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Year : 2015  |  Volume : 58  |  Issue : 2  |  Page : 170-174
Expression of Cathepsin L in tumor cells and tumor-associated macrophages in patients with Ewing sarcoma family of tumors: A pilot study

1 Department of Medical Oncology, Dr. B. R. Ambedkar Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India
2 Department of Pathology, Dr. B. R. Ambedkar Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India

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Date of Web Publication17-Apr-2015


Background: Cysteine protease Cathepsin L is involved in bone remodeling and expressed in activated macrophages. It is highly expressed in metastatic tumor tissue, especially with bone metastases. Aims: We evaluated immunohistochemical expression of Cathepsin L in tumor cells and tumor-associated macrophages (TAMs) in chemo-naive Ewing sarcoma. Settings and Design: Retrospective evaluation of archived specimens of Ewing sarcoma. Materials and Methods: Immunohistochemical staining was performed on archived blocks of chemo-naive patients with Ewing sarcoma treated with uniform chemotherapy at our institute between January 2009 and November 2011. Statistical Analysis: Immunohistochemical expression was co-related with baseline demographics and survival. Results: During the study period, we had evaluable baseline samples from 62 patients with median age 15 years (range: 2-40); 26 (42%) had metastases. Cathepsin L expression in tumor cells was observed in 8/62 (13%) specimens. None of the baseline clinical characteristics correlated with Cathepsin L expression. Cathepsin L positivity was associated with poor response to neoadjuvant chemotherapy (NACT) (P = 0.05), but did not influence either event-free-survival (EFS) or overall survival. Cathepsin L was expressed in TAMs in all specimens. Grade 3 TAMs (>10 TAMs/high power field) was associated with better response to NACT (P = 0.05). On univariate analysis Grade 3 TAMs predicted superior EFS (median EFS 28.5 months in those with Grade 3 TAMs versus 14.8 months in those with grade ½ TAMs [P = 0.04]). Conclusions: Cathepsin L expression by immunohistochemistry was low in our patient cohort, and it did not affect the outcome. In addition, Grade 3 TAMs with Cathepsin L expression was associated with improved EFS.

Keywords: Cathepsin L, Ewing′s sarcoma, immunohistochemistry, tumor-associated macrophage

How to cite this article:
Biswas B, Sharma MC, Mridha AR, Bakhshi S. Expression of Cathepsin L in tumor cells and tumor-associated macrophages in patients with Ewing sarcoma family of tumors: A pilot study. Indian J Pathol Microbiol 2015;58:170-4

How to cite this URL:
Biswas B, Sharma MC, Mridha AR, Bakhshi S. Expression of Cathepsin L in tumor cells and tumor-associated macrophages in patients with Ewing sarcoma family of tumors: A pilot study. Indian J Pathol Microbiol [serial online] 2015 [cited 2023 Sep 30];58:170-4. Available from:

   Introduction Top

Cathepsins are fundamental effectors of endolysosomal proteolysis, and they play a specific role in antigen presentation, bone remodeling, and epidermal homeostasis. [1],[2] Cathepsins have been reported to be involved in apoptosis, angiogenesis, cell proliferation, and invasion. Expression and activity levels of some Cathepsins are upregulated in human cancers. Cathepsin involves in bone remodeling and has been described in many studies to be expressed in many solid tumors especially with bone metastases. [2],[3] Cathepsin L is one of the mostly abundant Cathepsin in the human body and implicated for poor outcome in various malignancies. [2],[3],[4],[5]

Ewing sarcoma family of tumors (ESFT) is an aggressive malignancy in childhood and burdened with high incidence of metastasis, mostly to bone and lung. Metastatic disease at presentation is the most unfavorable prognostic factor for ESFT patients with long-term survival of <20%. The reason behind its metastatic predilection to bone and bone marrow is not fully understood.

Tumor-associated macrophages (TAMs) are pivotal members of tumor stroma. Continued endeavors at prognostication utilizing markers in the tumor environment have linked TAMs with poor prognosis in several cancers. [5],[6],[7] Cathepsin L has been reported to be expressed in activated macrophages. [1],[8],[9],[10]

We hypothesized that Cathepsin L expression will be high in ESFT patients' tumor cells especially those with bone metastasis and also in activated TAMs. Being an aggressive disease with a systemic nature, the Cathepsin positive activated TAMs may have prognostic effect on survival and outcome in ESFT patients. We here evaluated immunohistochemical expression of Cathepsin L in the diagnostic histopathology specimens of patients with ESFT and evaluated its association with baseline demographic factors and response to therapy.

   Materials and Methods Top


We studied the immunohistochemical expression of Cathepsin L in archived tissue specimens obtained at baseline prior to initiating chemotherapy in 62 consecutive ESFT patients that were treated at our institute from January 2009 to November 2011. ESFT of all sites (except brain) were included in this analysis. Patient's baseline clinico-pathological features, metastatic work-up, treatment modality, and outcome data were collected from case records. Ethical clearance was taken from institutional ethical review committee.

Diagnostic work-up

All patients underwent biopsy (tru-cut or incisional) of the primary lesion with immunohistochemistry. Diagnosis of ESFT required presence of small blue round cell tumor and positivity for CD99 (MIC-2), and/or synaptophysin or chromogranin. Other round cell tumors were ruled out by performing immunohistochemistry with leucocyte common antigen, desmin, and myogenin. Evaluation for translocation [t(11;22)(q24;q11.2-12)] was not performed in this cohort as it was not available routinely at our institute. Diagnosis of ESFT was made after review of histopathology and with the use of the immunohistochemistry panel. All patients underwent metastatic work-up including bone scan, computed tomography of chest and bone marrow biopsy.

Laboratory methods

For all eligible patients diagnosis was reconfirmed by review of baseline biopsies. Paraffin embedded archived tumor tissue of 5 μm thickness were deparaffinized and dehydrated. Immunohistochemical staining for Cathepsin L was performed according to Streptavidin-Biotin immunoperoxidase technique. [11] Antigen retrieval was done using the Heat Induced Epitope Retrieval method. Blocking of endogenous peroxidase activity was done by incubating the slides in 4% H 2 O 2 (in H 2 O) for 10-15 min. A volume of 100 μL of primary antibody (anti-Cathepsin L, clone CPL33/1; Abcam, Oxford, UK) was applied in 1:400 dilutions and incubated for 1 h at 37°C in a humid chamber. Secondary antibody (universal) coupled with biotin was applied and incubated for 15 min in room temperature. Slides were then incubated with 100 μL of streptavidin-peroxidase for 30 min at 15-25°C. Colour development was done with freshly prepared 3, 3'-Di-amino benzidene solution for 3-5 min under microscopic control. Counterstaining of nuclei was done with hematoxylin for 30 s and mounted in dibutyl phthalate xylone medium. Each batch of slides was immunostained with appropriate positive control (non-neoplastic breast tissue), and for negative control, primary antibody was omitted. Minimum 1000 tumor cells were counted in separate representative stained fields. Cytoplasmic staining intensity was semi-quantified on a scale of 0-3 (0, no staining; 1, weak; 2, moderate; 3, strong). When ≥30% of the tumor cells in a specimen were positively stained with ≥Grade 2 staining, the specimen was scored as positive. When <30% of the tumor cells were positively stained with ≥Grade 2 staining, the specimen was scored as negative. The macrophage staining with Cathepsin L was graded as follows - Grade 1: Less than 5 macrophages/high power field (HPF), Grade 2: 5-10 macrophages/HPF, Grade 3: >10 macrophages/HPF.

Treatment and response evaluation

Treatment protocol consisted of three phases: Neoadjuvant chemotherapy (NACT) for 9-12 weeks, local therapy, which was then followed with adjuvant chemotherapy (ACT). Chemotherapy was given alternating 3-weekly cycle of VAC (vincristine at 1.4 mg/m 2 , max = 2 mg; doxorubicin at 75 mg/m 2 or actinomycin-D at 1.25 mg/m 2 and cyclophosphamide at 1.2 g/m 2 with MESNA) with IE (ifosfamide at 9 g/m 2 with MESNA and etoposide at 500 mg/m 2 ) for total 48 weeks. [12] Local therapy was radiotherapy or surgery with or without postoperative radiotherapy. After local therapy all patients were subjected to receive ACT up to planned 48 weeks.

Disease response was done by appropriate radiology after NACT and after completion of local therapy. Complete remission (CR), partial response, stable disease, and progressive disease were defined as per Response Evaluation Criteria in Solid Tumors criteria wherever applicable. [13]

Statistical analysis

Descriptive statistics were used for demographics and clinical characteristics. Chi-square test was used to detect an association between categorical variables. Student's t-test was applied to compare continuous variables between groups. Survival was estimated by the Kaplan-Meier method and compared using log-rank test. Data were censored on 31 st December 2013. Event-free-survival (EFS) was calculated from the date of diagnosis to date of disease relapse or progression, death from any cause. Overall survival (OS) was calculated from the date of diagnosis to date of death from any cause. STATA/SE 9.0 (StataCorp LP, Texas, USA) was used for statistical analysis.

   Results Top

Clinical features, treatment, and outcome

Of the 62 recruited patients, median age was 15 years (range: 2-40 years) with male/female ratio of 49:13. Median tumor diameter was 10 cm (range: 3-20). The most common primary regions of disease included the extremity in 26 (42%), pelvis in 13 (21%), and thorax in 13 (21%) patients. Diagnostic work-up revealed metastatic disease in 26 patients (42%). All 62 patients were subjected to NACT at a median of 6 cycles (range: 3-10) and 44 patients (71%) received local therapy: Surgery was done in 20 patients and radical radiotherapy alone was administered in 24 (55%) patients. After median follow-up of 30.8 months (range: 2.5-55.6), median EFS and OS was 18.3 months and 27.1 months, respectively.

Immunohistochemical expression of Cathepsin L in tumor cells and tumor-associated macrophages and association with baseline clinical features and response to neoadjuvant chemotherapy

Cytoplasmic expression of Cathepsin L on IHC was detected in 8 patients (13%) [Figure 1]. Cathepsin L expression did not correlate with any of the baseline demographic features; however, patients with specimens having Cathepsin L positivity had a poor response to NACT (P = 0.05) [Table 1].
Figure 1: Cytoplasmic staining of tumor cells in Ewing sarcoma family of tumor patients with Cathepsin L (IHC, ×40) (a and b). Strong Cathepsin L positivity of tumor-associated macrophage on immunohistochemistry (×40) (c and d)

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Table 1: Comparison of baseline clinic-pathological factors between Cathepsin L positive (n = 8) and negative (n = 54) patients, and TAM grade 1 and 2 (n = 46) and grade 3 (n = 16) Cathepsin L expression

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In addition to staining of the tumor cell, there was strong Cathepsin L staining of the TAMs [Figure 2]. On grading of TAM staining, the result was as follows: Grade 1 in 13, Grade 2 in 33 and Grade 3 in 16 patients. It was observed that patients with Grade 3 TAM had higher CR rate than the other group (P = 0.05) [Table 1].
Figure 2: Event-free-survival estimate according to Cathepsin L expression in tumor cells in whole Ewing sarcoma family of tumor (ESFT) group (n = 62) (a), in ESFT with localized disease (n = 36) (b), in ESFT with metastatic disease (n = 26) (c). Event-free-survival estimate according to Cathepsin L expression in tumor-associated macrophages in whole ESFT group (n = 62) (d), in ESFT with localized disease (n = 36) (e), in ESFT with metastatic disease (n = 26) (f)

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Correlation of Cathepsin L with event-free-survival

When the whole cohort (n = 62) was analyzed, median EFS was 18.1 months vs 18.3 months (P = 0.94) between patients without and with Cathepsin L positivity, respectively [Figure 2]a. The same was 20.4 months vs 30.3 months (P = 0.59) and 6.8 months versus 7.7 months (P = 0.59) in ESFT patients with localized and metastatic disease, respectively [Figure 2]b and c.

Correlation of tumor-associated macrophages grade with event-free-survival

When the whole cohort (n = 62) was analyzed, median EFS was 14.8 months versus 28.5 months (P = 0.04) between ESFT patients with grade ½ TAM and Grade 3 TAM, respectively [Figure 2]d. The same was 20 months versus not reached (P = 0.17) and 6.8 months versus 22.1 months (P = 0.27) in patients with localized and metastatic disease, respectively [Figure 2]e and f.

   Discussion Top

We found Cathepsin L expression in 13% of our patients in contrary to our hypothesis that Cathepsin L expression will be high in ESFT patients, especially in those with bone metastasis. None of baseline factors correlated with Cathepsin L expression. Notably, patients with Cathepsin L positivity achieved lower CR rate when compared to those with negative expression after NACT; however, this did not significantly influence EFS and OS. In view small sample size and low positivity of Cathepsin L in our cohort, we could not make any definite conclusions regarding its predictive and prognostic role in ESFT.

Strong Cathepsin L positivity of TAM was an additional finding in our patients, which has not been evaluated in other solid tumors with Cathepsin expression as well as in ESFT. These Cathepsin positive TAMs may be the activated macrophages; previously it has been shown that Cathepsin L is expressed in activated macrophages. [8],[9],[10] In leiomyosarcoma, presence of Cathepsin expression in TAM predicts inferior outcome; [5] however, in colorectal cancer and gastric cancer, presence of TAMs in the tumor microenvironment predicts improved outcome. [14],[15] Grade 3 TAM predicted improved EFS in our cohort; one possible explanation may be that the higher proportion of activated TAM may result in stimulation of the adaptive immunity against tumor cells resulting in resultant tumor cell killing. Further, although TAMs are associated with invasive tumors, yet an alternative hypothesis could be that a vigorous macrophage response at sites of ongoing invasion is an important feature of the protective action of macrophages that may delay or, in some cases, even prevent further tumor spread. [16]

   Conclusion Top

Cathepsin L expression by immunohistochemistry was relatively lower as compared to other malignancies with propensity for bone metastases. In addition, TAMs with Grade 3 Cathepsin L expression was associated with improved outcome. This revelation needs validation in large prospective studies, and to perform functional studies of TAMs and other supporting cells in tumor stroma of patients with ESFT.

   References Top

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2. Leto G, Sepporta MV, Crescimanno M, Flandina C, Tumminello FM. Cathepsin L in metastatic bone disease: Therapeutic implications. Biol Chem 2010;391:655-64.  Back to cited text no. 2
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4. Jain M, Bakhshi S, Shukla AA, Chauhan SS. Cathepsins B and L in peripheral blood mononuclear cells of pediatric acute myeloid leukemia: Potential poor prognostic markers. Ann Hematol 2010;89:1223-32.  Back to cited text no. 4
5. Ganjoo KN, Witten D, Patel M, Espinosa I, La T, Tibshirani R, et al. The prognostic value of tumor-associated macrophages in leiomyosarcoma: A single institution study. Am J Clin Oncol 2011;34:82-6.  Back to cited text no. 5
6. Steidl C, Lee T, Shah SP, Farinha P, Han G, Nayar T, et al. Tumor-associated macrophages and survival in classic Hodgkin's lymphoma. N Engl J Med 2010;362:875-85.  Back to cited text no. 6
7. Farinha P, Masoudi H, Skinnider BF, Shumansky K, Spinelli JJ, Gill K, et al. Analysis of multiple biomarkers shows that lymphoma-associated macrophage (LAM) content is an independent predictor of survival in follicular lymphoma (FL). Blood 2005;106:2169-74.  Back to cited text no. 7
8. Takahashi H, Ishidoh K, Muno D, Ohwada A, Nukiwa T, Kominami E, et al. Cathepsin L activity is increased in alveolar macrophages and bronchoalveolar lavage fluid of smokers. Am Rev Respir Dis 1993;147:1562-8.  Back to cited text no. 8
9. Iwata Y, Mort JS, Tateishi H, Lee ER. Macrophage cathepsin L, a factor in the erosion of subchondral bone in rheumatoid arthritis. Arthritis Rheum 1997;40:499-509.  Back to cited text no. 9
10. Bühling F, Reisenauer A, Gerber A, Krüger S, Weber E, Brömme D, et al. Cathepsin K - A marker of macrophage differentiation? J Pathol 2001;195:375-82.  Back to cited text no. 10
11. Shi ZR, Itzkowitz SH, Kim YS. A comparison of three immunoperoxidase techniques for antigen detection in colorectal carcinoma tissues. J Histochem Cytochem 1988;36:317-22.  Back to cited text no. 11
12. Grier HE, Krailo MD, Tarbell NJ, Link MP, Fryer CJ, Pritchard DJ, et al. Addition of ifosfamide and etoposide to standard chemotherapy for Ewing's sarcoma and primitive neuroectodermal tumor of bone. N Engl J Med 2003;348:694-701.  Back to cited text no. 12
13. Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 2000;92:205-16.  Back to cited text no. 13
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15. Ohno S, Inagawa H, Dhar DK, Fujii T, Ueda S, Tachibana M, et al. The degree of macrophage infiltration into the cancer cell nest is a significant predictor of survival in gastric cancer patients. Anticancer Res 2003;23:5015-22.  Back to cited text no. 15
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Correspondence Address:
Prof. Sameer Bakhshi
Department of Medical Oncology, Dr. B. R. Ambedkar Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110 029
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.155307

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  [Figure 1], [Figure 2]

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