Every single step for all molecular tests must be controlled for completion to avoid false results. Appropriate controls must be run, especially for restriction enzyme digestions. Because XCI analysis is based on differential DNA methylation of one allele from X-chromosome genes (e.g., human androgen receptor gene) and relies on endonuclease digestion by methylation-sensitive restriction enzymes, suboptimal enzymatic digestion provides a changed clonality pattern in the case of monoclonal tissues. Likewise, abnormal methylation provides potentially the same false result in the case of hypermethylation, whereas hypomethylation could affect the clonality pattern in polyclonal tissues giving a pseudomonoclonal one (see Fig. 6). Additionally, methylation abnormalities occur during the course of malignant transformation (37). Hypomethylation has been described in relation to increased proliferation during early stages of neoplasms, whereas hypermethylation has been linked to late stages associated with a higher mutation rate and tumor progression (42).
PCR bias against one allele (especially the larger one) can result in preferential amplification of the other allele (usually the smaller) (24,49). An appropriate extraction method providing DNA of enough quality (3,19), and PCR designs including both long denaturation and extension during the first three cycles, and 7-deaza-deoxy-guanidine triphosphate (dGTP) in the amplification mixture to improve the amplification of CG-rich DNA regions reasonably avoids that bias (18,20,49).
Several chemical modifications are induced in tissues by fixation and processing, including cross-linking between basic amino acids of proteins and the amino groups on DNA bases. Nonspecific amplifications are caused mainly by primer-independent, but DNA polymerase-and cycling-dependent, incorporation of nucleotides into DNA, possibly related to DNA repair and/or internal priming (40). This is the reason for complete and intense protein digestion before DNA purification (19). Denaturing reagents can break the cross-linked strands but in turn provide short-length DNA strands, precluding their use in protocols that require DNA of 250 to 500 bp in length. The general DNA quality of the extracted DNA should be tested by gel electrophoresis of the protein-digested sample, universal DNA amplification using degenerated oligonucleotide primer–PCR, or amplifying β-globin gene with primer sets at least 100 bp lengthier than the final DNA target.
The easiest protocol giving the highest DNA quality is 55 to 60°C prolonged proteinase K digestion (5–7 days, with every-day enzyme replacement) (19). The standard phenol–chloroform purification protocol results in the best contaminant-free DNA for any PCR application. Negative amplification resulting from sample contamination (specific amplification lacking with no primer dimers) can be avoided by diluting the sample.
Certain applications need original DNA strands, such as those based on the genomic imprinting of XCI. The presence of methylated cytosine can be tested by sample digestion by methylation-sensitive restriction endonucleases. In any case, appropriate internal control should be included to prove complete digestion. The samples already show smear patterns, and even they may be undetectable using gel electrophoresis (especially for microdissected samples). A logical way to accomplish this issue is to include DNA mimickers in every sample undergoing restriction enzyme digestion. These mimickers are normally viral DNA (such as phages) and should fulfill some requirements. They must be linear and double stranded (like human genomic DNA), and they must contain base sequences recognized by the tested enzyme with reliable pre-and postdigestion patterns. In addition, no sequence similarity able to give nonspecific amplification in further PCR should be present. Xho-I-linearized φX174RII phage represents an ideal mimic for HhaI digestion used for XCI analysis.
XCI analysis tests the differential methylation level in a CpG island approximately 100 bp upstream of the CAG repeat (56). Different methylation-sensitive restriction enzymes have been used, especially HhaI and HpaII. The first provides more reliable results because of its activity with single-strand DNA (that activity has not been demonstrated for HpaII). We must keep in mind that the embedding process partly denatures DNA and, therefore, single-strand DNA is a normal component in archival material.
DNA samples from microdissected tissues are not ideal for reliable quantification and are generally run with unknown target DNA concentration. So, relative quantification of both allelic bands must be taken into consideration to determine whether LOH (or allelic imbalance) is present. This issue brings us to consider the labeling and detection method. 32P- and 33P-based radiolabeling represent the standard protocols, including both external (one primer is 5` labeled) and internal (labeled nucleotide in the PCR mixture) methods. Although the latter usually gives more background, it permits the highest sensitivity for microdissected paraffin-embedded samples. Allelic separation can be achieved by running the samples far enough into high-resolution denaturing polyacrylamide gels (variable concentrations of formamide and urea). Some other detection methods have been used, including fluorescent labeling (6,7) and silver staining of PCR products (38). The highest sensitivity is achieved by radioisotopic methods, which remains the standard for molecular detection of genetic alterations, especially in formalin-fixed, paraffin-embedded material. Additionally, the ratio between signal and initial DNA amount is highly variable for silver-stained gels, making the applications of this technique less reliable for quantification. Different technical approaches have been used to detect interstitial DNA deletion and single base changes (mutations/polymorphisms), including single-strand conformational polymorphism, denaturant gradient gel electrophoresis, mutant allele-specific amplification, ribonuclease (RNase) protection, etc. (27,50). Although the final proof for any mutation must be direct sequencing, one of the most sensitive methods for detecting single base changes is PCR/denaturant gradient gel electrophoresis, which is able to distinguish DNA strands differing in only one base (3,18).
Lastly, the linear ratio between radioactive emission and signal deposition can be maintained by film preflashing to get a 0.1 to 0.2-OD unit absorbance increase at 540 nm in the preflashed film. In addition, signal stabilization during autoradiogram development requires −70°C storage. The allelic ratio has to be quantitated in normalized samples to exclude any potential contamination with normal tissue (43). At that level, different computer software is available to aid in analysis.
Presented in part at the Xth International Congress of Histochemistry and Cytochemistry, Kyoto, Japan, August 18–23, 1996; and at the XXIst International Congress of the International Academy of Pathology, Budapest, Hungary, October 20–25, 1996.
1. Alman BA, Pajerski ME, Diaz–Cano S, Corboy K, Wolfe HJ. Aggressive fibromatosis (desmoid tumor) is a monoclonal disorder. Diagn Mol Pathol 1997; 6:98–101.
2. Boland CR. Setting microsatellites
free. Nat Med 1996; 2:972–4.
3. Brady SP, Magro CM, Diaz–Cano SJ, Wolfe HJ. Analysis of clonality
of atypical cutaneous lymphoid infiltrates associated with drug therapy by PCR/DGGE. Hum Pathol 1999; 30:130–6.
4. Brentnall TA. Microsatellite instability. Shifting concepts in tumorigenesis. Am J Pathol 1995; 147:561–3.
5. Califano J, van der Riet P, Westra W, et al. Genetic progression model for head and neck cancer: implications for field cancerization. Cancer Res 1996; 56:2488–92.
6. Cawkwell L, Bell SM, Lewis FA, Dixon MF, Taylor GR, Quirke P. Rapid detection of allele loss in colorectal tumours using microsatellites
and fluorescent DNA technology. Br J Cancer 1993; 67:1262–7.
7. Cawkwell L, Li D, Lewis FA, Martin I, Dixon MF, Quirke P. Microsatellite instability in colorectal cancer: improved assessment using fluorescent polymerase chain reaction. Gastroenterology 1995; 109:465–71.
8. Chen LC, Kurisu W, Ljung BM, Goldman ES, Moore DI, Smith HS. Heterogeneity for allelic loss in human breast cancer. J Natl Cancer Inst 1992; 84:506–10.
9. Cordon–Cardo C. Mutations of cell cycle regulators. Biological and clinical implications for human neoplasia. Am J Pathol 1995; 147:545–60.
10. Cossman J, Zehnbauer B, Garrett CT, et al. Gene rearrangements in the diagnosis of lymphoma/leukemia. Guidelines for use based on a multiinstitutional study. Am J Clin Pathol 1991; 95:347–54.
11. Cuatrecasas M, Matias–Guiu X, Prat J. Synchronous mucinous tumors of the appendix and the ovary associated with pseudomyxoma peritonei. A clinicopathologic study of six cases with comparative analysis of c-Ki-ras mutations. Am J Surg Pathol 1996; 20:739–46.
12. de Alava E, Lozano MD, Patino A, Sierrasesumaga L, Pardo–Mindan FJ. Ewing family tumors: potential prognostic value of reverse–transcriptase polymerase chain reaction detection of minimal residual disease in peripheral blood samples. Diagn Mol Pathol 1998; 7:152–7.
13. Deng G, Lu Y, Zlotnikov G, Thor AD, Smith HS. Loss of heterozygosity in normal tissue adjacent to breast carcinomas. Science 1996; 274:2057–9.
14. Diaz–Cano SJ. Designing a molecular analysis of clonality
in tumours. J Pathol 2000; 191:343–4.
15. Diaz–Cano SJ. Clonality
studies in the analysis of adrenal medullary proliferations: application principles and limitations. Endocr Pathol 1998; 9:301–16.
16. Diaz–Cano S. PCR-based alternative for diagnosis of immunoglobulin heavy chain gene rearrangement: principles, practice, and polemics. Diagn Mol Pathol 1996; 5:3–9.
17. Diaz–Cano SJ, Blanes A. Influence of intratumor heterogeneity in the interpretation of marker results in pheochromocytomas. J Pathol 1999; 189:627–8.
18. Diaz–Cano SJ, Blanes A, Rubio J, Matilla A, Wolfe HJ. Molecular evolution and intratumor heterogeneity by topographic compartments in muscle-invasive transitional cell carcinoma of the urinary bladder. Lab Invest 2000; 80:279–89.
19. Diaz–Cano SJ, Brady SP. DNA extraction from formalin-fixed, paraffin-embedded tissues
: protein digestion as a limiting step for retrieval of high-quality DNA. Diagn Mol Pathol 1997; 6:342–6.
20. Diaz–Cano SJ, de Miguel M, Blanes A, Tashjian R, Galera H, Wolfe HJ. Clonality
as expression of distinctive cell kinetics patterns in nodular hyperplasias and adenomas of the adrenal cortex. Am J Pathol 2000; 156:311–9.
20a. Diaz-Cano SJ, de Miguel M, Blanes A, Tashjian R, Galera H, Wolfe HJ. Clonal patterns in phaechromocytomas and MEN-2A adrenal medullary hyperplasias: histologic and kinetic correlates. J Pathol 2000; 192:221–8.
21. Diaz–Cano SJ, Wolfe HJ. Clonality
in Kaposi's sarcoma. N Engl J Med 1997; 337:571–2.
22. Fialkow PJ. Clonal origin of human tumors. Biochim Biophys Acta 1976; 458:283–321.
23. Fialkow PJ. Primordial cell pool size and lineage relationships of five human cell types. Ann Hum Genet 1973; 37:39–48.
24. Findlay I, Matthews P, Quirke P. Multiple genetic diagnoses from single cells using multiplex PCR: reliability and allele dropout. Prenat Diagn 1998; 18:1413–21.
25. Gaffey MJ, Iezzoni JC, Weiss LM. Clonal analysis of focal nodular hyperplasia of the liver. Am J Pathol 1996; 148:1089–96.
26. Gilliland DG, Blanchard KL, Levy J, Perrin S, Bunn HF. Clonality
in myeloproliferative disorders: analysis by means of the polymerase chain reaction. Proc Natl Acad Sci U S A 1991; 88:6848–52.
27. Housman DE. DNA on trial—the molecular basis of DNA fingerprinting. N Engl J Med 1995; 332:534–5.
28. Hugel A, Wernert N. Loss of heterozygosity (LOH), malignancy grade and clonality
in microdissected prostate cancer. Br J Cancer 1999; 79:551–7.
29. Jenkins RB, Qian J, Lieber MM, Bostwick DG. Detection of c-myc oncogene amplification and chromosomal anomalies in metastatic prostatic carcinoma by fluorescence in situ hybridization. Cancer Res 1997; 57:524–31.
30. Jones PA, Droller MJ. Pathways of development and progression in bladder cancer: new correlations between clinical observations and molecular mechanisms. Semin Urol 1993; 11:177–92.
31. Kappler JW. The 5-methylcytosine content of DNA: tissue specificity. J Cell Physiol 1971; 78:33–6.
32. Knudson AG. Antioncogenes and human cancer. Proc Natl Acad Sci U S A 1993; 90:10914–21.
33. Knudsen Jr. AG Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci U S A 1971; 68:820–3.
34. Knudsen Jr, AG Hethcote HW, Brown BW. Mutation and childhood cancer: a probabilistic model for the incidence of retinoblastoma. Proc Natl Acad Sci U S A 1975; 72:5116–20.
35. Koch WM, Brennan JA, Zahurak M, et al. p53 Mutation and locoregional treatment failure in head and neck squamous cell carcinoma. J Natl Cancer Inst 1996; 88:1580–6.
36. Koreth J, O'Leary JJ, McGee JOD. Microsatellites
and PCR genomic analysis. J Pathol 1996; 178:239–48.
37. Laird PW, Jaenisch R. DNA methylation and cancer. Hum Mol Genet 1994; 3:1487–95.
38. Lau DH, Yang B, Hu R, Benfield JR. Clonal origin of multiple lung cancers: K-ras and p53 mutations determined by nonradioisotopic single-strand conformation polymorphism analysis. Diagn Mol Pathol 1997; 6:179–84.
39. Lewis SM, Wu GE. The origin of V(D)J recombination. Cell 1997; 88:159–62.
40. Long AA, Komminoth P, Lee E, Wolfe HJ. Comparison of indirect and direct in-situ polymerase chain reaction in cell preparations and tissue sections. Detection of viral DNA, gene rearrangements and chromosomal translocations. Histochemistry 1993; 99:151–62.
41. Lyon MF. Some milestones in the history of X-chromosome inactivation. Annu Rev Genet 1992; 26:16–28.
42. Magewu AN, Jones PA. Ubiquitous and tenacious methylation of the CpG site in codon 248 of the p53 gene may explain its frequent appearance as a mutational hot spot in human cancer. Mol Cell Biol 1994; 14:4225–32.
43. Mutter GL, Boynton KA. X chromosome inactivation
in the normal female genital tract: implications for identification of neoplasia. Cancer Res 1995; 55:5080–4.
44. Nowell PC. The clonal evolution of tumor cell populations. Science 1976; 194:23–8.
45. Perren A, Roth J, Muletta–Feurer S, et al. Clonal analysis of sporadic pancreatic endocrine tumours. J Pathol 1998; 186:363–71.
46. Quade BJ, McLachlin CM, Soto–Wright V, Zuckerman J, Mutter GL, Morton CC. Disseminated peritoneal leiomyomatosis. Clonality
analysis by X chromosome inactivation
and cytogenetics of a clinically benign smooth muscle proliferation. Am J Pathol 1997; 150:2153–66.
47. Rabkin CS, Janz S, Lash A, et al. Monoclonal origin of multicentric Kaposi's sarcoma lesions. N Engl J Med 1997; 336:988–93.
48. Randerson J, Cawkwell L, Jack A, et al. Fluorescent polymerase chain reaction of a panel of CA repeats on chromosome 6 in the indolent phase of follicular centre cell lymphoma. Br J Cancer 1996; 74:942–6.
49. Ray PF, Handyside AH. Increasing the denaturation temperature during the first cycles of amplification reduces allele dropout from single cells for preimplantation genetic diagnosis. Mol Hum Reprod 1996; 2:213–18.
50. Rosenthal N. Molecular medicine. Recognizing DNA. N Engl J Med 1995; 333:925–7.
51. Sager R. Tumor suppressor genes
: the puzzle and the promise. Science 1989; 246:1406–12.
52. Shibata D, Schaeffer J, Li ZH, Capella G, Perucho M. Genetic heterogeneity of the c-K-ras locus in colorectal adenomas but not in adenocarcinomas. J Natl Cancer Inst 1993; 85:1058–63.
53. Sidransky D, Frost P, Von Eschenbach A, Oyasu R, Preisinger AC, Vogelstein B. Clonal origin bladder cancer. N Engl J Med 1992; 326:737–40.
54. Sinnock KL, Perez–Atayde AR, Boynton KA, Mutter GL. Clonal analysis of sacrococcygeal “teratomas.” Pediatr Pathol Lab Med 1996; 16:865–75.
55. Sleddens HF, Oostra BA, Brinkmann AO, Trapman J. Trinucleotide (GGN) repeat polymorphism in the human androgen receptor (AR) gene. Hum Mol Genet 1993; 2:493.
56. Sleddens HF, Oostra BA, Brinkmann AO, Trapman J. Trinucleotide repeat polymorphism in the androgen receptor gene (AR). Nucl Acid Res 1992; 20:1427.
57. Vogelstein B, Fearon ER, Hamilton SR, et al. Genetic alterations during colorectal-tumor development. N Engl J Med 1988; 319:525–32.
58. Vogelstein B, Fearon ER, Kern SE, et al. Allelotype of colorectal carcinomas. Science 1989; 244:207–11.
59. Wolman SR, Heppner GH. Genetic heterogeneity in breast cancer. J Natl Cancer Inst 1992; 84:469–70.
60. Zhuang Z, Lininger RA, Man YG, Albuquerque A, Merino MJ, Tavassoli FA. Identical clonality
of both components of mammary carcinosarcoma with differential loss of heterozygosity. Mod Pathol 1997; 10:354–62.