Validation of tumor markers in central nervous system germ cell tumors by real-time reverse transcriptase polymerase chain reaction using formalin-fixed paraffin-embedded tissues
- Authors:
- Published online on: October 23, 2012 https://doi.org/10.3892/mmr.2012.1144
- Pages: 337-341
Abstract
Introduction
With the emergence of versatile imaging modalities, stereotactic or endoscopic biopsy has become the gold standard for confirming brain tumors (1). However, in small biopsies, the tissue obtained may not truly represent the whole lesion due to the small amount of tissue compared to the excisional specimen or may not be diagnostic due to artifacts such as electrocauterization or pinching (2,3). As the therapeutic protocols for treatment of germinomas and non-germinomatous germ cell tumors (NGGCTs) are completely different, it is important to distinguish pure germinomas from NGGCTs (4–7). Immunohistochemistry (IHC) is broadly used as an essential ancillary test in order to reach an exact diagnosis. Despite the importance of IHC, in everyday practice problems such as misinterpretation of results and aberrant loss of immunoreactivity are frequently encountered. Reverse transcriptase polymerase reaction (RT-PCR) emerged in the mid-1980s and became routine in most laboratories due to its reliable amplification of target DNA sequences with relatively small quantities of starting deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) (8). In addition to dichotomous results from IHC, the recent introduction of more advanced real-time RT-PCR has enabled the quantitative analysis of levels of expression for target markers. Moreover, this quantitative analysis may reflect submorphologic alterations that were not observed in the usual hematoxylin and eosin (H&E) stained sections. To evaluate the possibilities of this new type of analysis as an alternative ancillary test for the diagnosis of brain germ cell tumors, we performed real-time RT-PCR on formalin-fixed paraffin-embedded (FFPE) tissues. After a meticulous literature search, we selected eight markers of germ cell tumors with relatively consistent expression levels for real-time RT-PCR. These markers were POU5F1, TGFB2, SLUG, NANOG, TWIST2 and cytokeratin 8, 18 and 19 (9–16). We performed this new PCR technique on the above molecular markers and statistically analyzed the results using SPSS.
Materials and methods
Patients and tissue samples
Samples of 30 pure germinomas and 30 NGGCTs from 60 patients (one case from each patient) treated between 1997 and 2010 were retrieved from the Pathology Department archive at Yonsei University Health System. Twenty-nine were biopsied tissue and 31 were removed tumor specimens <3 cm3. Two neuropathologists independently reviewed the slides and a consensus diagnosis was reached. Among the 30 NGGCTs, 22 were teratomas (including 2 immature teratomas), 4 were mixed germ cell tumors, 3 were yolk sac tumors and 1 was a choriocarcinoma. This study was approved by the Institutional Review Board (IRB) of Medicine of Yonsei University Severance Hospital, Seoul, Korea (IRB no. 4-2010-0060). Written informed consent was obtained from the patient or their family.
Quantitative real-time polymerase chain reaction
It is well-known that mRNAs of POU5F1 (Oct4), NANOG, KLF4, podoplanin and CD133 are preferentially expressed in fresh germinoma cells. On the contrary, mRNAs of cytokeratin 8, 18 and 19, LOX, PDGFR-α, TGF-β2, TWIST2 and SLUG are preferentially expressed in fresh tissue of NGGCTs (9–16).
Total RNA was isolated from FFPE blocks using the RNeasy Mini kit (cat no. 74404; Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The cDNA was synthesized from 1 μg total RNA using Maxime RT PreMix (cat no. 25082; Intron Biotechnology, Seongnam, Korea) and anchored oligo(dt)15-primers (Table I). Real-time PCR was performed with the 7300 Real-Time PCR System™ (Applied Biosystems, Foster City, CA, USA) using the SYBR-Green PCR Master mix™ (cat no. 4309155; Applied Biosystems). The relative amount of target mRNA was determined using an established comparative threshold (Ct) method which normalizes target mRNA Ct values (17,18).
Statistical analyses
Traditionally, central nervous system germ cell tumors are divided into two major categories: germinomas and NGGCTs. NGGCTs can be further classified into (mature) teratomas and malignant germ cell tumors (19,20). We decided to apply these clinically oriented classification schemes to our data. For the detailed analysis, the tumors were divided into three groups (germinoma, teratoma and malignant germ cell tumor) and multivariate analysis of variance (MANOVA) was performed, which mitigates type I errors by multiple executions of ANOVA. This was followed by Bonferroni’s method, which is known to be the most conservative and rigorous post-hoc test (21). The second discriminant analysis, which enables two-group discrimination, was applied to the above two major categories. Statistical analysis was performed with SPSS version 12.0 software (SPSS, Chicago, IL, USA). P-values of <0.05 were considered to indicate a statistically significant difference.
Results
Real-time RT-PCR
All samples were successfully analyzed by real-time RT-PCR (Table II). The majority of RNA samples were in good condition. They had optical density ratios 260/280 between 1.8 and 2.0 (22).
Multiple comparisons
Retrieved cases were assigned to three groups as follows: pure germinoma, mature teratoma and malignant germ cell tumor (Table II). MANOVA was performed and Bonferroni’s method was used as a post-hoc correction. The results are shown in Tables III and IV, respectively. Among the selected genes, POU5F1, TWIST2, NANOG and TGFB2 were statistically significant, and these results were consistent with the previous literature, which used conventional immunohistochemistry or other ancillary tests on germ cell tumors (9–16).
Two-group discriminant analysis
Retrieved cases were randomly assigned to two groups as follows: pure germinoma (group 1) and non-germinomatous germ cell tumor (group 2). Genes that were statistically significant in the previous MANOVA (POU5F1, TWIST2, NANOG and TGFB2) correctly discriminate 76.7% of original groups and 70% of cross-validation groups (Table V). In general, when discriminant functions classify >70% of cases correctly (hit ratio), it is considered to demonstrate good discriminating power (23).
Discussion
Real-time RT-PCR was introduced in the mid-1990s as a quantitative test for mRNA. Conventional IHC can be partly utilized as a semi-quantitative test for proteins, but there was no test for mRNA until this method emerged. Our primary aim was to introduce this quantitative test to FFPE-based pathology practice, particularly for the diagnosis of CNS germ cell tumors.
Data summarized in Table IV show markers that are useful to separate various germ cell tumors. POU5F1 is useful to discriminate mature teratomas from malignant germ cell tumors. TWIST2 is useful to discriminate pure germinomas from malignant germ cell tumors. NANOG and TGFB2 are useful to discriminate germ cell tumors with a benign clinical course from malignant germ cell tumors. Therefore, real-time RT-PCR may be applied more specifically to these particular situations. For example, in differentiating pure germinoma and malignant germ cell tumors, high -ΔCt of POU5F1 may be an indication of pure germinoma. For mature teratomas and malignant germ cell tumors, high -ΔCt of TWIST2 implies a malignant germ cell tumor. If one focuses on the benign vs. malignant problem, high -ΔCt of NANOG and TGFB2 are suggestive of malignancy. Although it is beyond the scope of this paper to determine the critical cut-off ΔCt value of the marker genes mentioned above, in difficult situations, the correct diagnosis may be made using a combination of the selected genes. Discriminant analysis also correctly sorts 70% of pure germinomas from other tumor types (Table V). All of our results suggest that real-time RT-PCR of FFPE tissues is a useful method for quantitative analysis in the diagnosis of CNS germ cell tumors.
In our opinion, although real-time RT-PCR cannot completely replace conventional immunohistochemistry in small biopsy specimens due to its labor-intensiveness, real-time RT-PCR is a second-line adjunctive test when IHC results are not informative.
There are several papers comparing the results of real-time RT-PCR from fresh tissue and from FFPE tissue such as lymph node, breast and lung cancer tissues (24–26). Although this type of validation is more direct and confirmative, it is not easy to obtain fresh brain tissue without hindrance to the correct diagnosis, so we decided to indirectly compare data from RT-PCR to data from the published literature, and have concluded that our results are consistent with previous results.
In conclusion, our experiment statistically validates real-time RT-PCR in FFPE-based pathology practice by using multivariate methods in neuropathology, which is in agreement with other papers that utilized RT-PCR on other types of pathology (27–31). Additionally, we showed that mRNAs of germ cell tumors were robust in FFPE tissue for a period of approximately 10 years. Real-time RT-PCR may be an alternative ancillary test, particularly when conventional immunohistochemistry is not available or not sufficient to make a correct diagnosis. With further study, it could be used as a primary diagnostic ancillary test in the future, similar to IHC.
Acknowledgements
This study was supported by the Yonsei University College of Medicine Research Fund to Se Hoon Kim (6-2011-0088).
References
|
Tirakotai W, Bozinov O, Sure U, Riegel T, Bertalanffi H, et al: The evolution of stereotactic guidance in neuroendoscopy. Child Nerv Syst. 20:790–795. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Luther N, Edgar MA, Dunkel IJ and Souweidane MM: Correlation of endoscopic biopsy with tumor marker status in primary intracranial germ cell tumors. J Neurooncol. 79:45–50. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Noh S, Kim DS, Kim J and Kim SH: Langerhans cell histiocytosis in endoscopic biopsy: marked pinching artifacts by endoscopy. Brain Tumor Pathol. 28:285–289. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Sawamura Y: Strategy of combined treatment of germ cell tumors. Prog Neurol Surg. 23:86–95. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Ogawa K, Toita T, Nakamura K, Uno T, Onishi H, et al: Treatment and prognosis of patients with intracranial nongerminomatous malignant germ cell tumors: a multiinstitutional retrospective analysis of 41 patients. Cancer. 98:369–376. 2003. View Article : Google Scholar | |
|
Kaur H, Singh D and Peereboom DM: Primary central nervous system germ cell tumors. Curr Treat Options Oncol. 4:491–498. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Kochi M, Itoyama Y, Shiraishi S, Kitamura I, Marubayashi T and Ushio YJ: Successful treatment of intracranial nongerminomatous malignant germ cell tumors by administering neoadjuvant chemotherapy and radiotherapy before excision of residual tumors. Neurosurg. 99:106–114. 2003. View Article : Google Scholar | |
|
King N: RT-PCR Protocols. 2nd edition. Humana Press; New York: 2010, View Article : Google Scholar | |
|
Lifschitz-Mercer B, Fogel M, Moll R, Jacob N, Kushnir I, Livoff A, et al: Intermediate filament protein profiles of human testicular non-seminomatous germ cell tumors: correlation of cytokeratin synthesis to cell differentiation. Differentiation. 48:191–198. 1991. View Article : Google Scholar : PubMed/NCBI | |
|
Hattab EM, Tu PH, Wilson JD and Cheng L: OCT4 immunohistochemistry is superior to placental alkaline phosphatase (PLAP) in the diagnosis of central nervous system germinoma. Am J Surg Pathol. 29:368–371. 2005. View Article : Google Scholar | |
|
Cossu-Rocca P, Jones TD, Roth LM, Eble JN, Zheng W, Karim FW, et al: Cytokeratin and CD30 expression in dysgerminoma. Hum Pathol. 37:1015–1021. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Santagata S, Hornick JL and Ligon KL: Comparative analysis of germ cell transcription factors in CNS germinoma reveals diagnostic utility of NANOG. Am J Surg Pathol. 30:1613–1618. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Biermann K, Heukamp LC, Nettersheim D, Steger K, Zhou H, et al: Embryonal germ cells and germ cell tumors. Verh Dtsch Ges Pathol. 91:39–48. 2007.(Article in German). | |
|
Biermann K, Heukamp LC, Steger K, Zhou H, Franke FE, et al: Genome-wide expression profiling reveals new insights into pathogenesis and progression of testicular germ cell tumors. Cancer Genomics Proteomics. 4:359–367. 2007.PubMed/NCBI | |
|
Looijenga LH: Human testicular (non)seminomatous germ cell tumours: the clinical implications of recent pathobiological insights. J Pathol. 218:146–162. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Idrees M, Saxena R, Cheng L, Ulbright TM and Badve S: Podoplanin, a novel marker for seminoma: a comparison study evaluating immunohistochemical expression of podoplanin and OCT3/4. Ann Diagn Pathol. 14:331–336. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI | |
|
Van der Velden VHJ, Szczepanski T and van Dongen JJM: Polymerase chain reaction, real-time quantitative. Encyclopedia of Genetics. Elsevier; Amsterdam: pp. 1503–1506. 2001 | |
|
Matsutani M, Sano K, Takakura K, Fujimaki T, Nakamura O, et al: Primary intracranial germ cell tumors: a clinical analysis of 153 histologically verified cases. J Neurosurg. 86:446–455. 1997. View Article : Google Scholar : PubMed/NCBI | |
|
Fujimaki T: Central nervous system germ cell tumors: classification, clinical features, and treatment with a historical overview. J Child Neurol. 24:1439–1445. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Streiner DL and Norman GR: Correction for multiple testing: is there a resolution? Chest. 140:16–18. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Becker C, Hammerle-Fickinger A, Riedmaier I and Pfaffi MW: mRNA and microRNA quality control for RT-qPCR analysis. Methods. 50:237–243. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Huberty CJ and Olejnik S: Applied MANOVA and Discriminant Analysis. 2nd edition. Balding DJ, Cressie NAC, Fisher NI, et al: Wiley; Hoboken, NJ: 2006, View Article : Google Scholar | |
|
Abrahamsen HN, Steiniche T, Nexo E, Hamilton-Dutoit SJ and Sorensen BS: Towards quantitative mRNA analysis in paraffin-embedded tissues using real-time reverse transcriptase-polymerase chain reaction: a methodological study on lymph nodes from melanoma patients. J Mol Diagn. 5:34–41. 2003. View Article : Google Scholar | |
|
Gjerdrum LM, Sorensen BS, Kjeldsen E, Sorensen FB, Nexo E and Hamilton-Dutoit S: Real-time quantitiative PCR of microdissected paraffin-embedded breast carcinoma: an altanative method for HER2/neu analysis. J Mol Diagn. 6:42–51. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Zhang F, Wang Z, Liu H, Bai Y, Wei S, et al: Application of RT-PCR in formalin-fixed and paraffin-embedded lung cancer tissues. Acta Pharmacol Sin. 31:111–117. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Lehmann U and Kreipe H: Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies. Methods. 5:409–418. 2001. View Article : Google Scholar : PubMed/NCBI | |
|
Garrel R, Dromard M, Costes V, Barbotte E, Comte F, Gardiner Q, et al: The diagnostic accuracy of reverse transcription-PCR quantification of cytokeratin mRNA in the detection of sentinel lymph node invasion in oral and oropharyngeal squamous cell carcinoma: a comparison with immunohistochemistry. Clin Cancer Res. 12:2498–2505. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Mullins M, Perreard L, Quackenbush JF, Gauthier N, Bayer S, et al: Agreement in breast cancer classification between microarray and quantitative reverse transcription PCR from fresh-frozen and formalin-fixed, paraffin-embedded tissues. Clin Chem. 53:1273–1279. 2007. View Article : Google Scholar | |
|
Rogerson L, Darby S, Jabbar T, Mathers ME, Leung HY, et al: Application of transcript profiling in formalin-fixed paraffin-embedded diagnostic prostate cancer needle biopsies. BJU Int. 102:364–370. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Lu X, van der Straaten T, Tiller M and Li X: Evidence for qualified quantitative mRNA analysis in formalin-fixed and paraffin-embedded colorectal carcinoma cells and tissue. J Clin Lab Anal. 25:166–173. 2011. View Article : Google Scholar : PubMed/NCBI |
