Journal of Cancer Research and Therapeutics, Vol. 1, No. 2, April-June, 2005, pp. 92-97
Use of Rt-PCR in detecting disseminated cancer cells after incisional biopsy among oral squamous cell carcinoma patients
Ramani Pratibha, Thomas George, Ahmed Shaheen
Department of Oral And Maxillofacial Pathology, Saveetha Dental College & Hospital, Chennai-77, Tamil Nadu
Code Number: cr05019
AbstractPURPOSE: Tumour metastasis is the most clinically significant and enigmatic aspect of tumour behavior and is an unequivocal hallmark of malignancy. Until recent years little has been known about the transportation phase of vascular dissemination during biopsy, because of the technical difficulties in demonstrating circulating cancer cells.
AIMS: This study examined whether cancer cell dissemination results from incisional biopsy in the peripheral blood by using Cytokeratin 19(CK-19) as the marker for Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR). In-house recipes without utilizing kits were employed to extract genomic and total RNA to make the procedure user friendly.
MATERIALS AND METHODS: The study population consisted of n=10patients who were clinically diagnosed for oral squamous cell carcinoma and who had not undergone any previous biopsies. 5 patients who were to undergo incisional biopsies for benign conditions served as controls.5 ml of blood aspirates were collected before and within 15 minutes after incisional biopsy. CK-19 gene and a positive control gene β actin were isolated to confirm the primers. Using the total RNA, RT-PCR was performed for β actin and Ck 19 gene expression. RESULTS: Rt-PCR did show any expression for the CK-19 gene.
CONCLUSION: In conclusion there was no evidence of dissemination of cancer cells in our study and the patients are on a regular follow up for the past one and half years. But larger sample size should be examined to make the procedure a diagnostic tool for cancer metastasis
Keywords: Cytokeratin19, β actin, Rt-pcr
Malignant neoplasms of the oral cavity are relatively rare in most parts of the world. In countries such as United Kingdom and the United States for example, malignant neoplasms comprise only 2% to 3% of total malignant tumours. There are exceptions however, and in various parts of India and with regard to Chennai, approximately 40% of all malignant neoplasms are oral in males and around 9% in females. The vast majority are epidermoid (squamous cell) carcinomas, accounting for 94.5% of all cancers of oral cavity.
The size of the clinical problem of oral squamous cell carcinoma is illustrated in [Table - 1], which shows the recent annual average number of new cancer registrations in our dental college & hospital only.
The role of histopathologist in the evaluation of tumour specimens has become complex in the last 10-20years and will continue to develop in the decade ahead. This is in part is attributable to the increasing sophistication of therapeutic approaches employed by oncologists, leading to requirements for more accurate identification of tumour groups that may vary in their natural progression and responsiveness to treatment
Tumour metastasis is the clinically significant and enigmatic aspect of tumour behavior, which can be defined as relentless progress of neoplasia resulting in death. It is believed that a few malignant cells are released into the blood stream during incisional biopsy of malignant epithelial tissues of the oral cavity and these free floating cells may establish metastasis elsewhere. Several authors have tried to show the dissemination phase of cancer cells in relation to cancers arising from other body tissues., ,  With the advent of molecular biology several studies have been successfully tested in detecting micrometastasis especially in relation to lung, breast and prostrate carcinomas., , 
Cytokeratins are one of the main families of intermediate filaments that belong to a multigene family distinguished by their cell type specific expression. Malignant cells general retain their intermediate filaments of their progenitor cell type and consequently cytokeratins have been used to characterize neoplastic cells.,  Among these, Cytokeratin 19, a 40-kDa epithelial cytoskeletol protein is considered as an epithelial marker as they are not expressed in normal haematopoietic tissue., 
As very few studies have been carried out in relation to oral cancer dissemination phase especially in Indian scenario, our objective was to test if we can detect these released cells by a powerful and sensitive method like RT-PCR, an adaptation of Polymerase Chain Reaction (PCR.)
MATERIALS AND METHODS
The study group consisted of n = 10 patients who were clinically diagnosed for oral cancer and were to undergo incisional biopsy [Table - 2]. The control group comprised of patients who were to undergo incisional biopsies for benign conditions (n = 5) [Table - 3]. The patients in the study group were clinically staged and an informed consent was obtained from all patients. Subsequently, a 17-gauge needle was placed in the median forearm vein. To avoid dermal contamination, initial 1 to 2 ml of blood was discarded and then subsequently 5ml of blood was collected. Samples of peripheral blood (5ml) were collected at various times viz. immediately before incision and within 15 minutes after biopsy. Subsequently 300 aliquots of whole blood was taken in two microfuge tubes for isolation of genomic DNA along with 900ml of red blood cell lysis buffer, incubated, centrifuged in microfuge and pellets of WBC were resuspended and then combined in a single tube. Then 600ml of ice-cold cell lysis buffer was added, homogenized with subsequent addition of Dnase-free Rnase and incubated at 37°c for 60 minutes. After adding 200ml of potassium acetate, the solution was vortexed and centrifuged at maximum speed (15,000 rpm) to obtain a visible pellet of the complex. The pellet was then dissolved in isopropanol and then reprecipitated with 70% ethanol. After centrifugation the DNA pellet was air dried and then finally redissolved in 100ml Tris EDTA (pH 7.6). After casting the gel the DNA was loaded onto the gel after mixing with gel loading buffer and electrophoresed at 5-8V/cm.
Standard precautionary steps were followed while isolating RNA; to avoid contamination with Rnases.Glass wares used were baked at 200°c for 5-6 hours. Strict standardization procedures according to the international norms were followed for the RNA extraction procedure including preparation of stock solutions, instrument usage and sterilization. The blood sample was centrifuged and the buffy coat obtained was immediately mixed with 2 ml guanidine isothiocyanate solution. Subsequently, total RNA was extracted using the acid guanidine isothiocyanate-phenol-chloroform (GITC) method.
RNA electrophoresis was carried on a formaldehyde agarose gel at 6-7 V/cm in the submarine gel electrophoresis system with 1X Formaldehyde gel running buffer. RNA was quantified to determine the quality and concentration for its subsequent use in RT-PCR, by 1ml in alkaline water and reading the A260 & A280 in a UV spectrophotometer.
In order to remove the contaminating DNA from the RNA, Dnase I treatment was given, as there should not be any false positive result due to contamination with the DNA.
The gene specific primers for CK-19 were taken according to Datta et al and were obtained from Hysel India Limited, New Delhi.
CK19 (Forward CK1)- 5′-AAG CTA ACC ATG CAG AAC CTC AAC GAC CGC -
1 mg of Dnase I treated RNA was used for reverse transcription along with the gene specific primers (CK19 1,CK19 2 and β actin 1 and β actin 2) in a PTC-150 Minicycler (MJ Research). The kit involves the usage of a blend of Moloney variant of Murine Leukemia Virus (M-MLV) with Avian Myeloblastosis virus (AMV) reverse transcriptase on the RNA at gradations of 47°C for 30 minutes, 50°C for 15 minutes and inactivated at 94°C for 2 minutes and then run at the PCR conditions for 35 cycles with appropriate positive and negative controls. Subsequently, the samples were loaded on a DNA gel and viewed in a transilluminator to appreciate any amplification.
The tissues obtained after the incisional biopsies were immediately processed and stained with haematoxylin and eosin, which confirmed squamous cell carcinoma with degrees of differentiation.
Amplification was obtained for the β actin RT-PCR that was set up for positive control among both the control and study population
Molecular biology has profound influence on the basic understanding of several disease processes. The study of genetics and its role in causing human disease is now widely recognised as being at the forefront of medical research. In itself, this approach has been a departure from conventional histopathology, where the broad tissue patterns and cytological appearance observed are a result of the combined expression of tens or thousands of genes.
There are two types of genes based on their expression patterns: Housekeeping genes that are expressed in all tissues (eg. β actin) and tissue specific genes, which have specific functions in specific tissues (eg. CK19 in oral epithelium). While tissue specific genes are present in every cell of all tissues, they are expressed by the formation of mRNA followed by the formation functional protein in specific cells or tissue. Cytokeratins are one of the main families of intermediate filaments that are predominantly expressed in epithelial cells where they show strict lineage and differentiation associated patterns of expression. Malignant cells generally retain their intermediate filaments of their progenitor type and consequently cytokeratins have been used to characterize neoplastic cells. Among these, cytokeratin19, a 19 Kda epithelial cytoskeletal protein is considered as an epithelial marker as they are not expressed in normal haematopoietic tissues. It is believed that malignant epithelial cells are released into the blood stream during incisional biopsy and these free floating cells may establish metastasis elsewhere. Our objective was to test if we can detect these released cells by a sensitive and powerful method like RT-PCR; an adaptation of PCR. PCR is a sensitive technique to detect the presence of specific DNA sequences. The preference of RT-PCR over routine PCR is due to the fact that the closeness of exons in mRNA reduces the segment to be amplified, especially in situations where the point of interest in the genome or gene to be amplified is too large.
Primer design is of paramount importance, as it should specifically detect the sequence of interest CK19 in this case and not with other non-specific entities. Further, they should be specific enough to distinguish between members of a family of genes or the functional gene and its pseudogene. Our primers were specific as shown by the amplification of the β actin and CK19 genes from the isolated genomic DNA and that our PCR technique is sensitive enough to detect the genes in the human genome. Under the same optimized conditions, RT-PCR was performed to detect mRNA expression of CK19 gene. However, no amplification was obtained for the same.
At this juncture, rationale thinking on non-amplification of CK19 gene expression by RT-PCR has thrown us with many possible considerations for the same.
Where have the Circulating Cells Gone?
Cancer cells after gaining access to the venous circulation are completely trapped in the vasculature of the organs of first encounter eg. The lung and vertebrae for cancers draining into the vena cava. Large numbers of circulating cancer cells may be killed in the microvasculature by haemodynamic destruction, by elements of the host surveillance system such as macrophages and natural killer cells. Nabil Hanna in her literature review on NK cells has mentioned the discovery of a fast clearance assay of circulating tumour cells invivo that selectively detects NK cell mediated tumouricidal activity.
Cytotoxic T Lymphocytes (CTL) recognise antigenic peptides only when they are presented in association with major histocompatibility ( MHC) class 1 molecules . A lack of MHC class 1 antigens on neoplastic cells would thus be as effective as a lack of tumour specific antigens in abrogating or evading immune surveillance.
The cytolytic conjugate between CTLs and tumour cells is stabilized by interactions between the LFA-1 integrin on the lymphocytes and the ICAM-1 ligand expressed by the target cell. Lack of expression of ICAM -1, would appear to permit circulating tumour cells to avoid establishing stable cytolytic conjugates, and might provide a mean of evading CTL mediated killing.
Moreover, metastasis is a dynamic and periodic event, and may not be ongoing at the time of a single blood flow. Interestingly many studies showed that the circulating cancer cells were transiently (15 minutes) detectable.
The time interval was followed accordingly in our study. Blood samples were drawn immediately before biopsy to check if there was any previous dissemination.
Time intervals between the samples may need to be defined for each tumour type or in relation to their metastatic potential, as the timing of samples in relation to invasive procedures may be critical.
Threshold of RT-PCR
Length of the Expected Fragment
Several authors have been successful in detecting occult metastasis especially in relation to breast carcinoma, lung carcinoma, and prostrate carcinoma using the CK 19 RTPCR assay.,, The same primer design has been followed in the present study as well .
Integrity of RNA
Cole et al\ discuss that the incisional biopsies for surface tumours such as oral cavity, bronchus may result in oozing of blood from the surface of the biopsy site. Since any bleeding from a surface biopsy is not confined, there should be no elevation of tissue pressure and therefore no possibility of intravasation of the cancer cells.
Even if there are viable tumour cells after the incisional biopsy, the first line of defence like the natural killer cells, nm23 gene expression and elaboration of natural inhibitor proteins such as TIMPs plasminogen inhibitors, will function as metastasis suppressors and prevent tumour cell invasion. Moreover neoplasms are biologically heterogeneous and the process of metastasis is selective and depends on a combination of factors., , , 
Genetic and Environmental Factors
Newer markers such asCK20 may be more fruitful in reliably detecting dissemination as few authors suggest that CK19 may not be a very powerful marker.
However, our patients in the study are so far free of any recurrence or metastasis after the treatment after being followed up for the past one and half years and hence suggesting that the molecular technique could have been accurate in assessing the dissemination status.
ConclusionThe results of our study show that there is no dissemination of cancer cells after incisional biopsy into the circulation in oral squamous cell carcinoma. The patients too are disease free after one and half years of follow up. However, larger sample size with the use of several markers would definitely increase the specificity and reliability of these PCR based assays.
Copyright 2005 - Journal of Cancer Research and Therapeutics
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