Detection of Gene Amplification by Multiplex Ligation-Dependent Probe Amplification in Comparison with In Situ Hybridization and Immunohistochemistry

  • Tabarestani, Sanaz (Cancer Research Center, Shahid Beheshti University of Medical Sciences) ;
  • Ghaderian, Sayyed Mohammad Hossein (Department of Medical Genetics, Faculty of Medicine, Shahid Beheshti University of Medical Sciences) ;
  • Rezvani, Hamid (Department of Hematology/Oncology, Taleghani Hospital, Shahid Beheshti University of Medical Sciences)
  • Published : 2015.12.03


Gene amplification is an important mechanism in the development and progression of cancer. Currently, gene amplification status is generally determined by in situ hybridization (ISH). Multiplex ligation-dependent probe amplification (MLPA) is a PCR-based method that allows copy number detection of up to 50 nucleic acid sequences in one reaction. The aim of the present study was to compare results for HER2, CCND1, MYC and ESR1 gene amplification detected by MLPA with fluorescent in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) as clinically approved methods. Tissue samples of 170 invasive breast cancers were collected. All were ER positive. Tissue samples had previously been tested for HER2 using immunohistochemistry. Amplification of the selected genes were assessed using MLPA, FISH and CISH and results were compared. HER2 MLPA and ISH results were also compared with HER2 immunohistochemistry (IHC) which detects protein overexpression. Amplification of HER2, CCND1, MYC and ESR1 by MLPA were found in 9%, 19%, 20% and 2% of samples, respectively. Amplification of HER2, CCND1, MYC and ESR1 by FISH was noted in 7%, 16%, 16% and 1% of samples, respectively. A high level of concordance was found between MLPA/FISH (HER2: 88%, CCND1: 88%, MYC: 86%, ESR1: 92%) and MLPA/CISH (HER2: 84%). Of all IHC 3+ cases, 91% were amplified by MLPA. In IHC 2+ group, 31% were MLPA amplified. In IHC 1+ group, 2% were MLPA amplified. None of the IHC 0 cases were amplified by MLPA. Our results indicate that there is a good correlation between MLPA, IHC and ISH results. Therefore, MLPA can serve as an alternative to ISH for detection of gene amplification.


  1. Al-Kuraya K, Schraml P, Torhorst J, et al (2004). Prognostic relevance of gene amplifications and coamplifications in breast cancer. Cancer Res, 64, 8534-40.
  2. Anzick SL, Kononen J, Walker RL, et al (1997). AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science, 277, 965-8.
  3. Bartlett J, Mallon E, Cooke T (2003). The clinical evaluation of HER-2 status: which test to use? J Pathol, 199, 411-7.
  4. Bartlett JM, Going JJ, Mallon EA, et al (2001). Evaluating HER2 amplification and overexpression in breast cancer. J Pathol, 195, 422-8.
  5. Bunyan DJ, Eccles DM, Sillibourne J, et al (2004). Dosage analysis of cancer predisposition genes by multiplex ligationdependent probe amplification. Br J Cancer, 91, 1155-9.
  6. Chen Y and Olopade OI (2008). MYC in breast tumor progression. Expert Rev Anticancer Ther, 8, 1689-98.
  7. Courjal F, Cuny M, Rodriguez C, et al (1996). DNA amplifications at 20q13 and MDM2 define distinct subsets of evolved breast and ovarian tumours. Br J Cancer, 74, 1984-9.
  8. Deming SL, Nass SJ, Dickson RB, et al (2000). C-MYC amplification in breast cancer: a meta-analysis of its occurrence and prognostic relevance. Br J Cancer, 83, 1688-95.
  9. Dowsett M, Bartlett J, Ellis IO, et al (2003). Correlation between immunohistochemistry (HercepTest) and fluorescence in situ hybridization (FISH) for HER-2 in 426 breast carcinomas from 37 centres. J Pathol, 199, 418-23.
  10. Ejlertsen B, Aldridge J, Nielsen KV, et al (2012). Prognostic and predictive role of ESR1 status for postmenopausal patients with endocrine-responsive early breast cancer in the Danish cohort of the BIG 1-98 trial. Ann Oncol, 23, 1138-44.
  11. Elsheikh S, Green AR, Aleskandarany MA, et al (2008). CCND1 amplification and cyclin D1 expression in breast cancer and their relation with proteomic subgroups and patient outcome. Breast Cancer Res Treat, 109, 325-35.
  12. Farshid G, Cheetham G, Davies R, et al (2011). Validation of the multiplex ligation-dependent probe amplification (MLPA) technique for the determination of HER2 gene amplification in breast cancer. Diagn Mol Pathol, 20, 11-17.
  13. Garcia-Caballero T, Grabau D, Green AR, et al (2010). Determination of HER2 amplification in primary breast cancer using dual-colour chromogenic in situ hybridization is comparable to fluorescence in situ hybridization: a European multicentre study involving 168 specimens. Histopathology, 56, 472-80.
  14. Ghaffari SR, Sabokbar T, Dastan J, et al (2011). HER2 amplification status in Iranian breast cancer patients: comparison of immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH). Asian Pac J Cancer Prev, 12, 1031-4.
  15. Gong Y, Sweet W, Duh YJ, et al (2009). Chromogenic in situ hybridization is a reliable method for detecting HER2 gene status in breast cancer: a multicenter study using conventional scoring criteria and the new ASCO/CAP recommendations. Am J Clin Pathol, 131, 490-7.
  16. Harvey JM, Clark GM, Osborne CK, et al (1999). Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol, 17, 1474-81.
  17. Holm K, Staaf J, Jonsson G, et al (2012). Characterisation of amplification patterns and target genes at chromosome 11q13 in CCND1-amplified sporadic and familial breast tumours. Breast Cancer Res Treat, 133, 583-94.
  18. Holst F, Moelans CB, Filipits M, et al (2012). On the evidence for ESR1 amplification in breast cancer. Nat Rev Cancer, 12, 149.
  19. Holst F, Stahl PR, Ruiz C, et al (2007). Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer. Nat Genet, 39, 655-60.
  20. Jimenez RE, Wallis T, Tabasczka P, et al (2000). Determination of Her-2/Neu status in breast carcinoma: comparative analysis of immunohistochemistry and fluorescent in situ hybridization. Mod Pathol, 13, 37-45.
  21. Lebeau A, Deimling D, Kaltz C, et al (2001). Her-2/neu analysis in archival tissue samples of human breast cancer: comparison of immunohistochemistry and fluorescence in situ hybridization. J Clin Oncol, 19, 354-363.
  22. Lundgren K, Brown M, Pineda S, et al (2012). Effects of cyclin D1 gene amplification and protein expression on time to recurrence in postmenopausal breast cancer patients treated with anastrozole or tamoxifen: a TransATAC study. Breast Cancer Res, 14, 57.
  23. Moelans CB, de Weger RA, Monsuur HN, et al (2010). Molecular profiling of invasive breast cancer by multiplex ligation-dependent probe amplification-based copy number analysis of tumor suppressor and oncogenes. Mod Pathol, 23, 1029-39.
  24. Moelans CB, de Weger RA, van Blokland MT, et al (2009). HER- 2/neu amplification testing in breast cancer by multiplex ligation-dependent probe amplification in comparison with immunohistochemistry and in situ hybridization. Cell Oncol, 31, 1-10.
  25. Moelans CB, de Weger RA, van Diest PJ (2010). Multiplex ligation-dependent probe amplification to detect HER2 amplification in breast cancer: new insights in optimal cutoff value. Cell Oncol, 32, 311-2.
  26. Moelans CB, Monsuur HN, de Pinth JH, et al (2010). ESR1 amplification is rare in breast cancer and is associated with high grade and high proliferation: a multiplex ligationdependent probe amplification study. Cell Oncol, 34, 489-94.
  27. Musgrove EA, Caldon CE, Barraclough J, et al (2011). Cyclin D as a therapeutic target in cancer. Nat Rev Cancer, 11, 558-72.
  28. Ooi A, Inokuchi M, Harada S, et al (2012). Gene amplification of ESR1 in breast cancers--fact or fiction? A fluorescence in situ hybridization and multiplex ligation-dependent probe amplification study. J Pathol, 227, 8-16.
  29. Owens MA, Horten BC, Da Silva MM (2004). HER2 amplification ratios by fluorescence in situ hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues. Clin Breast Cancer, 5, 63-9.
  30. Park K, Han S, Shin E, et al (2007). EGFR gene and protein expression in breast cancers. Eur J Surg Oncol, 33, 956-60.
  31. Pauletti G, Dandekar S, Rong H, et al (2000). Assessment of methods for tissue-based detection of the HER-2/neu alteration in human breast cancer: a direct comparison of fluorescence in situ hybridization and immunohistochemistry. J Clin Oncol, 18, 3651-64.
  32. Pazhoomand R, Keyhani E, Banan M, et al (2013). Detection of HER2 status in breast cancer: comparison of current methods with MLPA and real-time RT-PCR. Asian Pac J Cancer Prev, 14, 7621-8.
  33. Press MF, Hung G, Godolphin W, et al (1994). Sensitivity of HER-2/neu antibodies in archival tissue samples: potential source of error in immunohistochemical studies of oncogene expression. Cancer Res, 54, 2771-7.
  34. Ross JS, Fletcher JA (1998). The HER-2/neu Oncogene in Breast Cancer: Prognostic Factor, Predictive Factor, and Target for Therapy. Oncologist, 3, 237-52.
  35. Santarius T, Shipley J, Brewer D, et al (2010). A census of amplified and overexpressed human cancer genes. Nat Rev Cancer, 10, 59-64.
  36. Schechter AL, Hung MC, Vaidyanathan L, et al (1985). The neu gene: an erbB-homologous gene distinct from and unlinked to the gene encoding the EGF receptor. Science, 229, 976-8.
  37. Schlotter CM, Vogt U, Bosse U, et al (2003). C-MYC, not HER- 2/neu, can predict recurrence and mortality of patients with node-negative breast cancer. Breast Cancer Res, 5, R30-36.
  38. Schnitt SJ, Jacobs TW (2001). Current status of HER2 testing: caught between a rock and a hard place. Am J Clin Pathol, 116, 806-10.
  39. Schouten JP, McElgunn CJ, Waaijer R, et al (2002). Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res, 30, e57.
  40. Slamon DJ, Leyland-Jones B, Shak S, et al (2001). Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med, 344, 783-92.
  41. Tabarestani S, Ghaderian SM, Rezvani H, et al (2014). Prognostic and predictive value of copy number alterations in invasive breast cancer as determined by multiplex ligation-dependent probe amplification. Cell Oncol (Dordr), 37, 107-18.
  42. Tsuda H, Akiyama F, Terasaki H, et al (2001). Detection of HER-2/neu (c-erb B-2) DNA amplification in primary breast carcinoma. Interobserver reproducibility and correlation with immunohistochemical HER-2 overexpression. Cancer, 92, 2965-74.<2965::AID-CNCR10156>3.0.CO;2-A
  43. Tubbs RR, Pettay JD, Roche PC, et al (2001). Discrepancies in clinical laboratory testing of eligibility for trastuzumab therapy: apparent immunohistochemical false-positives do not get the message. J Clin Oncol, 19, 2714-21.
  44. Wolff AC, Hammond ME, Hicks DG, et al (2013). Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American society of clinical oncology/college of American Pathologists clinical practice guideline update. J Clin Oncol, 31, 3997-4013.
  45. Xu J, Chen Y, Olopade OI (2010). MYC and breast cancer. Genes Cancer, 1, 629-40.

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