|MINI SYMPOSIUM: BREAST CANCER
|Year : 2013 | Volume
| Issue : 1 | Page : 46-51
Effect of neoadjuvant chemotherapy on stromal CD10 antigens in breast cancer - A preliminary study
S Thomas1, RJ Babu1, K Agarwal2, V Puri2, M Jain2, M Andley1, SK Tudu1
1 Department of Surgery, Lady Hardinge Medical College, Shaheed Bhagat Singh Marg, New Delhi, India
2 Department of Pathology, Lady Hardinge Medical College, Shaheed Bhagat Singh Marg, New Delhi, India
|Date of Web Publication||20-May-2013|
Department of Surgery, Lady Hardinge Medical College, Shaheed Bhagat Singh Marg, New Delhi
Introduction: CD10 is a zinc-dependent peptidase (metalloproteinase). Stromal CD10 expression in breast cancer correlates with poor prognosis, oestrogen receptor negativity and higher grade. CD10 may be a potential target of new cancer therapies as it is involved in cleavage of doxorubicin. Aim: To evaluate the effect of neo-adjuvant anthracycline-based chemotherapy on status of stromal CD10 antigens in breast cancer. Materials and Methods: Patients with invasive breast cancer scheduled for anthracycline-based neo-adjuvant chemotherapy were included in the study. Tumor stromal CD10 expression was estimated before and after 3 cycles of chemotherapy, and change in its status was correlated with clinical response to chemotherapy. Results: 16 out of the 29 patients had strong CD10 expression; in these 16 patients, 14 (87.5%) were hormone receptor negative, and 14 (87.5%) had HER-2/neu overexpression. Stromal CD10 expression remained same in 13 out of 29 cases (44.83%) after chemotherapy. There was a change in CD10 expression in the remaining 16 cases (55.17%); in 13 cases (44.83%) it decreased from its pre-chemotherapy status, while its expression increased in 3 cases (10.34%). In cases of complete and partial clinical response, there was no increase in CD10 expression. Where CD10 expression had increased after chemotherapy, there was either a minor response or no response to chemotherapy. In 13 cases where CD10 expression had decreased, 12 cases had a clinical response to chemotherapy. Conclusions: Strong CD10 expression correlates with hormone receptor negativity and HER-2/neu overexpression. Stromal CD10 expression in breast cancer is not static and changes with neo-adjuvant anthracycline-based chemotherapy. A stable or decrease in CD10 expression correlates with complete or partial clinical response, while an increase in CD10 expression appears to correlate with poor clinical response. A larger series is required to determine the clinical significance of these changes. As stromal CD10 expression and its change with chemotherapy may have a prognostic significance, they should be documented in breast cancer patients before and after chemotherapy.
Keywords: Adjuvant chemotherapy, Anthracyclines, Breast cancer, CD10 antigen
|How to cite this article:|
Thomas S, Babu R J, Agarwal K, Puri V, Jain M, Andley M, Tudu S K. Effect of neoadjuvant chemotherapy on stromal CD10 antigens in breast cancer - A preliminary study. Indian J Cancer 2013;50:46-51
|How to cite this URL:|
Thomas S, Babu R J, Agarwal K, Puri V, Jain M, Andley M, Tudu S K. Effect of neoadjuvant chemotherapy on stromal CD10 antigens in breast cancer - A preliminary study. Indian J Cancer [serial online] 2013 [cited 2014 Jul 29];50:46-51. Available from: http://www.indianjcancer.com/text.asp?2013/50/1/46/112299
| » Introduction|| |
Neo-adjuvant chemotherapy has been the standard approach in the last quarter of a century for locally advanced breast cancer. In early stage breast cancer, it helps to shrink the tumour to a volume that makes them acceptable to breast conservation surgery.  Chemotherapy is also the mainstay of therapy and is considered for almost all patients with metastatic breast cancer. 
As a vast majority of the patients receive systemic therapy for breast cancer, it has led to an extensive search for new and more effective predictor factors. Estrogen receptor (ER) and Progesterone receptor (PR) status are the most important and helpful predictive factors currently available. Human Epidermal growth factor 2 (HER2) amplification or over expression has been associated with higher tumour grade and poorer prognosis.  Changes in status of hormone receptors and HER2 are seen after chemotherapy in studies, with ER and PR increasing after chemotherapy and HER2 decreasing after chemotherapy. ,
CD10 is a zinc-dependent peptidase (metalloproteinase). CD10 positivity has been reported in myoepithelial cells from normal breast tissue and benign myoepithelial tumours.  Stromal CD10 expression in invasive breast carcinoma correlates with poor prognosis, oestrogen receptor negativity and high grade. , Some experimental data indicates that CD10 may be a potential target of new cancer therapies as it is involved in cleavage of doxorubicin. 
There has been no study till date on the effect of chemotherapy on change in CD10 antigens in invasive breast carcinoma. The aim of our study was to evaluate the effect of neo-adjuvant anthracycline based chemotherapy on changes in status of CD10 antigens in invasive breast cancer.
| » Materials and Methods|| |
Patients with FNAC or biopsy proved invasive breast cancer attending the Surgery Outpatient Department of our hospital, and scheduled for anthracycline based neo-adjuvant chemotherapy were included in the study. Excluded from the study were patients not scheduled for, or unfit for anthracycline based neo-adjuvant chemotherapy, and those patients who had already received some form of neoadjuvant chemotherapy before presenting to the clinic. Pregnancy was also an exclusion criterion.
The study proposal was cleared by the Institutional Ethics Committee. An informed consent was taken from all the patients in the study.
All the patients satisfying the inclusion criteria underwent complete physical examination, complete blood count and biochemical tests, ECG, bilateral mammography, x-ray study of the chest, bone scan, and ultrasonography of abdomen.
The patients in the study were subjected to a trucut biopsy for assessing CD10 expression in the tumor stroma before starting neo-adjuvant anthracycline based chemotherapy. CD10 status was also estimated after completion of 3 cycles of chemotherapy in the mastectomy specimen. All patients were taken up for mastectomy after completing 3 cycles of neo adjuvant chemotherapy. Standard pathological markers / receptor status i.e., hormone receptors (estrogen and progesterone) and HER-2/neu overexpression, was also done in the trucut biopsy specimen before starting treatment.
Clinical response was evaluated by ultrasound measurement of the maximum diameter of the tumor before and after chemotherapy. The response was categorized as complete response (CR), partial response (PR - more than 50% reduction in size), minimal response (MR - less than 50% reduction in size), stable disease (SD - no change in size), and progressive disease (PD).
Chemotherapy schedule: Patients included in the study received the standard CAF regimen.  Their body surface area was measured and dosages were calculated accordingly (cyclophosphamide 500 mg/m2 on day 1, IV bolus, adriamycin 50 mg/m2 on day 1 as IV infusion over 2-3 hours to a maximum of 550 mg for total duration of chemotherapy, 5-flurouracil 500 mg/m2 on day 1, IV bolus). Patients received a total of 6 cycles of chemotherapy with interval of 21 days in between each cycle. Before every cycle, patients underwent complete physical examination, ECG and complete blood count. Estimation of CD10 was done before starting chemotherapy and after completing 3 cycles of chemotherapy.
Estimation of CD10: Estimation of CD10 was done in the Department of Pathology, Lady Hardinge Medical College. CD10 expression in tumour stroma was assessed by immuno-histochemistry. For this procedure, a slide which showed maximum tumour with minimal necrosis and hemorrhage was chosen. At least 10 serial sections were cut from the blocks and taken on slides coated with polyl lysine. Immunostaining was done by the streptavidin-biotin immunoperoxidase technique (LSAB) using monoclonal antibodies to the antigen.
Specimens with more than 10% of the stromal cells in tumor staining CD10 were taken as positive. If staining was between 10% to 30%, it was taken as weakly positive, and more than 30% staining was taken as strongly positive. 
The percentage of tumor cells expressing CD10 antigens was estimated and documented before and after chemotherapy. For each patient, it was seen if the percentage of CD10 expression had increased, decreased or remained the same after chemotherapy.
| » Results|| |
29 consecutive cases of newly diagnosed breast cancer patients who attended Surgery Outpatient Department of Lady Hardinge Medical College and were planned for anthracycline based neo-adjuvant chemotherapy were included in the study.
The age of the patients varied between 34 years to 55 years (mean age 44.483 years ± 6.202 SD, median age 44 years). There were 28 females and one male patient of carcinoma breast.
The duration of symptoms varied from 2 months to 24 months, with a mean of 7.139 months (±4.749 SD) and median of 5 months. The primary presenting complaint in all the patients was a breast lump detected by them.
On analysis of T staging of tumor, it was found that 25 cases (86.2%) were T4, 3 cases (10.34%) were T3 and 1 case (3.45%) was T2. 27 cases (93.1%) were infiltrating ductal carcinoma. There was one case (3.45%) of infiltrating ductal carcinoma with extensive squamous metaplasia and one case (3.45%) of mixed mucinous and ductal carcinoma with pagetoid nipple.
Out of 29 cases, 24 cases (82.76%) showed response to chemotherapy; 4 of these cases (13.79%) had complete response, 12 cases (41.38%) had partial response, and 8 cases (27.58%) had minor response. 1 case (3.45%) had no change and 4 cases (13.79%) had progressive disease. Complete pathological response was found in all the 4 cases with complete clinical response (13.79%).
Out of the 29 patients, 20 were hormone receptor negative, and 23 had HER-2/neu overexpression.
In the pre-chemotherapy samples, CD10 expression was positive in 24 cases (82.76%) [strongly positive in 16 cases (55.17%) and weakly positive in 8 cases (27.59%)], and negative in 5 cases (17.24%). In post-chemotherapy samples, out of 29 cases studied, CD10 expression was positive in 13 cases (44.83%) [strongly positive in 10 cases (34.82%) and weakly positive in 3 cases (10.34%)], and negative in 16 cases (55.17%). [Table 1]
On correlating CD10 expression with hormone receptor and HER-2/neu status, it was seen that out of the 16 patients who were strongly CD10 positive, 14 (87.5%) were hormone receptor negative, and 14 (87.5%) had HER-2/neu overexpression. [Table 2]
|Table 2: Correlation between CD10 expression, hormone receptors, and HER-3/neuexpression in patients (n = 29) before treatment|
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After neo-adjuvant chemotherapy, expression of stromal CD10 in the tumour remained same in 13 cases (44.83%). There was a change in expression of CD10 in the remaining 16 cases (55.17%); in 13 cases (44.83%) CD10 expression showed a decrease from its pre-chemotherapy status while its expression increased in 3 cases (10.34%). In complete responders, as there was no tumor tissue left, the CD10 was categorised as 'nil'. Thus, out of the 4 complete responders, one was negative for CD10 before treatment - this was recorded as 'no change'. The other 3 complete responders were positive for CD10 before starting chemotherapy - they were recorded as 'decreased'. [Table 1]
On correlation of the initial CD10 expression with response to neo-adjuvant chemotherapy; out of the 24 patients who were CD10 positive, 20 patients responded to chemotherapy (3 CR, 10 PR, and 7 MR), while 4 patients had progressive disease. Of the 5 patients who were CD10 negative at the onset, 4 patients responded to chemotherapy (1 CR, 2 PR, 1 MR), while 1 patient had stable disease. The numbers however were insufficient to draw any statistically significant conclusions.
In 4 cases that had a complete response to chemotherapy, there was no change in CD10 expression in 2 cases, whereas CD10 expression decreased in 2 cases. There was no increase in expression in this group.
In 12 cases that had partial response to chemotherapy, there was no change in expression in 6 cases and CD10 expression decreased in 6 cases. There was no case with an increased expression of CD10 in this group.
In 8 cases that had a minimal response to chemotherapy, CD10 expression increased in 2 cases. There was no change in expression in 2 cases and CD10 expression decreased in 4 cases.
In one case which showed no change in tumour size with chemotherapy, there was an increase in CD10 expression. In 4 cases of progressive disease, CD10 did not increase in any case, while there was no change in expression in 3 cases and CD10 decreased in one case.
On correlation of change in CD10 expression with clinical response, it was found that in cases of complete and partial clinical response there was no increase in expression of CD10. In cases where CD10 expression had increased with chemotherapy, there was either a minor response or no clinical response of the tumor to chemotherapy. In 13 cases, where CD10 expression decreased with chemotherapy, 12 cases had a clinical response to chemotherapy. But the sample size in the study is too small for these changes to be significant.
| » Discussion|| |
Breast cancer is a major public health problem for women throughout the world. In the United States, breast cancer remains the most frequent cancer in women and second most frequent cause of cancer death.  Breast cancer is also the most common form of cancer seen in Europe in 2006 with 429,000 new cases representing 13.5% of all new cancers.  Although breast cancer has traditionally been less common in developing countries, its incidence in these areas is increasing. In India breast is the second most common site of cancer in women after cervix uteri.  But in metropolitan cities like New Delhi and Mumbai, it is the most common form of cancer in women. 
Estrogen and progesterone receptor expression are the most important and useful predictive factors currently available for hormonal manipulation in breast carcinoma. Patients with invasive breast cancer whose tumor cells are totally lacking ERs and PRs do not respond to or derive benefit from hormonal manipulation. , ER/PR status also has some prognostic value. Compared to women with ER+/PR+ tumors, women with ER+/ PR-, ER-/PR+ or ER-/PR- tumors experienced higher risk of mortality, which were largely independent of various demographic and clinical tumor characteristics. ,
Human Epidermal growth factor 2 (HER2) is a member of Epidermal growth factor receptor family. HER2 amplification or over-expression has been shown to be associated with higher tumor grade, ER negativity, higher levels of indicators of tumor proliferation such as S-phase, MIB-1, Ki 67, and poorer prognosis.  HER2 status is the major predictive factor for benefit from trastuzumab (herceptin), the humanized monoclonal antibody against the Her-2 protein. ,
These receptors described above are not static. Primary neo-adjuvant chemotherapy can bring about changes in expression of various receptors and can have an influence on relapse free survival and overall survival. In a study of 420 tumors, 145 (35%) of which were hormone receptor (HR) negative and 275 (65%) of which were HR positive, it was found that HR status had changed in 98 patients (23%).  Those patients with HR-negative tumors who had switched to a positive status after neo-adjuvant chemotherapy (NCT) had better overall survival and disease-free survival than patients whose tumors remained HR negative after NCT. In this study, survival after NCT was related to the HR status of the residual disease. Furthermore, the high level of discordance in HR status between the initial biopsy and the remaining tumor at surgery underlines the importance of taking into account the HR status of the residual disease after NCT, rather than the HR status on the initial biopsy. 
This change was also noted in HER2/neu. In a study conducted with 39 locally advanced breast cancer patients receiving chemotherapy, HER2 scores decreased in 28.5% of patients.  They reported that HER-2/neu status may change in locally advanced breast cancer patients treated with neo-adjuvant chemotherapy and suggested that elimination of HER-2/neu positive tumor cells may account for development of resistance to chemotherapy in some cases of breast cancer. This may be a possible explanation for chemo-resistance following an initial period of chemo-sensitivity. They suggested that repeat testing for HER-2/neu status may be necessary in patients receiving neo-adjuvant chemotherapy especially if the tumor becomes refractory to chemotherapy or recurs. 
CD10 is a 90- to 110- kDa cell surface zinc dependent peptidase (metalloproteinase) that has also been called as neutral endopeptidase (NEP), enkephalinase, neprilysin and 'common acute lymphoblastic leukemia antigen' (CALLA). It is commonly expressed in bone marrow lymphoid stem cells, pro-B lymphoblasts, mature neutrophils, various lymphoma subtypes, renal cell carcinoma and endometrial stromal sarcoma cells. CD10 positive cells have been reported in the stroma of prostate, breast, colorectal and lung carcinomas.  Several reports indicate that stromal CD10 expression is associated with biological aggressiveness in various epithelial malignancies.  In gastric carcinoma CD10 positive stroma correlates with vascular invasion and metastasis.  Stromal CD10 is also expressed with higher frequency in malignant as opposed to borderline and benign phyllodes tumor of breast. 
Till date only two studies have examined the expression of CD10 in stromal cells around the infiltrating tumor cells in invasive breast carcinoma and have showed its association with worse prognosis. ,
In a study conducted by Iwaya et al.,  of 110 patients of invasive ductal breast carcinomas, 20 cases (18%) in which more than 10% of stromal cells stained positive throughout the cancer tissue were judged positive. This study addressed the prognostic significance of stromal CD10 expression in invasive carcinoma breast. It was shown that stromal CD10 expression was associated exclusively with invasive breast carcinoma, but not with carcinoma in situ and a higher frequency of stromal CD10 staining was seen primarily with lymph node positive breast cancers. There were no correlations between stromal CD10 expression, age, tumor size, histological grade, or clinical stage. CD10 positive subgroup of patients also had a shorter metastasis free interval, and CD10 was the single significant prognostic factor for overall survival in the univariate analysis. These results suggested that stromal expression of CD10 in breast cancer is an important novel prognostic factor. 
In another study conducted by Markretsov et al.,  of the 258 patients of invasive breast carcinoma, 21% of tumors stained negative for stromal CD10, 40% had a weak response and 39% had strong response. It was also found that strong response was more with higher grade tumors (59%) and estrogen receptor negativity compared to lower grade tumors (29%) and estrogen receptor positivity. There was no correlation between CD10 and lymph node status, tumor size, histological subtype, progesterone receptors and HER2 status. Stromal CD10 expression was associated with decreased long term disease specific and overall survival in the entire cohort and in lymph node negative group, but not lymph node positive subset of patients. CD10 expression was associated with relative risk of death of 2.8 compared to relative risk of 2.4 produced by lymph node positive status. Thus CD10 expression has a prognostic significance, particularly in lymph node negative subset of patients. They concluded that stromal CD10 expression in invasive carcinoma of the breast is associated with ER negativity, higher tumor grade and decreased survival and constitutes a potential prognostic marker and a target for development of novel therapies. 
Some experimental data indicates that CD10 may be a potential target for new cancer therapies as it is involved in cleavage of doxorubicin, which is a critical component in many chemotherapy protocols and thus results in chemo-resistance. Inhibition of CD10 enzymatic activity may thus enhance the anti-tumor efficacy of traditional chemotherapeutic regimens based on doxorubicin. Various novel CD10-cleavable peptide pro-drugs like CPI-0004Na are being developed which may be effective in a range of different tumor types which are expressing CD10. CPI-0004Na is a pro-drug of doxorubicin and has been shown to have improved anti-tumor efficacy profile with reduced toxicity compared to doxorubicin in tumor xenograft models. 
There has been no study till date on the effect of chemotherapy on change in CD10 receptors in invasive breast carcinoma.
In our study, we conclude that strong CD10 expression correlates with hormone receptor negativity and HER-2/neu overexpression. Stromal CD10 expression in breast cancer is not static and changes with neo-adjuvant anthracycline-based chemotherapy. A stable or decrease in CD10 expression correlates with complete or partial clinical response, while an increase in CD10 expression appears to correlate with poor clinical response. As stromal CD10 expression and its change with chemotherapy may have a prognostic significance, they should be documented in breast cancer patients before and after chemotherapy. A much larger series is required in order to determine whether these changes are statistically significant.
We suggest that, along with other receptor studies, stromal CD10 antigen status should also be documented in breast cancer patients before and after neoadjuvant chemotherapy for possible prognostic and predictive correlation, and an attempt should be made to correlate change in CD10 expression with clinical response to chemotherapy and patient disease-free interval and overall survival.
| » References|| |
|1.||Gianni L, Valagussa P, Zambetti M, Moliterni A, Capri G, Bonadonna G. Adjuvant and neoadjuvant treatment of breast cancer. Semin Oncol 2001;28:13-29. |
|2.||Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: Correlation of relapse and survival with amplification of HER2/neu oncogene. Science 1987;235:177-82. |
|3.||Tacca O, Penault-Llorca F, Abrial C, Mouret-Reynier MA, Raoelfils I, Durando X, et al. Changes in and prognostic value of hormone receptor status in a series of operable breast cancer patients treated with neoadjuvant chemotherapy. Oncologist 2007;12:636-43. |
|4.||Quddus RM, Sung JC, Zhang C, Pasqueriello T, Eklund M, Steinhoff MM. HER2/neu expression in locally advanced breast carcinomas: Pre-and post-neoadjuvant chemotherapy. Breast Cancer 2005;12:294-8. |
|5.||Kesse-Adu R, Shousha S. Myoepithelial markers are expressed in at least 29% of oestrogen receptor negative invasive breast carcinoma. Mod Pathol 2004;17:646-52. |
|6.||Iwaya K, Ogawa H, Izumi M, Kuroda M, Mukai K. Stromal CD10 expression in invasive breast carcinoma; a new predictor of clinical outcome. Virchows Arch 2002;440:589-93. |
|7.||Makretsov NA, Hayes M, Carter BA, Dabiri S, Gilks CB, Huntsman DG. CD10 expression in invasive breast carcinoma correlates with poor prognosis, estrogen receptor negativity, and high grade. Mod Pathol 2007;20:84-9. |
|8.||Pan C, Cardarelli PM, Nieder MH, Pickford LB, Gangwar S, King DJ, et al. CD10 is a key enzyme involved in the activation of tumor-activated peptide prodrug CPI-0004Na and novel analogues: Implications for the design of novel peptide prodrugs for therapy of CD10+ tumours. Cancer Res 2003;63:5526-31. |
|9.||Martin M, Villar A, Sole-Calvo A, Gonzalez R, Massuti B, Lizon J, et al.; GEICAM Group (Spanish Breast Cancer Research Group), Spain. Doxorubicin in combination with fluorouracil and cyclophosphamide versus methotrexate in combination with fluorouracil and cyclophosphamide as adjuvant chemotherapy for operable breast cancer: A study by the GEICAM group. Ann Oncol 2003;14:833-42. |
|10.||Chollet P, Amat S, Cure H, de Latour M, Le Bouedec G, Mouret-Reynier MA, et al. Prognostic significance of a complete pathological response after induction chemotherapy in operable breast cancer. Br J Cancer 2002;86:1041-6. |
|11.||American Cancer Society. Breast cancer facts and figures2012. World Wide Web URL http://www.cancer.org/Research/CancerFactsFigures/CancerFactsFigures/cancer-facts-figures-2012. |
|12.||Ferlay J, Autier P, Boniol M, Heanue M, Colombet M, Boyle P. Estimates of the cancer incidence and mortality in Europe in 2006. Ann Oncol 2007;18:581-92. |
|13.||Chopra R. The Indian Scene. J Clin Oncol 2001;19 (18 suppl):106S-11S. |
|14.||Murthy NS, Mathew A. Cancer epidemiology, prevention and control. Curr Sci 2004;86:518-27. |
|15.||Burstein HJ, Harris JR, Morrow M. Malignant tumors of breast. In: DeVita VT Jr, Lawrence TS, Steven A, editors. DeVita, Hellman and Rosenberg's Cancer: Principles and Practice of Oncology. 8 th ed. Philadelphia: Lippincott Williams and Wilkins; 2008. p. 1606-54. |
|16.||Dunnwald LK, Rossing MA, Li CI. Hormone receptor status, tumour characteristics, and prognosis: A prospective cohort of breast cancer patients. Breast Cancer Res 2007;9:R6. |
|17.||Huang WB, Zhou XJ, Chen JY, Zhang LH, Meng K, Ma HH, et al. CD10-positive stromal cells in gastric carcinoma: Correlation with invasion and metastasis. Jpn J Clin Oncol 2005;35:245-50. |
|18.||Tse GM, Tsang AK, Putti TC, Scolyer RA, Lui PC, Law BK, et al. Stromal CD10 expression in mammary fibroadenomas and phyllodes tumours. J Clin Pathol 2005;58:185-9. |
[Table 1], [Table 2]