissemination of normal mucosal cells into circulation by incision might also lead to false-positive results. To investigate this possibility, peripheral blood samples from ten patients with oral lesions (five submucous fibrosis and five leukoplakia) were examined for the presence of CK19 transcripts. All blood samples taken before and after incision were negative for CK19 transcripts suggesting that there was no dissemination of normal mucosal cells into circulation.

The presence of circulating carcinoma cells in peripheral blood of study group who were in stage III (two of eleven cases (18.18%)) and stage IV (two of eleven cases (18.18%)) 15 min after biopsy was demonstrated in the present study. Similar findings were observed in a study conducted on breast cancer by Datta et al, [7] they found that four of 19 patients with stage IV breast cancer were positive for CK19 transcripts. Authors suggested that lack of CK19 RT-PCR positivity in blood of stage IV patients may have been due to consistently low numbers of circulating carcinoma cells or sequestration of tumor cells to other sites. The dissemination of cancer cells also depends on the vascularity of the tumor. Two of four patients with peripheral blood positive for CK19 assays developed carcinomatous meningitis, and other two patients had distant sites of progressive disease.

Kusukawa J et al, [14] reported the incidence of neck metastases in stage I and II squamous cell carcinomas treated with excision biopsy was significantly lower than that in tumors excised after incision biopsy. They reported that one of the two patients who was positive for CK19 in peripheral blood 15 min after incision biopsy died because of lung metastases.

CK19 RT-PCR was negative for all the blood samples taken, 30 min after incision biopsy in the study group and control group. The results of our study are similar to the findings of Kusukawa et al. [10]

Interestingly, the circulating cancer cells were transiently detectable at 15 min after incision. After 30 min of incision biopsy, the cancer cells gain access to venous circulation where they are completely trapped in the vasculature of the organs, first encountered, that is, the lung and vertebrae for cancers draining into the venacava as suggested by Alexander P. [15] Large numbers of circulating cancer cells may be killed in the microvasculature by hemodynamic destruction or by elements of host surveillance system such as the macrophages and natural killer cells. [16] As metastasis is a dynamic and periodic event, and may not be an ongoing process at the time of a single blood draw, samples at different periods may increase the odds of detecting circulating tumor cells. Time intervals between the samples may need to be defined for each tumor type or in relation to their metastatic potential. The timing of blood drawn in relation to the invasive procedures may also be crucial.

Safour et al, [3] found metastatic spread to regional lymph nodes in approximately 50% of animals receiving incision biopsy. They suggested that lymph node metastasis may be due to trauma to the tumor nests, blood and lymph vessels, or may be due to inflammation of the vasculature at the site of injury.

When incision biopsy is necessary, chemotherapy should be administered immediately before and after biopsy. Kusukawa J and Kameyama T. [17] have reported that chemotherapy prior to biopsy is effective in preventing secondary neck metastases.

The possibility of missing clinically relevant micrometastatic disease based on tissue sampling or method of analysis has been acknowledged for quite some time. Further, more retrospective studies would suggest that biologic behavior of patients with occult micrometastases is similar to that of patients with histologically positive nodes. Currently, several technologies are being developed but traditional diagnostic techniques such as immunohistochemical staining continue to play a central role in diagnosis and characterization of cancer. It is likely that in a few years, newer techniques such as RT-PCR will play a central role in cancer diagnosis. Keratins are the most reliable markers of epithelial differentiation and are therefore more useful than the other intermediate filaments in characterization of neoplastic tissue.

We conclude that invasive surgical procedures for oral cancer such as incision biopsy might cause dissemination of cancer cells into circulation. Our study also reports that CK19 mRNA expression is seen in the peripheral blood samples of carcinoma patients 15 min after incision biopsy. The absence of CK19 mRNA expression in the peripheral blood sample of control group emphasizes the specificity of RT-PCR assay. We have demonstrated that RT-PCR of CK19 is a sensitive and specific technique for detecting circulating carcinoma cells in the peripheral blood.

 
   References Top

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2.Peters LJ. A study of the influence of various diagnostic and therapeutic procedures applied to a murine squamous carcinoma on its metastatic behaviour. Br J Cancer 1975;32:355-72.  Back to cited text no. 2  [PUBMED]  
3.Safour IM, Wood NK, Tsiklakis K, Doemling DB, Joseph G. Incisional biopsy and seeding in hamster cheek pouch carcinoma. J Dent Res 1984;63:1116-20.   Back to cited text no. 3  [PUBMED]  [FULLTEXT]
4.Ohtake K, Shingakib S, Nakajima. Effects of incision and irradiation on regional lymph node metastasis in carcinoma of the hamster tongue. Oral Surg Oral Med Oral Pathol 1990;70:62-9.  Back to cited text no. 4    
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6.Coltrera MD, Zarbo RJ, Sakr WA, Gown AM. Markers for dysplasia of the upper aerodigestive tract. Suprabasal expression of PCNA, p53 and CK 19 in alcohol fixed, embedded tissue. Am J Pathol 1992;141:817-25.   Back to cited text no. 6    
7.Datta YH, Adams PT, Drobyski WR, Ethier SP, Terry VH, Roth MS. Sensitive detection of occult breast cancer by reverse transcriptase polymerase chain reaction. J Clin Oncol 1994;12:475-82.   Back to cited text no. 7  [PUBMED]  [FULLTEXT]
8.Krismann M, Todt B, Schroder J, Gareis D, Mller KM, Seeber S, et al . Low specificity of cytokeratin 19 reverse transcriptase-polymerase chain reaction analyses for detection of hematogenous lung cancer dissemination. J Clin Oncol 1995;13:2769-75.   Back to cited text no. 8    
9.Traweek ST, Liu J, Battifora H. keratin gene expression in non-epithelial tissues: Detection with polymerase chain reaction. Am J Pathol 1993;142:1111-8.   Back to cited text no. 9  [PUBMED]  [FULLTEXT]
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11.Weiss L, Dimitrov DS, Angelova M. the hemodynamic destruction of intravascular cancer cells in relation to myocardial metastases. Proc Natl Acad Sci USA 1985;82:5737-41.   Back to cited text no. 11  [PUBMED]  [FULLTEXT]
12.Osborne MP, Asina S, Wong GY, Old LJ, Cote RJ. Immunofluroscent monoclonal antibody detection of breast cancer in bone marrow: Sensitivity in a model system. Cancer Res 1989;49:2510-3.   Back to cited text no. 12  [PUBMED]  [FULLTEXT]
13.Ruud P, Fostad O, Hovig E. identification of a novel cytokeratin 19 pseudogene that may interfere with reverse transcriptase polymerase chain reaction assay used to detect micrometastatic tumor cells. Int J Cancer 1999;80:119-25.  Back to cited text no. 13    
14.Kusukawa J, Nakamura Y, Kameyama T. evaluation of excisional biopsy for stage I and stage II SCC of the oral cavity. Int J Clin Oncol 1998;3:311-6.  Back to cited text no. 14    
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Correspondence Address:
Sunita Dyavanagoudar
Department of Oral Pathology and Microbiology, KLES's Institute of Dental Sciences, Belgaum, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.44534

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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]

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