- Email: [email protected]
The Stromal Microenvironment and Breast Cancer Biology
References 1. Sardanelli F, Podo F. Breast MR imaging in women at highrisk of breast cancer. Is something changing in early breast cancer detection? Eur Radiol. 2007;17:873-887. 2. Hillman BJ, Harms SE, Stevens G, et al. Diagnostic performance of a dedicated 1.5-T breast MR imaging system. Radiology. 2012;265:51-58. 3. Hollingsworth AB, Stough RG. Multicentric and contralateral invasive tumors identified with preoperative MRI in patients newly diagnosed with ductal carcinoma in situ of the breast. Breast J. 2012;18:420-427. 4. Holland R, Veling SH, Mravunac M, Hendriks JH. Histologic multifocality of Tis, T1-2 breast carcinomas. Implications for clinical trials of breast-conserving surgery. Cancer. 1985;56: 979-990. 5. Fisher B. Role of science in the treatment of breast cancer when tumor multicentricity is present. J Natl Cancer Inst. 2011;103:1292-1298.
8. Hollingsworth AB, Stough RG. Conflicting outcomes with preoperative breast MRI: differences in technology or methodology? Breast Diseases: A Year Book Quarterly. 2010;21:109-112. 9. Hollingsworth AB, Stough RG, O’Dell CA, Brekke CE. Breast magnetic resonance imaging for preoperative locoregional staging. Am J Surg. 2008;196:389-397. 10. Turnbull L, Brown S, Harvey I, et al. Comparative effectiveness of MRI in breast cancer (COMICE) trial: a randomised controlled trial. Lancet. 2010;375:563-571. 11. Houssami N, Hayes DF. Review of preoperative magnetic resonance imaging (MRI) in breast cancer: should MRI be performed on all women with newly diagnosed, early stage breast cancer? CA Cancer J Clin. 2009;59:290-302. 12. Fisher B, Bauer M, Margolese R, et al. Five-year results of a randomized clinical trial comparing total mastectomy and segmental mastectomy with or without radiation in the treatment of breast cancer. N Engl J Med. 1985;312:665-673.
6. Tuttle TM. Is breast conservation under siege? Breast Diseases: A Year Book Quarterly. 2012;23:128-131.
13. Anderson BO, Khalil el SA. Breast conservation without mammography? Oncology practice in the real world of limited resource countries. Breast. 2006;15:575-579.
7. Miller BT, Abbott AM, Tuttle TM. The influence of preoperative MRI on breast cancer treatment. Ann Surg Oncol. 2012;19:536-540.
14. Kuhn TS. The Structure of Scientific Revolutions. 2nd ed. Chicago, IL: University of Chicago Press; 1970.
The Stromal Microenvironment and Breast Cancer Biology Catherine C. Park, MD The basic architecture of the breast develops through dynamic interactions between the ectodermally derived ductal epithelium and stromal cells of mesenchymal origins.1 This interplay ensures the formation of a functional gland with the following developmental milestones throughout life: puberty, pregnancy, lactation, and involution. Despite dramatic changes in morphology during development, the basic structural integrity of the breast is maintained through normal interactions with the microenvironment. The concept that local stromal microenvironments contribute to cell fate decisions is a central tenet of classical embryology.2 It is now clear that the role of stromal cells in cancer progression is as important as their role in normal development. The evolution of breast cancers requires co-optation of the surrounding stromal tissues to facilitate progression and
support increased metabolic demand. Wound-like properties of the stroma have been shown in the earliest stages of breast cancer, ductal carcinoma in situ (DCIS), where proliferating malignant cells within the breast duct are associated with stromal remodeling outside of the duct that includes increased angiogenesis and microvessel density.3,4 In addition, there are striking changes in the cellular and non-cellular constituents of the activated cancerous stroma, including immune cell infiltrates,5 remodeling of the extracellular matrix,6 and physiologic changes in pH and oxygen tension.7 The past several years have witnessed an exciting increase in our understanding of the molecular and functional bases of these constituents, in part ensuing from advances in array and computational technology and imaging techniques. In particular, the use of genome-wide and gene arrayebased stromal signatures and novel applications of clinical imaging tools, which provide a means to analyze several biologic features in the context of tissue architecture simultaneously, have led to significant new hypotheses about the role of stroma in disease treatment. This editorial will highlight the recent findings in breast cancer biology, with respect to
Breast Diseases: A Year BookÒ Quarterly Vol 24 No 1 2013
23
the role of the stromal microenvironment, that have particular translational and therapeutic relevance. STROMAL GENE EXPRESSION It has been well over a decade since the seminal use of gene expression array technology revealed clinically relevant molecular phenotypes in breast cancer.8,9 Over time, advances in laser capture microdissection and the ability to process small amounts of RNA have been central to our investigation of the role of stromal cells in the co-evolution or progression of disease and to the determination of whether gene expression patterns or signatures are informative in prediction or prognosis. Although gene aberrations and loss of heterozygosity have been detected in the normal-appearing stromal tissue adjacent to invasive lesions,10 several questions remain as to the role of stroma in the evolution of cancers that arise in epithelial cells. To compare stromal gene expression changes in disease progression, Ma and colleagues11 used advanced laser-capture microdissection to isolate DCIS and invasive breast lesions and their adjacent stroma for expression array
analyses. These studies showed that stromal genes, particularly those related to invasion and extracellular matrix remodeling, were highly upregulated and that the most dramatic changes occurred in DCIS lesions, confirming that the tumor microenvironment participates in tumorigenesis well before there is evidence for histologic invasion or the emergence of malignancy. In elegant studies, which isolated cell lineages in normal and cancerous breast tissues, serial analysis of gene expression and expression array analysis showed that increased expression of cytokines that promote tumorigenesis occurs in the myoepithelial cells that are part of the barrier between epithelial and stromal cells in the normal breast.12 Thus, we have clear evidence that stromal gene expression dramatically changes in the early phases of cancer progression. One confounding issue has been the variations in the definitions of tumor stroma, which can include intratumoral stroma, extratumoral stroma, or both (Fig 1A). To address the controversy, Roma´n-Pe´rez and colleagues microdissected tissues adjacent to invasive cancer or DCIS to specifically investigate gene expression changes in the extratumoral
FIGURE 1.d(A) Schematic of tumor and stroma depicted, including intratumoral and extratumoral stroma. (B) Example of gene expression array from microdissected areas of stroma.
24
Breast Diseases: A Year BookÒ Quarterly Vol 24 No 1 2013
microenvironment (for example, Fig 1B).13 Distinct biological features were identified that were associated with cancer progression and a mesenchymal phenotype. Interestingly, an extratumoral stromal signature was identified that had prognostic value in estrogen receptorepositive breast cancer and was associated with inflammatory response, macrophage recruitment, fibrosis, and chemotaxis of cells. Other studies have also correlated stromal gene expression with treatment outcomes. Finak and colleagues14 first identified sets of genes derived from microdissected tumor stroma that had prognostic significance in several clinical data sets. Farmer and colleagues15 found that a stroma-related gene signature predicted resistance to treatment in breast cancer patients receiving neoadjuvant chemotherapy. In aggregate, studies of stromal gene expression indicate that a highly dynamic stromal environment arises early in the process of carcinogenesis and that specific biologic signaling patterns are seen repeatedly, including inflammation, remodeling, invasion, and angiogenesis. In addition, novel signatures with regard to specific pathways such as TWIST-1 and TGF-b also provide insight into the nature of the stromal contribution to malignancy.13 Questions for the future include how stromal gene expression differs in light of tumor molecular phenotypes and if there are specific stromal phenotypes, whether there is a clear transition between activated and benign stroma, and whether there is a consistent pattern of gene expression by stage or other pathologic features of disease. Further investigations and validation studies in larger prospective cohorts are needed to design robust prognostic signatures that could improve risk stratification.
and treatment decision-making in the future. However, understanding stromal biology could also improve the approach to local treatment. Decisions regarding breast surgery have been guided by imaging techniques developed to detect malignant tissues and by the post-surgical detection of microscopic cells at the resection margins as a surrogate for residual disease. However, the concept of an optimal resection volume remains elusive. We now know that the tumor stroma and extratumoral stroma have active signaling and molecular features of inflammation and progression; however, our challenges for the future are to understand the extent of these changes and whether they can be classified into subtypes, have correlations with features of the primary tumor, or have prognostic or predictive importance. Is there a “zone or niche of activation” that has biological significance and can be detected non-invasively? And, ultimately, can the detection of stromal abnormalities guide local resection and radiotherapy? A better understanding of stromal changes could improve re-excision rates and certainly help guide local radiotherapy procedures, especially as partial-breast radiotherapy becomes more developed. We have attempted to find ways to map enhancement patterns in the normal-appearing stroma of breast cancer patients. Taking advantage of the ability to measure gadolinium-based enhancement values in normal-appearing breast tissues, we asked whether an activated stroma could possibly be detected via stromal enhancement patterns. We created topographic enhancement maps where enhancement of each voxel (0.7 mm3) in the stroma was measured and mapped to the edge of the tumor (Fig 2). This showed that in unifocal small tumors, there was a region of high stromal
IMAGING OF STROMAL FEATURES To further our understanding of the functional and molecular aspects of stromal tissue biology, the structural and architectural context must be studied more closely. Microdissection has allowed us to investigate spatial relationships and tissue architecture. Recent developments in computational microscopy imaging have exponentially expanded the number of proteins or features that can be evaluated in a topographic and temporal fashion.16,17 In a study of pathology specimens, systematic analysis of thousands (>6000) of morphologic features from breast cancer tissues using a computational platform, C-Path,16 found that 3 of the 11 features most associated with outcomes were derived from the stroma. A major stromal feature was inflammation, which is consistent with a theme that has been observed by others. The findings we have discussed thus far are based on molecular and histologic material that with more rigorous and prospective validation could enhance risk assessment
FIGURE 2.dBreast MRI with schematic tumor labeled and normalappearing stroma in concentric volumetric shells around the tumor.
Breast Diseases: A Year BookÒ Quarterly Vol 24 No 1 2013
25
enhancement, associated with increased microvessel density, that then decreased with distance from the edge of the tumor.18 In addition, we asked whether global stromal enhancement was associated with clinical outcomes. In a pilot study of patients receiving neoadjuvant chemotherapy, we found that stromal enhancement was associated with disease-free survival, but surprisingly, patients with higher enhancement had significantly better outcomes.19 We postulated that this could be a reflection of improved baseline vascular physiology and chemotherapy penetrance; however, clearly, the picture is much more complicated. The findings from these studies indicate that there is a detectable stromal pattern of enhancement in the normal-appearing fibroglandular breast tissue beyond the malignant tumor; however, further studies correlating the underlying biology of these stromal patterns and outcomes are ongoing. SUMMARY Gene expression studies of the intra- and extratumoral stroma have consistently shown changes in gene expression in these tissue compartments, indicating their active participation in the early evolution of breast tumors. Several lines of evidence from studies in microdissected clinical specimens consistently point to stromal changes in the expression of genes involving inflammation and tissue and extracellular matrix remodeling. As in tumors, there is evidence of heterogeneity among patients, and specific expression signatures have not yet been validated. Mapping of morphologic cancer features using advanced imaging and computational tools also have revealed stromal features that show promise in helping to improve patient risk stratification. Early studies using magnetic resonance imaging technology have attempted to map stromal enhancement in normal-appearing breast tissues surrounding breast cancers; these studies show promise for the ability to detect regions of abnormality. In the future, rigorous correlations between imaging platforms, biology, and outcomes are needed and hold great promise for creating an entirely new way of incorporating stromal biology into cancer care.
References 1. Anbazhagan R, Osin PP, Bartkova J, Nathan B, Lane EB, Gusterson BA. The development of epithelial phenotypes in the human fetal and infant breast. J Pathol. 1998;184:197-206. 2. Bissell MJ, Radisky D. Putting tumours in context. Nat Rev Cancer. 2001;1:46-54. 3. Dvorak HF. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N Engl J Med. 1986;315:1650-1659.
26
Breast Diseases: A Year BookÒ Quarterly Vol 24 No 1 2013
4. Guidi AJ, Fischer L, Harris JR, Schnitt SJ. Microvessel density and distribution in ductal carcinoma in situ of the breast. J Natl Cancer Inst. 1994;86:614-619. 5. DeNardo DG, Coussens LM. Inflammation and breast cancer. Balancing immune response: crosstalk between adaptive and innate immune cells during breast cancer progression. Breast Cancer Res. 2007;9:212. 6. Lu P, Weaver VM, Werb Z. The extracellular matrix: a dynamic niche in cancer progression. J Cell Biol. 2012;196:395-406. 7. Erler JT, Giaccia AJ. Lysyl oxidase mediates hypoxic control of metastasis. Cancer Res. 2006;66:10238-10241. 8. Perou CM, Sørlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature. 2000;406:747-752. 9. Sørlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 2001; 98:10869-10874. 10. Deng G, Lu Y, Zlotnikov G, Thor AD, Smith HS. Loss of heterozygosity in normal tissue adjacent to breast carcinomas. Science. 1996;274:2057-2059. 11. Ma XJ, Dahiya S, Richardson E, Erlander M, Sgroi DC. Gene expression profiling of the tumor microenvironment during breast cancer progression. Breast Cancer Res. 2009; 11:R7. 12. Allinen M, Beroukhim R, Cai L, et al. Molecular characterization of the tumor microenvironment in breast cancer. Cancer Cell. 2004;6:17-32. 13. Roma´n-Pe´rez E, Casbas-Herna´ndez P, Pirone JR, et al. Gene expression in extratumoral microenvironment predicts clinical outcome in breast cancer patients. Breast Cancer Res. 2012;14:R51. 14. Finak G, Bertos N, Pepin F, et al. Stromal gene expression predicts clinical outcome in breast cancer. Nat Med. 2008;14: 518-527. 15. Farmer P, Bonnefoi H, Anderle P, et al. A stromarelated gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer. Nat Med. 2009;15: 68-74. 16. Beck AH, Sangoi AR, Leung S, et al. Systematic analysis of breast cancer morphology uncovers stromal features associated with survival. Sci Transl Med. 2011;3: 108ra113. 17. Schubert W, Bonnekoh B, Pommer AJ, et al. Analyzing proteome topology and function by automated multidimensional fluorescence microscopy. Nat Biotechnol. 2006;24:1270-1278. 18. Nabavizadeh N, Klifa C, Newitt D, et al. Topographic enhancement mapping of the cancer-associated breast stroma using breast MRI. Integr Biol (Camb). 2011;3: 490-496.
19. Hattangadi J, Park C, Rembert J, et al. Breast stromal enhancement on MRI is associated with response to
When a Doctor Becomes a Patient Chitra Viswanathan, MD I was recently asked to participate in a panel facilitated by my mentor and friend, Dr. Warren Holleman, on the topic “When the Doctor Becomes the Patient.” The panel was a noontime program for faculty and staff of The University of Texas MD Anderson Cancer Center, where we both work. The panel consisted of 4 current or former members of our faculty, all of whom have been recently diagnosed with cancer. The topic of doctors being patients was not new to me. In 1998, when I was a fourth-year medical student, I had a spinal cord injury as a result of a rock climbing accident. I broke my neck at C7-T1, which resulted in C8 quadriplegia, paralysis, and loss of sensation from the mid-chest down. Since then I have used a wheelchair for mobility, as I cannot walk. I had spent the previous 4 years learning how to care for others, and now being on the other side of the treatment equation felt so limiting. There was so much about living a life with a disability that I had not learned in my medical training. Thankfully, I have wonderful family and friends, and I had mentors at Baylor College of Medicine and MD Anderson who helped me advance my career and my life past the accident. Since 2006, I have worked as a chest and body radiologist at MD Anderson. Over the years, I settled into a routine of managing my disability and leading my version of a productive, fruitful life. Actually, I thought I had settled into a routine. In December 2011, I was diagnosed with early-stage breast cancer and began a second journey on the other side of the treatment equation. When Dr. Holleman invited me to serve on the panel, I had mixed emotions; I thought, I have never shared this with other people, but this is something I certainly know about! Many questions arose during the panel discussion, and I was very honored to be asked by my radiation oncologist, Dr. Thomas Buchholz, to share some of them in this editorial, along with my reflections, then and since. 1. Tell us about your illness. I felt a mass in my right breast at the beginning of December. I really thought I might be overreacting, that it could be a rib callus, but I showed it to my gynecologist. I was 38 years old and had never had a mammogram. I didn’t think it was likely to be cancer, and my gynecologist thought it might be glandular tissue, but we decided to
neoadjuvant chemotherapy. AJR Am J Roentgenol. 2008;190: 1630-1636.
get imaging e a mammogram and an ultrasound. I had these tests done at MD Anderson, on a day I was scheduled to work. I was nervous about having the tests done while in my wheelchair, but my friend, the mammographer, and the technologists put me at ease and helped with the proper positioning. After the mammogram was finished, I went back to my reading room to look at the images. Once I saw the mammogram, I became worried. It showed a 1.5-cm spiculated mass. I had to wait only 2-3 hours for the ultrasound, but all I could think was how lucky I was to have it the same day. I remembered how, during my residency, some women had to wait days, weeks, and even months for follow-up studies. The ultrasound confirmed the same findings, along with a cyst in the left breast. The core biopsy results came back the following Monday, and I was diagnosed with invasive ductal carcinoma, grade I-II. I was originally scheduled to have surgery and radiation therapy, but my surgical pathology results showed a 2-mm lymph node metastasis in the sentinel node. That changed my path to post-surgical chemotherapy and radiation therapy. 2. As a physician, what has it been like for you to be on the other side of the treatment equation? I quickly realized that I am not as rational as I thought. In our medical training, we always think about the “what-if” scenario. What if it happened to us? In the case of breast cancer, I was fairly dogmatic; I would have both breasts removed and be done with it. However, when the diagnosis came, I completely changed my tune, which surprised me. I was fortunate, given my early diagnosis and BRCA status, to be able to have breastconserving surgery and oncoplastic rearrangement. The hardest thing about treatment for me was losing “control” of my time and my body. In the medical profession, our lives are dictated by schedules, mostly to ensure the continuity of excellent care. Patients and other physicians rely on us to be on time and to function at a high level. Treatment puts a halt to all of this. The side effects became my guide. When I was on Taxol, I knew that every Monday of therapy would be a day spent in the hospital and that on Tuesday and Wednesday, the bone pain would begin. By Friday afternoon, I would be better, but the following Monday the routine would be repeated, for a total of 12 weeks. On Mondays, the only thing I knew was that it was chemotherapy day. I tried not to have much planned for that day, and I let people know that I would not be
Breast Diseases: A Year BookÒ Quarterly Vol 24 No 1 2013
27
8. Hollingsworth AB, Stough RG. Conflicting outcomes with preoperative breast MRI: differences in technology or methodology? Breast Diseases: A Year Book Quarterly. 2010;21:109-112. 9. Hollingsworth AB, Stough RG, O’Dell CA, Brekke CE. Breast magnetic resonance imaging for preoperative locoregional staging. Am J Surg. 2008;196:389-397. 10. Turnbull L, Brown S, Harvey I, et al. Comparative effectiveness of MRI in breast cancer (COMICE) trial: a randomised controlled trial. Lancet. 2010;375:563-571. 11. Houssami N, Hayes DF. Review of preoperative magnetic resonance imaging (MRI) in breast cancer: should MRI be performed on all women with newly diagnosed, early stage breast cancer? CA Cancer J Clin. 2009;59:290-302. 12. Fisher B, Bauer M, Margolese R, et al. Five-year results of a randomized clinical trial comparing total mastectomy and segmental mastectomy with or without radiation in the treatment of breast cancer. N Engl J Med. 1985;312:665-673.
6. Tuttle TM. Is breast conservation under siege? Breast Diseases: A Year Book Quarterly. 2012;23:128-131.
13. Anderson BO, Khalil el SA. Breast conservation without mammography? Oncology practice in the real world of limited resource countries. Breast. 2006;15:575-579.
7. Miller BT, Abbott AM, Tuttle TM. The influence of preoperative MRI on breast cancer treatment. Ann Surg Oncol. 2012;19:536-540.
14. Kuhn TS. The Structure of Scientific Revolutions. 2nd ed. Chicago, IL: University of Chicago Press; 1970.
The Stromal Microenvironment and Breast Cancer Biology Catherine C. Park, MD The basic architecture of the breast develops through dynamic interactions between the ectodermally derived ductal epithelium and stromal cells of mesenchymal origins.1 This interplay ensures the formation of a functional gland with the following developmental milestones throughout life: puberty, pregnancy, lactation, and involution. Despite dramatic changes in morphology during development, the basic structural integrity of the breast is maintained through normal interactions with the microenvironment. The concept that local stromal microenvironments contribute to cell fate decisions is a central tenet of classical embryology.2 It is now clear that the role of stromal cells in cancer progression is as important as their role in normal development. The evolution of breast cancers requires co-optation of the surrounding stromal tissues to facilitate progression and
support increased metabolic demand. Wound-like properties of the stroma have been shown in the earliest stages of breast cancer, ductal carcinoma in situ (DCIS), where proliferating malignant cells within the breast duct are associated with stromal remodeling outside of the duct that includes increased angiogenesis and microvessel density.3,4 In addition, there are striking changes in the cellular and non-cellular constituents of the activated cancerous stroma, including immune cell infiltrates,5 remodeling of the extracellular matrix,6 and physiologic changes in pH and oxygen tension.7 The past several years have witnessed an exciting increase in our understanding of the molecular and functional bases of these constituents, in part ensuing from advances in array and computational technology and imaging techniques. In particular, the use of genome-wide and gene arrayebased stromal signatures and novel applications of clinical imaging tools, which provide a means to analyze several biologic features in the context of tissue architecture simultaneously, have led to significant new hypotheses about the role of stroma in disease treatment. This editorial will highlight the recent findings in breast cancer biology, with respect to
Breast Diseases: A Year BookÒ Quarterly Vol 24 No 1 2013
23
the role of the stromal microenvironment, that have particular translational and therapeutic relevance. STROMAL GENE EXPRESSION It has been well over a decade since the seminal use of gene expression array technology revealed clinically relevant molecular phenotypes in breast cancer.8,9 Over time, advances in laser capture microdissection and the ability to process small amounts of RNA have been central to our investigation of the role of stromal cells in the co-evolution or progression of disease and to the determination of whether gene expression patterns or signatures are informative in prediction or prognosis. Although gene aberrations and loss of heterozygosity have been detected in the normal-appearing stromal tissue adjacent to invasive lesions,10 several questions remain as to the role of stroma in the evolution of cancers that arise in epithelial cells. To compare stromal gene expression changes in disease progression, Ma and colleagues11 used advanced laser-capture microdissection to isolate DCIS and invasive breast lesions and their adjacent stroma for expression array
analyses. These studies showed that stromal genes, particularly those related to invasion and extracellular matrix remodeling, were highly upregulated and that the most dramatic changes occurred in DCIS lesions, confirming that the tumor microenvironment participates in tumorigenesis well before there is evidence for histologic invasion or the emergence of malignancy. In elegant studies, which isolated cell lineages in normal and cancerous breast tissues, serial analysis of gene expression and expression array analysis showed that increased expression of cytokines that promote tumorigenesis occurs in the myoepithelial cells that are part of the barrier between epithelial and stromal cells in the normal breast.12 Thus, we have clear evidence that stromal gene expression dramatically changes in the early phases of cancer progression. One confounding issue has been the variations in the definitions of tumor stroma, which can include intratumoral stroma, extratumoral stroma, or both (Fig 1A). To address the controversy, Roma´n-Pe´rez and colleagues microdissected tissues adjacent to invasive cancer or DCIS to specifically investigate gene expression changes in the extratumoral
FIGURE 1.d(A) Schematic of tumor and stroma depicted, including intratumoral and extratumoral stroma. (B) Example of gene expression array from microdissected areas of stroma.
24
Breast Diseases: A Year BookÒ Quarterly Vol 24 No 1 2013
microenvironment (for example, Fig 1B).13 Distinct biological features were identified that were associated with cancer progression and a mesenchymal phenotype. Interestingly, an extratumoral stromal signature was identified that had prognostic value in estrogen receptorepositive breast cancer and was associated with inflammatory response, macrophage recruitment, fibrosis, and chemotaxis of cells. Other studies have also correlated stromal gene expression with treatment outcomes. Finak and colleagues14 first identified sets of genes derived from microdissected tumor stroma that had prognostic significance in several clinical data sets. Farmer and colleagues15 found that a stroma-related gene signature predicted resistance to treatment in breast cancer patients receiving neoadjuvant chemotherapy. In aggregate, studies of stromal gene expression indicate that a highly dynamic stromal environment arises early in the process of carcinogenesis and that specific biologic signaling patterns are seen repeatedly, including inflammation, remodeling, invasion, and angiogenesis. In addition, novel signatures with regard to specific pathways such as TWIST-1 and TGF-b also provide insight into the nature of the stromal contribution to malignancy.13 Questions for the future include how stromal gene expression differs in light of tumor molecular phenotypes and if there are specific stromal phenotypes, whether there is a clear transition between activated and benign stroma, and whether there is a consistent pattern of gene expression by stage or other pathologic features of disease. Further investigations and validation studies in larger prospective cohorts are needed to design robust prognostic signatures that could improve risk stratification.
and treatment decision-making in the future. However, understanding stromal biology could also improve the approach to local treatment. Decisions regarding breast surgery have been guided by imaging techniques developed to detect malignant tissues and by the post-surgical detection of microscopic cells at the resection margins as a surrogate for residual disease. However, the concept of an optimal resection volume remains elusive. We now know that the tumor stroma and extratumoral stroma have active signaling and molecular features of inflammation and progression; however, our challenges for the future are to understand the extent of these changes and whether they can be classified into subtypes, have correlations with features of the primary tumor, or have prognostic or predictive importance. Is there a “zone or niche of activation” that has biological significance and can be detected non-invasively? And, ultimately, can the detection of stromal abnormalities guide local resection and radiotherapy? A better understanding of stromal changes could improve re-excision rates and certainly help guide local radiotherapy procedures, especially as partial-breast radiotherapy becomes more developed. We have attempted to find ways to map enhancement patterns in the normal-appearing stroma of breast cancer patients. Taking advantage of the ability to measure gadolinium-based enhancement values in normal-appearing breast tissues, we asked whether an activated stroma could possibly be detected via stromal enhancement patterns. We created topographic enhancement maps where enhancement of each voxel (0.7 mm3) in the stroma was measured and mapped to the edge of the tumor (Fig 2). This showed that in unifocal small tumors, there was a region of high stromal
IMAGING OF STROMAL FEATURES To further our understanding of the functional and molecular aspects of stromal tissue biology, the structural and architectural context must be studied more closely. Microdissection has allowed us to investigate spatial relationships and tissue architecture. Recent developments in computational microscopy imaging have exponentially expanded the number of proteins or features that can be evaluated in a topographic and temporal fashion.16,17 In a study of pathology specimens, systematic analysis of thousands (>6000) of morphologic features from breast cancer tissues using a computational platform, C-Path,16 found that 3 of the 11 features most associated with outcomes were derived from the stroma. A major stromal feature was inflammation, which is consistent with a theme that has been observed by others. The findings we have discussed thus far are based on molecular and histologic material that with more rigorous and prospective validation could enhance risk assessment
FIGURE 2.dBreast MRI with schematic tumor labeled and normalappearing stroma in concentric volumetric shells around the tumor.
Breast Diseases: A Year BookÒ Quarterly Vol 24 No 1 2013
25
enhancement, associated with increased microvessel density, that then decreased with distance from the edge of the tumor.18 In addition, we asked whether global stromal enhancement was associated with clinical outcomes. In a pilot study of patients receiving neoadjuvant chemotherapy, we found that stromal enhancement was associated with disease-free survival, but surprisingly, patients with higher enhancement had significantly better outcomes.19 We postulated that this could be a reflection of improved baseline vascular physiology and chemotherapy penetrance; however, clearly, the picture is much more complicated. The findings from these studies indicate that there is a detectable stromal pattern of enhancement in the normal-appearing fibroglandular breast tissue beyond the malignant tumor; however, further studies correlating the underlying biology of these stromal patterns and outcomes are ongoing. SUMMARY Gene expression studies of the intra- and extratumoral stroma have consistently shown changes in gene expression in these tissue compartments, indicating their active participation in the early evolution of breast tumors. Several lines of evidence from studies in microdissected clinical specimens consistently point to stromal changes in the expression of genes involving inflammation and tissue and extracellular matrix remodeling. As in tumors, there is evidence of heterogeneity among patients, and specific expression signatures have not yet been validated. Mapping of morphologic cancer features using advanced imaging and computational tools also have revealed stromal features that show promise in helping to improve patient risk stratification. Early studies using magnetic resonance imaging technology have attempted to map stromal enhancement in normal-appearing breast tissues surrounding breast cancers; these studies show promise for the ability to detect regions of abnormality. In the future, rigorous correlations between imaging platforms, biology, and outcomes are needed and hold great promise for creating an entirely new way of incorporating stromal biology into cancer care.
References 1. Anbazhagan R, Osin PP, Bartkova J, Nathan B, Lane EB, Gusterson BA. The development of epithelial phenotypes in the human fetal and infant breast. J Pathol. 1998;184:197-206. 2. Bissell MJ, Radisky D. Putting tumours in context. Nat Rev Cancer. 2001;1:46-54. 3. Dvorak HF. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N Engl J Med. 1986;315:1650-1659.
26
Breast Diseases: A Year BookÒ Quarterly Vol 24 No 1 2013
4. Guidi AJ, Fischer L, Harris JR, Schnitt SJ. Microvessel density and distribution in ductal carcinoma in situ of the breast. J Natl Cancer Inst. 1994;86:614-619. 5. DeNardo DG, Coussens LM. Inflammation and breast cancer. Balancing immune response: crosstalk between adaptive and innate immune cells during breast cancer progression. Breast Cancer Res. 2007;9:212. 6. Lu P, Weaver VM, Werb Z. The extracellular matrix: a dynamic niche in cancer progression. J Cell Biol. 2012;196:395-406. 7. Erler JT, Giaccia AJ. Lysyl oxidase mediates hypoxic control of metastasis. Cancer Res. 2006;66:10238-10241. 8. Perou CM, Sørlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature. 2000;406:747-752. 9. Sørlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 2001; 98:10869-10874. 10. Deng G, Lu Y, Zlotnikov G, Thor AD, Smith HS. Loss of heterozygosity in normal tissue adjacent to breast carcinomas. Science. 1996;274:2057-2059. 11. Ma XJ, Dahiya S, Richardson E, Erlander M, Sgroi DC. Gene expression profiling of the tumor microenvironment during breast cancer progression. Breast Cancer Res. 2009; 11:R7. 12. Allinen M, Beroukhim R, Cai L, et al. Molecular characterization of the tumor microenvironment in breast cancer. Cancer Cell. 2004;6:17-32. 13. Roma´n-Pe´rez E, Casbas-Herna´ndez P, Pirone JR, et al. Gene expression in extratumoral microenvironment predicts clinical outcome in breast cancer patients. Breast Cancer Res. 2012;14:R51. 14. Finak G, Bertos N, Pepin F, et al. Stromal gene expression predicts clinical outcome in breast cancer. Nat Med. 2008;14: 518-527. 15. Farmer P, Bonnefoi H, Anderle P, et al. A stromarelated gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer. Nat Med. 2009;15: 68-74. 16. Beck AH, Sangoi AR, Leung S, et al. Systematic analysis of breast cancer morphology uncovers stromal features associated with survival. Sci Transl Med. 2011;3: 108ra113. 17. Schubert W, Bonnekoh B, Pommer AJ, et al. Analyzing proteome topology and function by automated multidimensional fluorescence microscopy. Nat Biotechnol. 2006;24:1270-1278. 18. Nabavizadeh N, Klifa C, Newitt D, et al. Topographic enhancement mapping of the cancer-associated breast stroma using breast MRI. Integr Biol (Camb). 2011;3: 490-496.
19. Hattangadi J, Park C, Rembert J, et al. Breast stromal enhancement on MRI is associated with response to
When a Doctor Becomes a Patient Chitra Viswanathan, MD I was recently asked to participate in a panel facilitated by my mentor and friend, Dr. Warren Holleman, on the topic “When the Doctor Becomes the Patient.” The panel was a noontime program for faculty and staff of The University of Texas MD Anderson Cancer Center, where we both work. The panel consisted of 4 current or former members of our faculty, all of whom have been recently diagnosed with cancer. The topic of doctors being patients was not new to me. In 1998, when I was a fourth-year medical student, I had a spinal cord injury as a result of a rock climbing accident. I broke my neck at C7-T1, which resulted in C8 quadriplegia, paralysis, and loss of sensation from the mid-chest down. Since then I have used a wheelchair for mobility, as I cannot walk. I had spent the previous 4 years learning how to care for others, and now being on the other side of the treatment equation felt so limiting. There was so much about living a life with a disability that I had not learned in my medical training. Thankfully, I have wonderful family and friends, and I had mentors at Baylor College of Medicine and MD Anderson who helped me advance my career and my life past the accident. Since 2006, I have worked as a chest and body radiologist at MD Anderson. Over the years, I settled into a routine of managing my disability and leading my version of a productive, fruitful life. Actually, I thought I had settled into a routine. In December 2011, I was diagnosed with early-stage breast cancer and began a second journey on the other side of the treatment equation. When Dr. Holleman invited me to serve on the panel, I had mixed emotions; I thought, I have never shared this with other people, but this is something I certainly know about! Many questions arose during the panel discussion, and I was very honored to be asked by my radiation oncologist, Dr. Thomas Buchholz, to share some of them in this editorial, along with my reflections, then and since. 1. Tell us about your illness. I felt a mass in my right breast at the beginning of December. I really thought I might be overreacting, that it could be a rib callus, but I showed it to my gynecologist. I was 38 years old and had never had a mammogram. I didn’t think it was likely to be cancer, and my gynecologist thought it might be glandular tissue, but we decided to
neoadjuvant chemotherapy. AJR Am J Roentgenol. 2008;190: 1630-1636.
get imaging e a mammogram and an ultrasound. I had these tests done at MD Anderson, on a day I was scheduled to work. I was nervous about having the tests done while in my wheelchair, but my friend, the mammographer, and the technologists put me at ease and helped with the proper positioning. After the mammogram was finished, I went back to my reading room to look at the images. Once I saw the mammogram, I became worried. It showed a 1.5-cm spiculated mass. I had to wait only 2-3 hours for the ultrasound, but all I could think was how lucky I was to have it the same day. I remembered how, during my residency, some women had to wait days, weeks, and even months for follow-up studies. The ultrasound confirmed the same findings, along with a cyst in the left breast. The core biopsy results came back the following Monday, and I was diagnosed with invasive ductal carcinoma, grade I-II. I was originally scheduled to have surgery and radiation therapy, but my surgical pathology results showed a 2-mm lymph node metastasis in the sentinel node. That changed my path to post-surgical chemotherapy and radiation therapy. 2. As a physician, what has it been like for you to be on the other side of the treatment equation? I quickly realized that I am not as rational as I thought. In our medical training, we always think about the “what-if” scenario. What if it happened to us? In the case of breast cancer, I was fairly dogmatic; I would have both breasts removed and be done with it. However, when the diagnosis came, I completely changed my tune, which surprised me. I was fortunate, given my early diagnosis and BRCA status, to be able to have breastconserving surgery and oncoplastic rearrangement. The hardest thing about treatment for me was losing “control” of my time and my body. In the medical profession, our lives are dictated by schedules, mostly to ensure the continuity of excellent care. Patients and other physicians rely on us to be on time and to function at a high level. Treatment puts a halt to all of this. The side effects became my guide. When I was on Taxol, I knew that every Monday of therapy would be a day spent in the hospital and that on Tuesday and Wednesday, the bone pain would begin. By Friday afternoon, I would be better, but the following Monday the routine would be repeated, for a total of 12 weeks. On Mondays, the only thing I knew was that it was chemotherapy day. I tried not to have much planned for that day, and I let people know that I would not be
Breast Diseases: A Year BookÒ Quarterly Vol 24 No 1 2013
27