Correspondence

Breast-Cancer Stromal Cells with TP53 Mutations

N Engl J Med 2008; 358:1634-1636April 10, 2008

Article

To the Editor:

Patocs et al. (Dec. 20, 2007, issue)1 report a frequency of TP53 mutations in fibroblasts associated with sporadic breast carcinoma of 27.4%, and they show that mutation status is associated with regional nodal metastasis. If confirmed, this finding would represent an important discovery.

We sought to confirm their findings by direct sequencing of exons 4 through 9 of TP53 in microdissected areas of stroma (<5 mm from the epithelial cancer interface) from 10 fresh-frozen sporadic breast-cancer specimens and 7 primary breast-carcinoma–associated fibroblast cultures.2,3 No mutation was detected in any of these 17 samples. The reason for this discrepancy is unclear, although we note that Patocs et al. used DNA derived from formalin-fixed, paraffin-embedded tumor tissue, which is notorious for generating polymerase-chain-reaction (PCR) artifacts.4

Ian G. Campbell, Ph.D.
Wen Qiu, B.Sc.
Peter MacCallum Cancer Centre, East Melbourne 3002, Australia
ian.campbell@petermac.org

Kornelia Polyak, M.D., Ph.D.
Dana–Farber Cancer Institute, Boston, MA 02115

Izhak Haviv, Ph.D.
Peter MacCallum Cancer Centre, East Melbourne 3002, Australia

Dr. Polyak reports receiving consulting fees from Novartis, Pfizer, and AVEO Pharmaceuticals, holding stock in AVEO Pharmaceuticals, receiving lecture fees from Biogen Idec, and receiving grant support from Novartis and Biogen Idec. No other potential conflict of interest relevant to this letter was reported.

4 References

1. 1

   Patocs A, Zhang L, Xu Y, et al. Breast-cancer stromal cells with TP53 mutations and nodal metastases. N Engl J Med 2007;357:2543-2551
   Full Text | Web of Science | Medline

2. 2

   Allinen M, Beroukhim R, Cai L, et al. Molecular characterization of the tumor microenvironment in breast cancer. Cancer Cell 2004;6:17-32
   CrossRef | Web of Science | Medline

3. 3

   Lebret SC, Newgreen DF, Thompson EW, Ackland ML. Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors. Breast Cancer Res 2007;9:R19-R19
   CrossRef | Web of Science | Medline

4. 4

   Kern SE, Winter JM. Elegance, silence and nonsense in the mutations literature for solid tumors. Cancer Biol Ther 2006;5:349-359
   CrossRef | Web of Science | Medline

To the Editor:

Patocs et al. report TP53 mutations in breast-cancer stromal and epithelial cells. The pattern of these mutations is very unusual, since the frequency of TP53 mutations in sporadic breast cancer was 54%, as compared with 20% in the literature,1,2 and the frequency of tumors with double mutations was exceptionally high (23%, vs. 1 to 2%). In addition, several mutations have not previously been described — for example, the Pro89Ser mutation identified in 21 samples in the study by Patocs et al. has never been reported among the 3000 sporadic and familial breast carcinomas included in the universal mutation database — for p53 (p53.free.fr/).3 The distribution of the loss-of-activity TP53 mutant is out of range as compared with previous studies of breast carcinoma (P<0.001).4 Overall, the pattern of p53 mutations described in this study is consistent with either technical problems — commonly encountered with the use of paraffin-embedded tissue — or a mutator phenotype associated with random passenger mutations.

Cecilia Soussi Zander, M.D., Ph.D.
Uppsala University Hospital, S-75185 Uppsala, Sweden

Thierry Soussi, Ph.D.
Karolinska Institutet, S-17176 Stockholm, Sweden
thierry.soussi@ki.se

4 References

1. 1

   Williams C, Norberg T, Ahmadian A, et al. Assessment of sequence-based p53 gene analysis in human breast cancer: messenger RNA in comparison with genomic DNA targets. Clin Chem 1998;44:455-462
   Web of Science | Medline

2. 2

   Borresen-Dale AL. TP53 and breast cancer. Hum Mutat 2003;21:292-300
   CrossRef | Web of Science | Medline

3. 3

   Soussi T, Ishioka C, Claustres M, Beroud C. Locus-specific mutation databases: pitfalls and good practice based on the p53 experience. Nat Rev Cancer 2006;6:83-90
   CrossRef | Web of Science | Medline

4. 4

   Soussi T, Asselain B, Hamroun D, et al. Meta-analysis of the p53 mutation database for mutant p53 biological activity reveals a methodologic bias in mutation detection. Clin Cancer Res 2006;12:62-69
   CrossRef | Web of Science | Medline

To the Editor:

Among 32 specimens from patients with hereditary breast cancer with TP53 mutations, Patocs et al. reported that 11 specimens had mutations in stroma alone and 10 had mutations in both epithelium and stroma. Fourteen of these 32 mutations predicted Pro89Ser, of which 5 were simultaneously encountered in epithelium and stroma, arguing for a common genetic lineage. Pro89Ser is an infrequent mutation that appeared only twice in the universal mutation database for p531 and three times among the 24,810 mutations compiled in the International Agency for Research on Cancer p53 database.2,3 This mutation is predicted to be neutral for its effects on p53 protein structure.4 The mutant protein is able to transactivate eight different p53-dependent promoters in yeast functional assay. Therefore, the role of Pro89Ser as a loss-of-function mutation in carcinogenesis appears to be questionable. Indeed, in the study by Patocs et al., such a mutation was not found among the 74 TP53 mutations in the group of patients with sporadic breast cancer. We feel that the absence of an association of stromal TP53 mutations with a positive nodal status in hereditary breast cancer could be the consequence of the inclusion of such a mutation in the statistical analysis.

Gérard Zalcman, M.D., Ph.D.
Emmanuel Bergot, M.D.
Caen University Hospital, 14033 Caen CEDEX 05, France
zalcman-g@chu-caen.fr

Pierre Hainaut, Ph.D.
International Agency for Research on Cancer, 69372 Lyon CEDEX 08, France

4 References

1. 1

   Soussi T, Asselain B, Hamroun D, et al. Meta-analysis of the p53 mutation database for mutant p53 biological activity reveals a methodologic bias in mutation detection. Clin Cancer Res 2006;12:62-69
   CrossRef | Web of Science | Medline

2. 2

   International Agency for Research on Cancer. IARC TP53 mutation database. (Accessed March 21, 2008, at http://www-p53.iarc.fr/MutantDetail.asp?TypeGraph=MutantValidation&Mutant=P89S.)
   

3. 3

   Olivier M, Eeles R, Hollstein M, Khan MA, Harris CC, Hainaut P. The IARC TP53 database: new online mutation analysis and recommendations to users. Hum Mutat 2002;19:607-614
   CrossRef | Web of Science | Medline

4. 4

   Kato S, Han SY, Liu W, et al. Understanding the function-structure and function-mutation relationships of p53 tumor suppressor protein by high-resolution missense mutation analysis. Proc Natl Acad Sci U S A 2003;100:8424-8429
   CrossRef | Web of Science | Medline

To the Editor:

The study by Patocs et al. cannot exclude biases regarding hereditary breast cancer. The number of patients with BRCA1 mutations (25 patients) or BRCA2 mutations (16 patients) was small, and the authors included these two distinctly different groups1,2 within the same heterogeneous group.

Dimitrios H. Roukos, M.D.
Ioannina University School of Medicine, 45110 Ioannina, Greece
droukos@cc.uoi.gr

2 References

1. 1

   Roukos DH, Murray S, Briasoulis E. Molecular genetic tools shape a roadmap towards a more accurate prognostic prediction and personalized management of cancer. Cancer Biol Ther 2007;6:308-312
   CrossRef | Web of Science | Medline

2. 2

   Roukos DH, Briasoulis E. Individualized preventive and therapeutic management of hereditary breast ovarian cancer syndrome. Nat Clin Pract Oncol 2007;4:578-590
   CrossRef | Web of Science | Medline

Author/Editor Response

In their study, which they concede was underpowered, Campbell et al. were unable to find TP53 mutations in 10 frozen “stroma” samples near breast-cancer epithelium, and they did not detect TP53 mutations in the epithelium. The latter result suggests a systematic error, since 10 samples should yield one or more somatic TP53 mutations in carcinomatous epithelium. A challenge of working with archival or frozen templates is to avoid artifacts. Although we selected intratumoral stromal fibroblasts, it is unclear whether Campbell et al. did actually obtain intratumoral stroma or normal stroma near the tumor. This group previously reported no genomic alterations or mutations in CD10-positive stroma, but in fact they had selected only for myoepithelial cells and rare intratumoral myofibroblasts.1 When we used this selection, we also did not find genomic alterations or mutations from either archival or frozen template–derived DNA. Furthermore, almost all our germ-line DNA samples were also procured from archived templates of normal breast epithelium and normal stroma distinct from tumor after laser-capture microdissection (see Table 1 of the Supplementary Appendix, available with the full text of this letter at www.nejm.org). We always perform a series of quality-control measures for each study (see Table 1 of the Supplementary Appendix), as we report in our article. Finding the association between stromal TP53 mutation status and lymph-node status would be extremely unlikely if the mutations were obtained by artifact. In addition, we found that in the absence of a TP53 mutation, the loss of heterozygosity at five loci in stroma was associated with a positive lymph-node status. Genes that encode proteins in the p53 pathway lie in three of these five loci, again corroborating the biology behind our data.

With regard to the comments by Zander and Soussi and by Zalcman et al., comparisons of somatic TP53 mutational spectra from databases with our compartment-specific spectra are comparisons of apples with oranges. Existing databases register somatic mutations derived from analyses of variable numbers of exons from whole breast tumors (a variable admixture of epithelium, stroma, and germ-line cells), using mutation-detection techniques of variable sensitivities. Thus, no rigorous conclusions can be drawn from these databases.

Finally, with regard to the comments of Roukos, we suspect that intratumoral stromal genetic alterations might be found in other tumors. Stromal genetic alterations and mutations have been independently described in carcinomas of the head and neck, colon, bladder, and cervix and in inflammatory bowel disease.2-4

Attila Patocs, M.D., Ph.D.
Petra Platzer, Ph.D.
Charis Eng, M.D., Ph.D.
Cleveland Clinic Foundation, Cleveland, OH 44195
engc@ccf.org

4 References

1. 1

   Allinen M, Beroukhim R, Cai L, et al. Molecular characterization of the tumor microenvironment in breast cancer. Cancer Cell 2004;6:17-32
   CrossRef | Web of Science | Medline

2. 2

   Moinfar F, Man YG, Arnould L, Bratthauer GL, Ratschek M, Tavassoli FA. Concurrent and independent genetic alterations in the stromal and epithelial cells of mammary carcinoma: implications for tumorigenesis. Cancer Res 2000;60:2562-2566
   Web of Science | Medline

3. 3

   Bian Y, Knobloch TJ, Sadim M, et al. Somatic acquisition of TGFBR1*6A by epithelial and stromal cells during head and neck and colon cancer development. Hum Mol Genet 2007;16:3128-3135
   CrossRef | Web of Science | Medline

4. 4

   Paterson RF, Ulbright TM, MacLennan GT, et al. Molecular genetic alterations in the laser-capture-microdissected stroma adjacent to bladder carcinoma. Cancer 2003;98:1830-1836
   CrossRef | Web of Science | Medline

Citing Articles (16)

Citing Articles

1. 1

   Judith Leibovici, Orit Itzhaki, Monica Huszar, Judith Sinai. (2011) The tumor microenvironment: part 1. Immunotherapy 3:11, 1367-1384
   CrossRef

2. 2

   Hasan Korkaya, Suling Liu, Max S. Wicha. (2011) Breast cancer stem cells, cytokine networks, and the tumor microenvironment. Journal of Clinical Investigation 121:10, 3804-3809
   CrossRef

3. 3

   Ian Campbell, Wen Qiu, Izhak Haviv. (2011) Genetic changes in tumour microenvironments. The Journal of Pathology 223:4, 450-458
   CrossRef

4. 4

   Thierry Soussi. 2011. TP53 Mutations in Human Cancer: Database Reassessment and Prospects for the Next Decade. , 107-139.
   CrossRef

5. 5

   Michael Allen, J Louise Jones. (2011) Jekyll and Hyde: the role of the microenvironment on the progression of cancer. The Journal of Pathology 223:2, 163-177
   CrossRef

6. 6

   José María Vera-Román. (2010) Implicaciones clinicopatológicas del estadio preinvasivo del cáncer de mama. Medicina Clínica 135:15, 695-696
   CrossRef

7. 7

   Thierry Soussi, Dalil Hamroun, Linn Hjortsberg, Jean Michel Rubio-Nevado, Jean Louis Fournier, Christophe Béroud. (2010) MUT-TP53 2.0: a novel versatile matrix for statistical analysis of TP53 mutations in human cancera. Human Mutation 31:9, 1020-1025
   CrossRef

8. 8

   JeanMarie Houghton, Hanchen Li, Xueli Fan, Yingwang Liu, Jian Hua Liu, Varada P. Rao, Theofilos Poutahidis, Christie L. Taylor, Erin A. Jackson, Christine Hewes, Stephen Lyle, Anna Cerny, Glennice Bowen, Jan Cerny, Nathan Moore, Evelyn A. Kurt-Jones, Susan E. Erdman. (2010) Mutations in Bone Marrow-Derived Stromal Stem Cells Unmask Latent Malignancy. Stem Cells and Development 19:8, 1153-1166
   CrossRef

9. 9

   Takahiro Hasebe, Motoki Iwasaki, Sadako Akashi-Tanaka, Takashi Hojo, Tatsuhiro Shibata, Yuko Sasajima, Takayuki Kinoshita, Hitoshi Tsuda. (2010) p53 expression in tumor-stromal fibroblasts forming and not forming fibrotic foci in invasive ductal carcinoma of the breast. Modern Pathology 23:5, 662-672
   CrossRef

10. 10

    M Bauer, G Su, C Casper, R He, W Rehrauer, A Friedl. (2010) Heterogeneity of gene expression in stromal fibroblasts of human breast carcinomas and normal breast. Oncogene 29:12, 1732-1740
    CrossRef

11. 11

    Tamas A. Gonda, Andrea Varro, Timothy C. Wang, Benjamin Tycko. (2010) Molecular biology of cancer-associated fibroblasts: Can these cells be targeted in anti-cancer therapy?. Seminars in Cell & Developmental Biology 21:1, 2-10
    CrossRef

12. 12

    Jair Bar, Neta Moskovits, Moshe Oren. (2010) Involvement of stromal p53 in tumor-stroma interactions. Seminars in Cell & Developmental Biology 21:1, 47-54
    CrossRef

13. 13

    Takahiro Hasebe, Nao Okada, Nobuko Tamura, Takashi Houjoh, Sadako Akashi-Tanaka, Histoshi Tsuda, Tatsuhiro Shibata, Yuko Sasajima, Motoki Iwasaki, Takayuki Kinoshita. (2009) p53 expression in tumor stromal fibroblasts is associated with the outcome of patients with invasive ductal carcinoma of the breast. Cancer Science 100:11, 2101-2108
    CrossRef

14. 14

    Ran Brosh, Varda Rotter. (2009) When mutants gain new powers: news from the mutant p53 field. Nature Reviews Cancer
    CrossRef

15. 15

    J Bar, R Feniger-Barish, N Lukashchuk, H Shaham, N Moskovits, N Goldfinger, D Simansky, M Perlman, M Papa, A Yosepovich, G Rechavi, V Rotter, M Oren. (2009) Cancer cells suppress p53 in adjacent fibroblasts. Oncogene 28:6, 933-936
    CrossRef

16. 16

    Kornelia Polyak, Izhak Haviv, Ian G. Campbell. (2009) Co-evolution of tumor cells and their microenvironment. Trends in Genetics 25:1, 30-38
    CrossRef