, Volume 70, Issue 10, pp 647–659 | Cite as

HLA class I alterations in breast carcinoma are associated with a high frequency of the loss of heterozygosity at chromosomes 6 and 15

  • María A. Garrido
  • Teresa Rodriguez
  • Svitlana Zinchenko
  • Isabel Maleno
  • Francisco Ruiz-Cabello
  • Ángel Concha
  • Nicolás Olea
  • Federico GarridoEmail author
  • Natalia AptsiauriEmail author
Original Article


HLA class I (HLA-I) molecules play a crucial role in the presentation of tumor antigenic peptides to CD8+ T cells. Tumor HLA-I loss provides a route of immune escape from T cell-mediated killing. We analyzed HLA-I expression in 98 cryopreserved breast cancer tissues using a broad panel of anti-HLA-I antibodies. Genomic HLA-I typing was performed using DNA obtained from autologous normal breast tissue. Analysis of the loss of heterozygosity (LOH) in the HLA-I region of chromosome 6 (LOH-6) and in the β2-microglobulin (B2M) region of chromosome 15 (LOH-15) was done by microsatellite amplification of DNA isolated from microdissected tumor areas. B2M gene sequencing was done using this DNA form HLA-I-negative tumors. Immunohistological analysis revealed various types of HLA-I alterations in 79 tumors (81%), including total HLA-I loss in 53 cases (54%) and partial loss in 16 samples (14%). In 19 cases (19%), HLA-I expression was positive. Using microsatellite analysis, we detected LOH in 36 cases out of 92 evaluated (39%), including 15 samples with only LOH-6, 14 with LOH-15, and seven tumors with LOH-6 and LOH-15 at the same time. Remarkably, we detected LOH-6 in eight tumors with positive HLA-I immunolabeling. We did not find any B2M mutations in HLA-I-negative breast tumors. In conclusion, LOH at chromosomes 6 and 15 has a high incidence in breast cancer and occurs in tumors with different HLA-I immunophenotypes. This common molecular mechanism of HLA-I alterations may reduce the ability of cytotoxic T lymphocytes  to kill tumor cells and negatively influence the clinical success of cancer immunotherapy.


HLA class I Breast cancer Loss of heterozygosity Cancer immune escape 



Human leukocite antigens


Major histocompatibility antigens


Loss of heterozygosity




Formalin fixed paraffin embedded


Infiltrating ductal carcinoma


Infiltrating lobular carcinoma




Estrogen receptor


Progesterone receptor


Sequence-specific oligonucleotide analysis


Short tandem repeat



We would like to thank Antonia Martín Casares for the technical support in HLA typing and Amanda Rocío González-Ramírez for the statistical analysis of the obtained results.


This work was supported by the grants from Spanish Institute of Health Carlos III (ISCIII, Instituto Carlos III) co-financed by European Union (FEDER-Fondo Europeo de Desarrollo Regional) (PI12/02031, PI08/1265, PI11/01022, PI11/01386, RETIC RD 06/020, RD09/0076/00165, PT13/0010/0039, PI14/01978, PI16/00752, PI17/00197), the Junta de Andalucía in Spain (Groups CTS-143), and Beckman-Coulter. This study is part of the doctoral thesis of Maria A. Garrido.

Compliance with ethical standards

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

The study protocol was approved by the ethical committee of the Virgen de las Nieves University Hospital and Instituto de Investigación Biosanitaria “ibs. Granada” (Comité de Ética de la Investigación de Centro de Granada (CEI Granada), number 2014-22/12). Signed informed consent approved by the Ethics Committee of our institution was obtained from all the patients.

Competing interest

The authors declare that they have no competing interests.


  1. Aptsiauri N, Cabrera T, Mendez R, Garcia-Lora A, Ruiz-Cabello F, Garrido F (2007) Role of altered expression of HLA class I molecules in cancer progression. Adv Exp Med Biol 601:123–131CrossRefGoogle Scholar
  2. Aptsiauri N, Garcia-Lora A, Garrido F (2014) ‘Hard’ and ‘soft’ loss of MHC class I expression in cancer cells. In Tumor Immunology and Immunotherapy. Edited by Rees RC. Oxford University Press, 63–78Google Scholar
  3. Barnstable CJ, Bodmer WF, Brown G, Galfre G, Milstein C, Williams AF, Ziegler A (1978) Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis. Cell 14(1):9–20CrossRefGoogle Scholar
  4. Bernal M, Ruiz-Cabello F, Concha A, Paschen A, Garrido F (2012) Implication of the β2-microglobulin gene in the generation of tumor escape phenotypes. Cancer Immunol Immunother 61(9):1359–1371CrossRefGoogle Scholar
  5. Boesen M, Svane IM, Engel AM, Rygaard J, Thomsen AR, Werdelin O (2000) CD8+ T cells are crucial for the ability of congenic normal mice to reject highly immunogenic sarcomas induced in nude mice with 3-methylcholantrene. Clin Exp Immunol 121:210–215CrossRefGoogle Scholar
  6. Burrone OR, Kefford RF, Gilmore D, Milstein C (1985) Stimulation of HLA-A,B,C by IFN-alpha. The derivation of Molt 4 variants and the differential expression of HLA-A,B,C subsets. EMBO J 4(11):2855–2860CrossRefGoogle Scholar
  7. Cabrera T, Ruiz-Cabello F, Lopez MA, de la Higuera B, Sanchez M, Garrido F (1986) Characterization of monoclonal antibodies directed against HLA class II molecules. Hybridoma 5(3):191–197CrossRefGoogle Scholar
  8. Cabrera T, Fernandez MA, Sierra A, Garrido A, Herruzo A, Escobedo A, Fabra A, Garrido F (1996) High frequency of altered HLA class I phenotypes in invasive breast carcinomas. Hum Immunol 50:127–134CrossRefGoogle Scholar
  9. Cabrera T, Lopez-Nevot MA, Gaforio JJ, Ruiz-Cabello F, Garrido F (2003) Analysis of HLA expression in human tumor tissues. Cancer Immunol Immunother 52:1–9PubMedGoogle Scholar
  10. Carretero R, Cabrera T, Sáenz-López P, Maleno I, Aptsiauri N, Cózar JM, Garrido F (2011) Bacillus Calmette-Guerin immunotherapy of bladder cancer induces selection of human leukocyte antigen class I-deficient tumor cells. Int J Cancer 129(4):839–846CrossRefGoogle Scholar
  11. Concha A, Cabrera T, Ruiz-Cabello F, and Garrido F. Can the HLA phenotype be used as a prognostic factor in breast carcinomas? Int J Cancer, 1991a; Suppl 6, 146–54CrossRefGoogle Scholar
  12. Concha A, Esteban F, Cabrera T, Ruiz-Cabello F, Garrido F (1991b) Tumor aggressiveness and MHC class I and II antigens in laryngeal and breast cancer. Semin Cancer Biol 2:47–54PubMedGoogle Scholar
  13. Coulie PG, Van den Eynde BJ, van der Bruggen P, Boon T (2014) Tumour antigens recognized by T lymphocytes: at the core of cancer immunotherapy. Nat Rev Cancer 14(2):135–146CrossRefGoogle Scholar
  14. Del Mar Valenzuela-Membrives M, Perea-García F, Sanchez-Palencia A, Ruiz-Cabello F, Gómez-Morales M, Miranda-León MT, Galindo-Angel I, Fárez-Vidal ME (2016) Progressive changes in composition of lymphocytes in lung tissues from patients with non-small-cell lung cancer. Oncotarget 7:71608–71619PubMedPubMedCentralGoogle Scholar
  15. Garrido F, Algarra I (2001) MHC antigens and tumor escape from immune surveillance. Adv Cancer Res 83:117–158CrossRefGoogle Scholar
  16. Garrido F, Cabrera T, Concha A, Glew S, Ruiz-Cabello F, Stern PL (1993) Natural history of HLA expression during tumour development. Immunol Today 14:491–499CrossRefGoogle Scholar
  17. Garrido F, Ruiz-Cabello F, Cabrera T, Perez-Villar JJ, Lopez-Botet M, Duggan-Keen M, Stern PL (1997a) Implications for immunosurveillance of altered HLA class I phenotypes in human tumours. Immunol Today 18:89–95CrossRefGoogle Scholar
  18. Garrido, T. Cabrera, R.S. Accola, J.C. Bensa, W. Wodmer, G. Dohr, B. Drenou, M. Drouet, R. Fauchet, G.B. Ferrara, S. Ferrone, P. Giacomini, T. Kageshita, L. Koopman, M. Maio, F. Marincola, C. Mazzilli, P.A. Morell, A. Murray, Crh. Papasteriades, L. Salvaneschi, P.L. Stern, A. Ziegler. HLA and cancer: 12th International Histocompatibility workshop study. In: Genetic diversity of HLA. Functional and Medical Implications. EDK (Ed. by D. Charron) vol. I, 1997b; 445–452Google Scholar
  19. Garrido F, Algarra I, García-Lora AM (2010a) The escape of cancer from T lymphocytes: immunoselection of MHC class I loss variants harboring structural-irreversible "hard" lesions. Cancer Immunol Immunother 59(10):1601–1606CrossRefGoogle Scholar
  20. Garrido F, Cabrera T, Aptsiauri N (2010b) ‘Hard’ and ‘soft’ lesions underlying the HLA class I alterations in cancer cells: implications for immunotherapy. Int J Cancer 127:249–256PubMedGoogle Scholar
  21. Garrido F, Aptsiauri N, Doorduijn EM, Garcia Lora AM, van Hall T (2016) The urgent need to recover MHC class I in cancers for effective immunotherapy. Curr Opin Immunol 39:44–51CrossRefGoogle Scholar
  22. Garrido F, Ruiz-Cabello F, Aptsiauri N (2017a) Rejection versus escape: the tumor MHC dilemma. Cancer Immunol Immunother 66:259–271CrossRefGoogle Scholar
  23. Garrido F, Perea F, Bernal M, Sánchez-Palencia A, Aptsiauri N, Ruiz-Cabello F. The escape of cancer from T cell-mediated immune surveillance: HLA class I loss and tumor tissue architecture. Vaccines (Basel). 2017b Feb 27;5(1)Google Scholar
  24. Herrmann F, Lehr HA, Drexler I, Sutter G, Hengstler J, Wollscheid U, Seliger B (2004) HER-2/neu-mediated regulation of components of the MHC class I antigen-processing pathway. Cancer Res 64:215–220CrossRefGoogle Scholar
  25. Kaneko K, Isihigami S, Kijima Y, Funasako Y, Hirata M, Okumura H, Shinchi H, Koriyama C, Ueno S, Yoshinaka H, Natsugoe S (2011) Clinical implication of HLA class I expression in breast cancer. BMC Cancer 11:454–459CrossRefGoogle Scholar
  26. de Kruijf EM, van Nes JG, Sajet A, Tummers QR, Putter H, Osanto S, Speetjens FM, Smit VT, Liefers GJ, van de Velde CJ, Kuppen PJ (2010) The predictive value of HLA class I tumor cell expression and presence of intratumoral Tregs for chemotherapy in patients with early breast cancer. Clin Cancer Res 16:1272–1280CrossRefGoogle Scholar
  27. del Campo AB, Kyte JA, Carretero J, Zinchencko S, Mendez R, Gonzalez-Aseguinolaza G, Ruiz-Cabello F, Aamdal S, Gaudernack G, Garrido F, Aptsiauri N (2014) Immune escape of cancer cells with beta2- microglobulin loss over the course of metastatic melanoma. Int J Cancer 134:102–113CrossRefGoogle Scholar
  28. López Nevot MA, Ruiz-Cabello F, Huelin C, Cabrera A, Garrido F (1986) A monoclonal antibody produced against the surface immunoglobulin of B-prolymphocytic leukemia. Sangre (Barc) 31(6):751–758Google Scholar
  29. Lopez-Nevot MA, Esteban F, Ferron A, Gutierrez J, Oliva MR, Romero C, Huelin C, Ruiz-Cabello F, Garrido F (1989) HLA class I gene expression on human primary tumours and autologous metastases: demonstration of selective losses of HLA antigens on colorectal, gastric and laryngeal carcinomas. Br J Cancer 59:221–226CrossRefGoogle Scholar
  30. Madjd Z, Spendlove I, Pinder SE, Ellis IO, Durant L (2005) Total loss of MHC class I is an independent indicator of good prognosis in breast cancer. Int J Cancer 117:248–255CrossRefGoogle Scholar
  31. Maleno I, Aptsiauri N, Cabrera T, Gallego A, Paschen A, López-Nevot MA, Garrido F (2001) Frequent loss of heterozygosity in the □2-microglobulin region of chromosome 15 in primary human tumors. Immunogenetics 63(2):65–71CrossRefGoogle Scholar
  32. Maleno I, Cabrera CM, Cabrera T, Paco L, Lopez-Nevot MA, Collado A, Ferron A, Garrido F (2004) Distribution of HLA class I altered phenotypes in colorectal carcinomas: high frequency of HLA haplotype loss associated with loss of heterozygosity in chromosome region 6p21. Immunogenetics 56:244–253CrossRefGoogle Scholar
  33. Maleno I, Romero JM, Cabrera T, Paco L, Aptsiauri N, Cozar JM, Tallada M, Lopez-Nevot MA, Garrido F (2006) LOH at 6p21.3 region and HLA class altered phenotypes in bladder carcinomas. Immunogenetics 58(7):503–510CrossRefGoogle Scholar
  34. Marincola FM, Jafee EM, Hicklin DJ, Ferrone S (2000) Escape of human solid tumors from T cell recognition: molecular mechanisms and functional significance. Adv Immunol 74:181–273CrossRefGoogle Scholar
  35. McGranaham N, Rosenthal R, Hiley C, Rowan AJ, Watkins T, Wilson G, Birkbak N, Veeriah S, Van Loo P, Herrero J, Swanton C, TRACERx Consortium (2017) Allele-specific HLA loss and immune escape in lung cancer evolution. Cell 171(6):1259–1271CrossRefGoogle Scholar
  36. Neefjes JJ, Ploegh HL (1988) Allele and locus-specific differences in cell surface expression and the association of HLA class I heavy chain with beta 2-microglobulin: differential effects of inhibition of glycosylation on class I subunit association. Eur J Immunol 18(5):801–810CrossRefGoogle Scholar
  37. Pedersen MH, Hood BL, Beck HC, Conrads TP, Ditzel HJ, Leth-Larsen R (2017) Downregulation of antigen presentation-associated pathway proteins is linked to poor outcome in triple-negative breast cancer patient tumors. 6(5):e1305531. eCollection 2017CrossRefGoogle Scholar
  38. Perea F, Bernal M, Sanchez-Palencia A, Carretero J, Torres C, Bayarri C, Gomez-Morales GF, Ruiz-Cabello F (2017) The absence of HLA class I expression in non-small cell lung cancer correlates with the tumor tissue structure and the pattern of T cell infiltration. Int J Cancer 140:888–899CrossRefGoogle Scholar
  39. Perez M, Cabrera T, Lopez Nevot MA, Gomez M, Peran F, Ruiz-Cabello F, Garrido F (1986) Heterogeneity of the expression of class I and II HLA antigens in human breast carcinomas. J Immunogenet 13:247–253CrossRefGoogle Scholar
  40. Ramal LM, Feenstra M, van der Zwan AW, Collado A, Lopez-Nevot MA, Tilanus M, Garrido F (2000) Criteria to define HLA haplotype loss in human solid tumors. Tissue Antigens 55:443–448CrossRefGoogle Scholar
  41. Romero P, Coulie P (2014) Adaptive T-cell immunity and tumor antigen recognition. Tumor Immunology and Immunotherapy. Edited by Rees RC. Oxford University Press, In, pp 1–14Google Scholar
  42. Ryschich E, Notzel T, Hinz U, Autschbach F, Ferguson J, Simon I, Weitz J, Frohlich B, Klar E, Buchler MW, Schmidt J (2005) Control of T-cell-mediated immune response by HLA class I in human pancreatic carcinoma. Clin Cancer Res 11(Pt 1):498–504PubMedGoogle Scholar
  43. Sade-Feldman M, Jiao YJ, Chen JH, Rooney MS, Barzily-Rokni M, Eliane JP, Bjorgaard SL, Hammond MR, Vitzthum H, Blackmon SM, Frederick DT, Hazar-Rethinam M, Nadres BA, Van Seventer EE, Shukla SA, Yizhak K, Ray JP, Rosebrock D, Livitz D, Adalsteinsson V, Getz G, Duncan LM, Li B, Corcoran RB, Lawrence DP, Stemmer-Rachamimov A, Boland GM, Landau DA, Flaherty KT, Sullivan RJ, Hacohen N (2017) Resistance to checkpoint blockade therapy through inactivation of antigen presentation. Nature Communications 8(1):1136–1143CrossRefGoogle Scholar
  44. Seliger B, Kiessling R (2013) The two sides of HER2/neu: immune escape versus surveillance. Trends Mol Med 19(11):677–684CrossRefGoogle Scholar
  45. Seliger B, Cabrera T, Garrido F, Ferrone S (2002) HLA class I antigen abnormalities and immune escape by malignant cells. Semin Cancer Biol 12:3–13CrossRefGoogle Scholar
  46. Sobin L, Gospodarowiaz M, Wittekind CH. TNM classification of malignant tumours. UICC 2009, 7th edn. Chichester: Wiley, 2010; 310pGoogle Scholar
  47. Spear BT, Kornbluth J, Strominger JL, Wilson DB (1985) Evidence for a shared HLA-A intralocus determinant defined by monoclonal antibody 131. J Exp Med 162(6):1802–1810CrossRefGoogle Scholar
  48. Vertuani S, Triulzi C, Roos AK, Charo J, Norell H, Lemonnier F, Pisa P, Seliger B, Kiessling R (2009) HER-2/neu mediated down-regulation of MHC class I antigens processing prevents CTL-mediated tumor recognition upon DNA vaccination in HLA-A2 transgenic mice. Cancer Immunol Immunother 58:653–654CrossRefGoogle Scholar
  49. Wang E, Worschech A, Marincola FM (2008) The immunological constant of rejection. Trends Immunol 29:256–262CrossRefGoogle Scholar
  50. Zacharakis N, Chinnasamy H, Black M, Xu H, Lu YC, Zheng Z, Pasetto A, Langhan M, Shelton T, Prickett T, Gartner J, Jia L, Trebska-McGowan K, Somerville RP, Robbins PF, Rosenberg SA, Goff SL, Feldman SA (2018 Jun) Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer. Nat Med 24(6):724–730CrossRefGoogle Scholar
  51. Zaretsky JM, Garcia-Diaz A, Shin DS, Escuin-Ordinas H, Hugo W, Hu-Lieskovan S, Torrejon DY, Abril-Rodriguez G, Sandoval S, Barthly et al (2016) Mutations associated with acquired resistance to PD-1 blockade in melanoma. N Engl J Med 375(9):819–829CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • María A. Garrido
    • 1
  • Teresa Rodriguez
    • 2
  • Svitlana Zinchenko
    • 2
  • Isabel Maleno
    • 2
  • Francisco Ruiz-Cabello
    • 2
    • 3
    • 4
  • Ángel Concha
    • 5
  • Nicolás Olea
    • 6
    • 3
  • Federico Garrido
    • 2
    • 3
    • 4
    Email author
  • Natalia Aptsiauri
    • 3
    • 4
    Email author
  1. 1.Servicio de Radiología, UGC de RadiologíaHospital Universitario Virgen de las NievesGranadaSpain
  2. 2.Servicio de Análisis Clínicos e Inmunología, UGC de Laboratorio ClinicoHospital Universitario Virgen de las NievesGranadaSpain
  3. 3.Instituto de Investigación Biosanitaria ibs. GranadaGranadaSpain
  4. 4.Departamento de Bioquímica, Biología Molecular III e Inmunología, Facultad de MedicinaUniversidad de GranadaGranadaSpain
  5. 5.Servicio de Anatomía PatológicaHospital Juan CanalejoA CoruñaSpain
  6. 6.Servicio de Radiología, UGC de RadiologíaHospital del CampusGranadaSpain

Personalised recommendations