SEARCH

SEARCH BY CITATION

Keywords:

  • cell lines;
  • inflammatory breast cancer;
  • radiation;
  • surgery;
  • chemotherapy;
  • history;
  • stem cells;
  • mammospheres

Abstract

  1. Top of page
  2. Abstract
  3. Biological Basis for Radiation Resistance of IBC
  4. CONFLICT OF INTEREST DISCLOSURES
  5. REFERENCES

The clinical-pathological features of inflammatory breast cancer include enrichment of factors that have been previously associated with radioresistant disease, including negative hormone receptor status and a phenotype enriched for relatively radioresistant breast cancer stem/progenitor cells. The risks and benefits of accelerated postmastectomy radiation treatment regimens in the multimodality management of inflammatory breast cancer were reviewed at the first International Inflammatory Breast Cancer Conference at The University of Texas M. D. Anderson Cancer Center. The biological basis for radiation resistance and strategies to radiosensitize these tumors were also presented. The prevalent basal phenotype of inflammatory breast cancer makes it an ideal clinical model to examine stem cell hypotheses, which the authors believe can help guide future trials to continue making incremental progress against this aggressive disease. Cancer 2010;116(11 suppl):2840–5. © 2010 American Cancer Society.

Currently, the most widely referenced definition of inflammatory breast cancer (IBC) is that of the American Joint Committee on Cancer,1 which states in part that inflammatory carcinoma is a clinicopathologic entity characterized by diffuse erythema and edema (peau d'orange) of the breast, often without an underlying mass. These clinical findings should involve the majority of the breast. It is important to remember that inflammatory carcinoma is primarily a clinical diagnosis. Involvement of the dermal lymphatics alone does not indicate inflammatory carcinoma in the absence of clinical findings.

The unique features of the primary disease in IBC make optimal local/regional management a critical component of the multidisciplinary care of IBC. The current standard or care in the management of IBC includes neoadjuvant chemotherapy ± trastuzumab for appropriate patients, modified radical mastectomy, and high-dose postmastectomy radiation targeting the chest wall skin and soft tissue as well as the draining lymphatics. Clearly the skin is a target in IBC, and care must be taken to ensure adequate dose and skin reaction. Importantly, “despite the rapid progressive nature of IBC, 70% of patients present with local/regional disease without distant metastases at diagnosis,”2 highlighting the potential for improved cure rates with adequate local/regional disease control.

Before the introduction of effective chemotherapy in the management of solid tumors, the 5-year actuarial overall survival for IBC was <5%, with a median survival of only 15 months. Modified radical mastectomy was not technically possible for many patients with IBC because of the extent of disease on the chest wall at presentation. It was often difficult to achieve negative margins, and there was little or no benefit to a positive margin surgery. This led to the use of primary radiotherapy as the sole treatment modality yielding excellent symptom palliation, with modest short-term local control rates of 50%.

In this era, The University of Texas M. D. Anderson Cancer Center initiated a pilot study of altered-fractionation radiation therapy with the intent of intensifying the radiation dose and improving local control. Among 80 patients studied, 69 patients received standard fractionation of 2 gray (Gy) per day to the chest wall and draining lymphatics. With this regimen, patients received a total of 50 Gy + a 10- to 16-Gy boost to gross disease. Eleven patients received twice-daily fractionation treatment of 1.5 Gy per treatment to a total of 51 Gy + a 15- to 20-Gy boost to gross disease. Although this was a small study, the local control was 54% in the once-a-day arm versus 73% in the altered fractionation arm.3 In 1974, the introduction of doxorubicin chemotherapy yielded another significant gain in IBC outcome. Although the addition of chemotherapy had no effect on local control (73% vs 74%3), the 5-year survival improved from <5% to 30%-50% because of the reduction in distant metastasis. Importantly, the addition of chemotherapy rendered more patients operable, and modified radical mastectomy was reintroduced. Several series subsequently reported the local control benefit of mastectomy in the setting of systemic chemotherapy, demonstrating an absolute increase in local control of 24% to 45%.4-6 In the postmastectomy setting, however, total radiation dose was lowered from a gross disease dose of 66 to 70 Gy to a microscopic disease dose of a standard 60 Gy. Although this lowered the complication rates related to radiotherapy, local failure remained a challenge, with local failure rates exceeding 20%.7 Demonstrating that this was a problem of radiation resistance to this lowered dose, the majority of these local failures were centered in the radiation field on the chest wall. This provided the rationale for dose escalation in the setting of postmastectomy radiation, and in 1987, a dose escalation regimen was introduced for IBC patients at The University of Texas M. D. Anderson Cancer Center consisting of 51 Gy to the chest wall and draining lymphatics, and a 15-Gy boost to the chest wall (to 66 Gy total) delivered at 1.5 Gy/fraction twice daily. Treating twice a day reduced the overall treatment time from 6 to 4½ weeks, an example of the early dose-dense strategy in radiation therapy.

In 2008, a retrospective analysis was performed of all patients with nonmetastatic IBC cancer treated at The University of Texas M. D. Anderson Cancer Center over the progression of these treatment strategies from 1977 to 2004.8 Patients who were initially intended to receive neoadjuvant chemotherapy, modified radical mastectomy, and postmastectomy radiation were included regardless of whether they were able to complete all therapy and grouped by those who completed all therapy or those who did not, often because of progressive disease. Among 256 patients analyzed, 192 completed all therapy (Group 1), whereas 64 did not and received either preoperative radiation (n = 21) or definitive radiation (n = 21). An additional 22 patients developed a local recurrence before radiation therapy commenced. For the entire cohort of 256 patients, the 5-year actuarial distant metastasis-free survival, local control, and overall survival were 40%, 76%, and 44%, respectively.8 Not unexpectedly, 5-year overall survival was significantly improved for patients who completed all therapy, 51% versus 24% (P = .0001), and the local/regional control rate was 84% for patients who completed all therapy. This rate of local/regional control was higher than historical rates. Examination of the factors that predicted for local recurrence after radiation among patients in Group 1 revealed that age <45 years, less than partial response to chemotherapy, ≥4 positive lymph nodes, lack of taxane use, and positive or unknown surgical margins were all associated with increased risk of local/regional recurrence. Patients with any 1 of these factors had higher rates of local/regional recurrence, ranging from 21% to 52%. Dose escalation (60 vs 66 Gy) appeared to have the most significant benefit in these high-risk groups, with age <45, positive/unknown margins, and disease unresponsive to chemotherapy. Given the higher rate of grade 3 toxicity at 5 years with dose escalation (29% vs 15%, P = .08), we now reserve dose escalation for patients with high-risk features predictive of local failure.

Biological Basis for Radiation Resistance of IBC

  1. Top of page
  2. Abstract
  3. Biological Basis for Radiation Resistance of IBC
  4. CONFLICT OF INTEREST DISCLOSURES
  5. REFERENCES

The clinical-pathological features of IBC include enrichment of factors that have been previously associated with radioresistant disease, including negative receptor status (estrogen and progesterone9, 10) and a phenotype enriched for relatively radioresistant breast cancer stem/progenitor cells.11-13 In general, it is clear that although treatment advances have been made, clinical experience demonstrates a relative radioresistance of IBC tumors compared with non-IBC breast tumors, where local failure rates after contemporary postmastectomy radiation are <10%.14 Although improved local control can be achieved in IBC with dose escalation, the cost in terms of toxicity can be high, and alternative approaches to radiosensitize these tumor cells are desirable.

Laboratory investigations of available IBC cell lines SUM-149 and SUM-190 demonstrate relative radioresistance of these lines (data not shown), making them good models for investigation of radioresistance mechanisms. Radiation resistance is classically assessed by clonogenic cell survival curves, which are generated by counting colonies produced from cells that survive radiation and are capable of dividing clonally. The primary mechanism of radiation cell killing in solid tumors is mitotic cell death rather than apoptosis, and as such, this long-term assessment of replicative potential eliminates cells that may persist briefly after radiation but are destined to die after a few cell divisions and thus are not likely to be capable of mediating recurrence.

Clonogenicity has been commonly assessed in monolayer culture in serum containing media that may diminish the stem or progenitor cells' potential in the culture system.15, 16 Because normal and cancer stem/progenitor cells from mammary gland have been shown to be resistant to radiation compared with bulk, differentiated cells,11, 12, 17 we have compared clonogenicity of IBC cell lines assessed in 2-dimensional (2D) monolayer clonogenic assays as well as in 3-dimensional serum-free growth factor-enriched clonogenic assays designed to support stem/progenitor cells in in vitro culture.15 Exposure to stem cell-promoting media increases the percentage of tumor-initiating cells that have been prospectively identified as CD44high/CD24low18 (Fig. 1) from human breast cancers. These tumor-initiating cell populations are more resistant to radiation in clonogenic assays compared with the adherent population (Fig. 2). This assay system provides an effective model to test pharmacologic and genetic approaches to inhibit putative cancer stem cell survival factors in the search for selective cancer stem cell radiosensitizers in IBC.

thumbnail image

Figure 1. Flow cytometric analysis of expression of CD24 and CD44 in SUM149 inflammatory breast cancer (IBC) cells is shown. SUM149 IBC cells were cultured under adherent (2-dimentional [2D]) or tumor-initiating cells enrichment condition (3D). Cells were stained with CD24 and CD44 antibodies conjugated with different dyes and subjected to cytometric analysis. (A) The distribution of CD24 and CD44 expression in 1 representative experiment is shown: (A1) cells cultured in 2D condition stained with CD24 only; (A2) cells cultured in 3D condition stained with CD44 only; (A3) cells grown in 2D condition stained with both CD24 and CD44; (A4) cells grown in 3D condition stained with both CD24 and CD44. (B) The relative increase of CD44 + CD24low population in cells cultured in 3D condition compared with that in 2D condition is shown. The result is a summary of 5 independent experiments. *P < .05.

Download figure to PowerPoint

thumbnail image

Figure 2. Tumor-initiating cells from inflammatory breast cancer (IBC) cell lines are more resistant to radiation than monolayer (2-dimensional [2D]) cells. Clonogenic survival assay of IBC cells derived from (A) SUM149 and (B) SUM190 cultured under 2D or mammosphere (3D) cultures is shown. Cells in 3D are more resistant to radiation than cells grown in 2D. Cells were grown in 2D or 3D culture conditions and irradiated with single, increasing doses (0, 2, 4, 6 gray [Gy]) of radiation.

Download figure to PowerPoint

Clinical Radiosensitizers in IBC

Although novel targeted cancer stem cell radiosensitizers are desirable, they are still in early in clinical development, and it will be some time before optimal strategies are defined. In the meantime, building on published and institutional experience with the oral prodrug of 5-fluoruracil, capecitabine,19 we have activated a new protocol for patients with progressive primary IBC to radiosensitize the primary tumors during radiation using capecitabine.

On the basis of the long history of 5-fluorouracil chemotherapy for breast cancer and the encouraging safety and efficacy results for preoperative concurrent capecitabine and radiation in rectal cancer,20, 21 this approach was advocated for carefully selected breast cancer patients with inoperable breast cancer who had no better treatment options at The University of Texas M. D. Anderson Cancer Center. In a retrospective review of 55 patients treated with concurrent radiation and capecitabine at The University of Texas M. D. Anderson Cancer Center for inoperable breast cancer (IBC and non-IBC), concurrent chemoradiation with capecitabine demonstrated 91% of these patients converted to having operable disease after treatment.22 The clinical complete response rate was 33%; moreover, the overall pathological complete response rate was 20%. Only 1 patient had progressive disease. The 5-year overall survival, local recurrence-free survival, and distant metastasis-free survival rates were 48%, 85%, and 37%. Sixteen (29%) patients had a grade 3 or higher complication (acute yet resolving skin toxicity).22

In a phase 2 study of radiation and capecitabine in women with locally advanced breast cancer who had failed first-line anthracycline-based neoadjuvant therapy, Gaui et al. studied the concomitant use of radiation therapy and capecitabine, to determine the toxicity and efficacy of this regimen as a second-line neoadjuvant treatment.19 Twenty-eight patients with inoperable locally advanced breast cancer refractory to first-line anthracycline-based treatment were enrolled between January 2003 and May 2004. Patients received radiation therapy (total dose 5000 cGy) and concomitant capecitabine (850 mg/m2) twice daily for 14 days every 3 weeks. This treatment rendered 23 (82%) of the 28 patients operable. The 5 remaining patients did not undergo surgery because of disease progression. The median clinical tumor size decreased from 80 to 49 cm2. Microscopic residual disease was observed in 3 (13%) patients, and another patient achieved a complete pathologic response. The median number of involved lymph nodes was 2, and treatment was well tolerated, with no grade 3 or 4 events. These results indicate that second-line neoadjuvant treatment with radiation therapy and capecitabine is feasible, well tolerated, and effective in patients with locally advanced breast cancer refractory to primary anthracycline-based treatment.

To assess the efficacy and toxicity prospectively, to set the bar for upcoming novel radiosensitizers, and to permit the correlation of breast cancer stem cells in the primary tumor pre- and post-radiation therapy to outcome, we have recently activated a single-arm phase 2 study to quantify response, conversion to operable among nonoperable patients, local control of gross disease not resected at the time of surgery, and toxicity. Patients consenting to optional procedures will have a core biopsy performed before and at the conclusion of radiotherapy to be assessed for cancer stem cells before and after radiotherapy. We believe that advances in technology for radiation therapy targeting and delivery make this the right time for strategies exploring aggressive local/regional management in patients with progressive IBC. Although we anticipate preoperative radiation with capecitabine will provide disease control and conversion to operability in some patients, patient selection and consent are critical before embarking on any new therapy with significant potential toxicity. These data will provide a baseline against which to compare new pipeline IBC targets. The prevalent basal phenotype of IBC makes it an ideal clinical model to examine stem cell hypotheses, which we believe can help guide future trials to continue making incremental progress against this profoundly aggressive disease.

Conclusions

Acceleration of radiotherapy regimens, giving increased dose in less time, has been an effective strategy against the aggressive biology of IBC. Higher-risk patient subsets have been identified that gain the greatest benefit from these aggressive strategies, but local control has not yet been maximized for all patients, and safer, less toxic approaches are needed. Greater understanding of the biology of radiation resistance in IBC is providing new insights to guide the design of new trials with novel radiosensitizers to improve tumor control in IBC.

Methods

Clonogenic assay

To evaluate radiosensitivity, specified numbers of cells derived from monolayer cultures were seeded into individual wells of a 6-well tissue culture plate (for 2D) or ultralow attachment plates (for 3D). Cells from both 2D and 3D were exposed to increasing doses of radiation (2, 4, or 6 Gy) 4 hours after plating. 2D plates were incubated for 10 to 14 days, and colonies were stained with crystal violet. For 3D, cells were incubated in mammosphere media for 7 days, and spheres were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to improve visualization and counted using a Gelcount colony counter (Oxford Optronix, Oxford, UK). Clonogenic survival curves were generated using Sigmaplot 8.0.

Flow cytometry

SUM149 and SUM190 cells cultured under mammosphere and adherent conditions were harvested with 10 mM ethylenediaminetetraacetic acid, centrifuged, and resuspended in phosphate-buffered saline (PBS) (105 cells/mL). They were then incubated with fluorescein isothiocyanate (FITC)-conjugated CD44 and phycoerythrin (PE)-conjugated CD24 antibodies (BD Biosciences, San Diego, Calif) for 30 minutes at concentrations recommended by the manufacturer. Cells incubated in PBS, FITC, or PE alone served as controls. Cell analysis for the expression of CD44 and CD24 was performed using a Beckman Coulter (Fullerton, Calif) machine, and the data files were analyzed using FlowJo software (Treestar, Ashland, Ore).

CONFLICT OF INTEREST DISCLOSURES

  1. Top of page
  2. Abstract
  3. Biological Basis for Radiation Resistance of IBC
  4. CONFLICT OF INTEREST DISCLOSURES
  5. REFERENCES

This supplement was sponsored by the Houston Affiliate of Susan G. Komen for the Cure, the National Cancer Institute, and the State of Texas Rare and Aggressive Breast Cancer Research Program. The First International Inflammatory Breast Cancer Conference was supported in part by GlaxoSmithKline, Pfizer, Eli Lilly and Company, and Cardinal Health. Supported by the Morgan Welch Inflammatory Breast Cancer Research Program and Clinic; the State of Texas Grant for Rare and Aggressive Cancers; and The University of Texas Institutional Research Grants; KL2 RR024149 and R01CA138239-01.

REFERENCES

  1. Top of page
  2. Abstract
  3. Biological Basis for Radiation Resistance of IBC
  4. CONFLICT OF INTEREST DISCLOSURES
  5. REFERENCES
  • 1
    Singletary SE, Allred C, Ashley P, et al. Staging system for breast cancer: revisions for the 6th edition of the AJCC Cancer Staging Manual. Surg Clin North Am. 2003; 83: 803-819.
  • 2
    Wingo PA, Jamison PM, Young JL, Gargiullo P. Population-based statistics for women diagnosed with inflammatory breast cancer (United States). Cancer Causes Control. 2004; 15: 321-328.
  • 3
    Barker JL, Montague ED, Peters LJ. Clinical experience with irradiation of inflammatory carcinoma of the breast with and without elective chemotherapy. Cancer. 1980; 45: 625-629.
  • 4
    Fleming RY, Asmar L, Buzdar AU, et al. Effectiveness of mastectomy by response to induction chemotherapy for control in inflammatory breast carcinoma. Ann Surg Oncol. 1997; 4: 452-461.
  • 5
    Panades M, Olivotto IA, Speers CH, et al. Evolving treatment strategies for inflammatory breast cancer: a population-based survival analysis. J Clin Oncol. 2005; 23: 1941-1950.
  • 6
    Perez CA, Fields JN, Fracasso PM, et al. Management of locally advanced carcinoma of the breast. II. Inflammatory carcinoma. Cancer. 1994; 74( 1 suppl): 466-476.
  • 7
    Thoms WW Jr, McNeese MD, Fletcher GH, Buzdar AU, Singletary SE, Oswald MJ. Multimodal treatment for inflammatory breast cancer. Int J Radiat Oncol Biol Phys. 1989; 17: 739-745.
  • 8
    Bristol IJ, Woodward WA, Strom EA, et al. Locoregional treatment outcomes after multimodality management of inflammatory breast cancer. Int J Radiat Oncol Biol Phys. 2008; 72: 474-484.
  • 9
    Kyndi M, Sorensen FB, Knudsen H, Overgaard M, Nielsen HM, Overgaard J. Estrogen receptor, progesterone receptor, HER-2, and response to postmastectomy radiotherapy in high-risk breast cancer: the Danish Breast Cancer Cooperative Group. J Clin Oncol. 2008; 26: 1419-1426.
  • 10
    Woodward WA, Buchholz TA. The role of locoregional therapy in inflammatory breast cancer. Semin Oncol. 2008; 35: 78-86.
  • 11
    Phillips TM, McBride WH, Pajonk F. The response of CD24(-/low)/CD44+ breast cancer-initiating cells to radiation. J Natl Cancer Inst. 2006; 98: 1777-1785.
  • 12
    Woodward WA, Chen MS, Behbod F, Alfaro MP, Buchholz TA, Rosen JM. WNT/beta-catenin mediates radiation resistance of mouse mammary progenitor cells. Proc Natl Acad Sci U S A. 2007; 104: 618-623.
  • 13
    Xiao Y, Ye Y, Yearsley K, Jones S, Barsky SH. The lymphovascular embolus of inflammatory breast cancer expresses a stem cell-like phenotype. Am J Pathol. 2008; 173: 561-574.
  • 14
    Greenbaum MP, Strom EA, Allen PK, et al. Low locoregional recurrence rates in patients treated after 2000 with doxorubicin based chemotherapy, modified radical mastectomy, and post-mastectomy radiation. Radiother Oncol.In press.
  • 15
    Dontu G, Abdallah WM, Foley JM, et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 2003; 17: 1253-1270.
  • 16
    Woodward WA, Bristow RG. Radiosensitivity of cancer-initiating cells and normal stem cells (or what the Heisenberg uncertainly principle has to do with biology). Semin Radiat Oncol. 2009; 19: 87-95.
  • 17
    Zhang M, Behbod F, Atkinson RL, et al. Identification of tumor-initiating cells in a p53-null mouse model of breast cancer. Cancer Res. 2008; 68: 4674-4682.
  • 18
    Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003; 100: 3983-3988.
  • 19
    Gaui MF, Amorim G, Arcuri RA, et al. A phase II study of second-line neoadjuvant chemotherapy with capecitabine and radiation therapy for anthracycline-resistant locally advanced breast cancer. Am J Clin Oncol. 2007; 30: 78-81.
  • 20
    Dunst J, Debus J, Rudat V, et al. Neoadjuvant capecitabine combined with standard radiotherapy in patients with locally advanced rectal cancer: mature results of a phase II trial. Strahlenther Onkol. 2008; 184: 450-456.
  • 21
    Krishnan S, Janjan NA, Skibber JM, et al. Phase II study of capecitabine (Xeloda) and concomitant boost radiotherapy in patients with locally advanced rectal cancer. Int J Radiat Oncol Biol Phys. 2006; 66: 762-771.
  • 22
    Perkins GH, Middleton LP, Tran R, et al. Concurrent chemoradiation with capecitabine achieves meritable response and local control for inoperable and recurrent neoadjuvant chemotherapy refractory breast cancer. Paper presented at: San Antonio Breast Cancer Symposium; December 13-16, 2007; San Antonio, Tex. Paper 4074.