Molecular and Clinical Analysis of Locally Advanced Dermatofibrosarcoma Protuberans Treated With Imatinib: Imatinib Target Exploration Conso
http://www.100md.com
《临床肿瘤学》
the Peter MacCallum Cancer Centre, East Melbourne, Australia
Dana-Farber Cancer Institute and Harvard Medical School, Sarcoma Center
Department of Pathology, Brigham & Women's Hospital, Boston, MA
Universiteit Ziekenhuis Gasthuisberg dienst oncology
Cytogenetics and Molecular Genetics of Human Malignancies, Department of Human Genetics, Catholic University of Leuven, Leuven, Belgium
Departments of Medicine and Pathology, Oregon Health Sciences University Cancer Institute and Portland Veterans Affairs Medical Center, Portland, OR
Clinical Research Oncology, Novartis Pharma AG, Basel, Switzerland
ABSTRACT
PATIENTS AND METHODS: We analyzed the objective radiologic and clinical response to imatinib at 400 mg twice daily in eight patients with locally advanced DFSP and two patients with metastatic disease.
RESULTS: Each of eight patients with locally advanced DFSP had evidence of t(17;22) and showed a clinical response to imatinib. Four of these patients had complete clinical responses. The two patients with metastatic disease had fibrosarcomatous histology and karyotypes that were substantially more complex than those typically associated with localized DFSP. One patient with metastatic DFSP and an associated t(17;22) had a partial response to imatinib but experienced disease progression after 7 months of therapy. In contrast, the other patient with metastatic disease had a tumor lacking t(17;22), and there was no clinical response to imatinib. Unexpectedly, there was minimal platelet-derived growth factor receptor–beta phosphorylation in the untreated DFSP, despite the documented presence of a PDGFB autocrine mechanism.
CONCLUSION: Imatinib has clinical activity against both localized and metastatic DFSP with t(17;22). However, fibrosarcomatous variants of DFSP lacking t(17;22) may not respond to imatinib.
INTRODUCTION
More than 90% of DFSP feature a translocation involving distinct regions of chromosomes 17 and 22.6 Most commonly, the t(17;22) breakpoint region is tandemly repeated within a supernumerary ring chromosome.7 The translocation breakpoint generally involves the second exon of the platelet-derived growth factor-B (PDGFB) gene on chromosome 22, which is fused with the strongly expressed collagen 1 alpha 1 (COL1A1) gene on chromosome 17. This distinctive translocation mechanism results in transcriptional upregulation of the PDGFB gene, in the form of a COL1A1-PDGFB fusion oncogene. The associated COL1A1-PDGF-B fusion protein8-10 is post-translationally processed to yield mature and fully functional PDGFB.11 Therefore, the t(17;22) results in PDGFB-mediated activation of platelet-derived growth factor receptor–beta (PDGFRB) by autocrine and paracrine production of a functional ligand for PDGFRB. Because PDGFRB is the major PDGFR isoform expressed on DFSP,12,13 the t(17;22) most likely activates PDGFRB rather than PDGFR-alpha (PDGFRA).11
The identification of deregulated expression of PDGFB as a result of the t(17;22) led to the hypothesis that inhibitors of PDGFRB, such as imatinib, might have activity in DFSP. After supportive preclinical studies,11,12 there have been several reports of clinical activity of imatinib in DFSP.14-16 Here we report a series of 10 patients with DFSP who received imatinib for locally advanced or metastatic DFSP. Strikingly, all eight patients with locally advanced disease responded to imatinib. Unexpectedly, these dramatic clinical responses were seen even in DFSPs expressing relatively low amounts of activated PDGFRB.
PATIENTS AND METHODS
Patients were treated with 800 mg of imatinib daily (400 mg twice daily) with dose reductions for grade 3 toxicity or for recurrent grade 2 toxicity after an initial 1-week treatment break. The initial dose reduction was to 600 mg, with a second dose reduction to 400 mg allowed for further grade 3 toxicity or recurrent grade 2 toxicity after a 1-week treatment break. Full details of the clinical trial will be published separately.
Study investigations were performed after approval by a local human investigations committee and for United States centers in accord with an assurance filed with and approved by the United States Department of Health and Human Services. Informed consent was obtained from each patient.
Eligibility
Patients with primary or metastatic DFSP, Eastern Cooperative Oncology Group performance status of 0 to 2, and adequate end organ function were eligible for treatment. Prior systemic therapy for DFSP was allowed.
Efficacy Assessments
Objective responses to imatinib were evaluated using traditional Southwest Oncology Group criteria (pre-Response Evaluation Criteria In Solid Tumors). Partial response was defined as a ≥ 50% decrease from baseline in the sum of products of perpendicular diameters of all measurable lesions. Complete response was defined as complete disappearance of all measurable and nonmeasurable but assessable disease.
All patients were evaluated with either tumor imaging with computed tomography or magnetic resonance imaging scans (patients 9 and 10), clinical photography in the case of locally advanced tumors with superficially visible disease (patients 1, 2, 4, and 6), or clinical examination by palpation with measurements confirmed by ultrasound (patients 3, 5, 7, and 8). Measurable disease in the skin was defined as lesions with at least one diameter ≥ 0.5 cm that could be assessed by medical photography or two diameters ≥ 2 cm that could be assessed by palpation with measurements confirmed by ultrasound. Data presented include follow-up to March 1, 2004.
Western Blot Analyses
Frozen tumor specimens were homogenized in ice-cold cell lysis buffer using a Tissue Tearor unit (Biospec, Bartlesville, OK). The lysates were then rocked at 4°C for 30 minutes, cleared by centrifugation, and quantitated using the BioRad protein assay (Hercules, CA). Electrophoresis was performed using 30 μg of DFSP cell lysate per lane, and with inclusion of control lanes containing PDGFA- and PDGFB-stimulated NIH3T3 cells, and a PDGFRA mutant gastrointestinal stromal tumor, as positive controls for phospho PDGFRB and phospho PDGFRA, respectively. The gels were blotted to polyvinylidenedifluoride membranes, then stained for phospho PDGFRs, using a rabbit phosphoPDGFRA Y754 antibody (which binds to both phosphoPDGFRA and phosphoPDGFRB) from Santa Cruz Biotechnology Inc (SC-12911; Santa Cruz Biotechnology Inc, Santa Cruz, CA). Detection was performed with enhanced chemiluminescence, and protein expression was quantitated using a FUJI LAS1000-plus chemiluminescence imaging system (Fuji; Stamford, CT).
Karyotyping
DFSP karyotyping was performed after 3 to 6 days in tissue culture, and the metaphase cells were stained and analyzed by Giemsa-trypsin banding according to standard methods.17
Fluorescence In Situ Hybridization
Fluorescence in situ hybridization (FISH) was performed by labeling bacterial artificial chromosomes (BACs) centromeric (RP11-1149B8 and RP11-348I17) and telomeric (RP11-101B10 and RP11-434E5) to the PDGFB locus with biotin and digoxigenin, respectively.18 FISH evaluations of cytogenetic DFSP metaphase material were performed by standard methods, using either the DFSP BAC probes or using painting probes for chromosomes 17 and 22. FISH evaluations of paraffin sections were performed after pretreating 4-μm sections by microwaving and digestion with Digest All-III (Zymed, South San Francisco, CA), then applying the DFSP BAC probes and codenaturing the probe and section in a polymerase chain reaction machine. Detection of the biotinylated and digoxigenin-labeled probes was performed with streptavidin Alexa 594 (Molecular Probes, Eugene, OR) and fluorescein isothiocyanate antidigoxigenin (Roche, Indianapolis, IN), respectively.
RESULTS
All patients were commenced on imatinib at 800 mg daily (400 mg bid). In this patient population, 800 mg daily was well tolerated, with only one patient requiring dose reduction to 600 mg daily. Four patients received tumor biopsies before and approximately 28 days after commencement of imatinib. Responses were evaluated using traditional Southwest Oncology Group criteria (pre-Response Evaluation Criteria in Solid Tumors). Notably, all eight patients with locally advanced DFSP had partial (four patients) or complete (four patients) responses to imatinib (Table 2). Two patients (patients 7 and 8) who proceeded to surgical resection after initial clinical response had pathologically confirmed complete responses. In four further cases (patients 1, 2, 3, and 5), the patients were rendered disease-free by surgical resection after partial response to imatinib. Of the two patients with metastatic disease and complex cytogenetics, patient 10, whose DFSP lacked the t(17;22), had no clinical response to imatinib by either conventional imaging or fluorodeoxyglucose positron emission tomography scan at day 19 of imatinib therapy. However, follow-up on this patient was quite short because of the patient's death on day 32. By contrast, patient 9, whose metastatic DFSP featured the t(17;22) and other cytogenetic aberrations, had a robust partial response (Table 2 and Fig 2). The clinical responses to imatinib were sustained in all four patients treated with imatinib for greater than 6 months. However, two of these patients subsequently experienced disease progression. Patient 1, with locally advanced DFSP on the cheek, responded for just less than 2 years before the tumor progressed, at which point the patient was rendered disease-free after resection using Mohs surgical technique. Patient 9 with metastatic disease experienced disease progression after 7 months. Patients rendered free of disease by surgical resection had no evidence of progression at 4, 18, 12, and 1 months after the resection.
Several patients had matched pretreatment and on-therapy tumor biopsy specimens. Biopsy specimens obtained during imatinib therapy had reduced cellularity and the development of hyaline changes (Fig 3). PDGFB FISH analysis showed persistence of the t(17;22) in the spindle-cell component of the resected stable disease. However, the t(17;22) was found in only 20% of the spindle cells, suggesting that most of the residual spindle cells were either reactive or were DFSP cells lacking t(17;22).
Tumor tissue was examined for expression and activation of PDGFRA and PDGFRB by immunoblot using antisera to the active phosphorylated forms of the PDGF-receptor. Interestingly, PDGFR activation was low in five DFSPs evaluated before imatinib therapy (two examples are shown in Fig 4). Indeed, PDGFR activation was at least 10-fold lower than in gastrointestinal stromal tumors (GISTs) expressing PDGFR with an intrinsic gain-of-function mutation (Fig 4). The weak baseline activation in the DFSP precluded accurate quantification of the reduction in activated PDGFRB inhibition in response to imatinib.
DISCUSSION
The high response rate to imatinib therapy in DFSP supports the hypothesis that DFSP cells are dependent on aberrant activation of PDGFRB for cellular proliferation and survival. Our clinical results are consistent with studies of DFSP cell culture and animal models, in which t(17;22)-mediated PDGFRB autocrine activation is effectively inhibited by imatinib. Unexpectedly, the level of PDGFRB activation as assessed by receptor autophosphorylation was substantially lower than PDGFR activation in a GIST with intrinsic PDGFRA oncogenic mutation. Therefore, although mutation of a serine/threonine kinase such as BRAF in melanoma21 or mutation or amplification of a receptor tyrosine kinase (RTK) such as PDGFRA in GIST tumors,22 epidermal growth factor receptor in epithelial tumors,23 or ERB-B2 in breast cancer24 leads to strong activation of the involved oncogenic RTKs, it seems that autocrine/paracrine oncogenic mechanisms—as seen in DFSP—can be associated with substantially lower levels of RTK activation. These novel observations indicate that neither high levels of RTK activation nor RTK overexpression are required for clinical response to therapeutic inhibition of receptor signaling. Rather, inhibition of low-level RTK activation can be clinically effective, providing that the tumor cells are dependent on that signaling mechanism.
Our findings demonstrating low activation of PDGFRB in DFSP tumors contrast with those of Sjblom et al,12 who readily detected phosphorylation of PDGFRB in cultured cells derived from DFSP tumors. We propose two possible explanations for this apparent discrepancy: first, the culture conditions used by Sjblom et al included the use of fetal calf serum, a source of exogenous PDGFs. It is possible that these culture conditions induced greater phosphorylation of PDGFRB than we observed in tumor biopsy specimens. Second; Sjblom et al used immunoprecipitation rather than direct immunoblotting to detect phosphorylation of PDGFRB. It is likely that immunoprecipitation is more sensitive than immunoblotting at detecting phosphorylated PDGFRB. Nonetheless, there is a clear and substantial difference between receptor phosphorylation in GISTs or PDGFA/B-stimulated NIH3T3 cells compared with the DFSP tumors (Fig 4). Moreover, in our experience, only weak activation of PDGFRB is detectable using immunoprecipitation of protein lysates purified from fresh DFSP tumors (J.A.F., unpublished data).
We treated two patients with metastatic disease; both patients had fibrosarcomatous histology associated with complex karyotypes. In one case (patient 10), both the locally recurrent and metastatic lesions lacked t(17;22), suggesting that this DFSP-like tumor was not necessarily dependent on signaling through PDGFRs. Consistent with this prediction, the second metastatic DFSP (patient 9) had an associated t(17;22) and had a partial clinical response to imatinib, although the DFSP progressed after 7 months of therapy. Four other patients with metastatic DFSP have been reported in the literature with clinical responses observed in all of these cases,14-16 although one patient had only a transient response of some but not all lesions.14 Notably, this patient had fibrosarcomatous histology with an associated complex karyotype but no evidence of t(17;22).14 Only one of the three other patients whose metastatic DFSP had a significant clinical response to imatinib had cytogenetic evaluation of their tumor; this case had evidence of PDGFB rearrangement by FISH. However, neither a ring chromosome or t(17;22) were present, and the PDGFB rearrangement involved an unidentified translocation partner. The limited clinical experience in metastatic DFSP suggests that imatinib therapy has a role in the management of advanced disease. Given the ineffectiveness of cytotoxic chemotherapy for this disease, we conclude that a trial of imatinib therapy is clinically indicated in patients with metastatic disease. Further studies may be helpful in determining the usefulness of cytogenetics and/or PDGFB FISH in predicting the likelihood of clinical response of metastatic DFSP to imatinib therapy.
Authors' Disclosures of Potential Conflicts of Interest
NOTES
Presented in part at the 39th Annual Meeting of the American Society of Clinical Oncology, Chicago, IL, May 31-June 3, 2003.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
1. Gloster HM Jr: Dermatofibrosarcoma protuberans. J Am Acad Dermatol 35:355-374, 1996
2. Rutgers EJ, Kroon BB, Albus-Lutter CE, et al: Dermatofibrosarcoma protuberans: Treatment and prognosis. Eur J Surg Oncol 18:241-248, 1992
3. Mark RJ, Bailet JW, Tran LM, et al: Dermatofibrosarcoma protuberans of the head and neck: A report of 16 cases. Arch Otolaryngol Head Neck Surg 119:891-896, 1993
4. Bowne WB, Antonescu CR, Leung DH, et al: Dermatofibrosarcoma protuberans: A clinicopathologic analysis of patients treated and followed at a single institution. Cancer 88:2711-2720, 2000
5. Gloster HM Jr, Harris KR, Roenigk RK: A comparison between Mohs micrographic surgery and wide surgical excision for the treatment of dermatofibrosarcoma protuberans. J Am Acad Dermatol 35:82-87, 1996
6. Sandberg AA, Bridge JA: Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: Dermatofibrosarcoma protuberans and giant cell fibroblastoma. Cancer Genet Cytogenet 140:1-12, 2003
7. Naeem R, Lux ML, Huang SF, et al: Ring chromosomes in dermatofibrosarcoma protuberans are composed of interspersed sequences from chromosomes 17 and 22. Am J Pathol 147:1553-1558, 1995
8. Simon MP, Pedeutour F, Sirvent N, et al: Deregulation of the platelet-derived growth factor B-chain gene via fusion with collagen gene COL1A1 in dermatofibrosarcoma protuberans and giant-cell fibroblastoma. Nat Genet 15:95-98, 1997
9. O'Brien KP, Seroussi E, Dal Cin P, et al: Various regions within the alpha-helical domain of the COL1A1 gene are fused to the second exon of the PDGFB gene in dermatofibrosarcomas and giant-cell fibroblastomas. Genes Chromosomes Cancer 23:187-193, 1998
10. Wang J, Hisaoka M, Shimajiri S, et al: Detection of COL1A1-PDGFB fusion transcripts in dermatofibrosarcoma protuberans by reverse transcription-polymerase chain reaction using archival formalin-fixed, paraffin-embedded tissues. Diagn Mol Pathol 8:113-119, 1999
11. Shimizu A, O'Brien KP, Sjoblom T, et al: The dermatofibrosarcoma protuberans-associated collagen type Ialpha1/platelet-derived growth factor (PDGF) B-chain fusion gene generates a transforming protein that is processed to functional PDGF-BB. Cancer Res 59:3719-3723, 1999
12. Sjoblom T, Shimizu A, O'Brien KP, et al: Growth inhibition of dermatofibrosarcoma protuberans tumors by the platelet-derived growth factor receptor antagonist STI571 through induction of apoptosis. Cancer Res 61:5778-5783, 2001
13. Linn SC, West RB, Pollack JR, et al: Gene expression patterns and gene copy number changes in dermatofibrosarcoma protuberans. Am J Pathol 163:2383-2395, 2003
14. Maki RG, Awan RA, Dixon RH, et al: Differential sensitivity to imatinib of 2 patients with metastatic sarcoma arising from dermatofibrosarcoma protuberans. Int J Cancer 100:623-626, 2002
15. Rubin BP, Schuetze SM, Eary JF, et al: Molecular targeting of platelet-derived growth factor B by imatinib mesylate in a patient with metastatic dermatofibrosarcoma protuberans. J Clin Oncol 20:3586-3591, 2002
16. Labropoulos S, Papadopoulos S, Hadjiyiassemi L, et al: Response of metastatic dermatofibrosarcoma protuberans to imatinib mesylate. Proc Am Soc Clin Oncol 830:830, 2003 (abstr 3334)
17. Fletcher JA, Kozakewich HP, Hoffer FA, et al: Diagnostic relevance of clonal cytogenetic aberrations in malignant soft-tissue tumors. N Engl J Med 324:436-442, 1991
18. Hsi BL, Xiao S, Fletcher JA: Chromogenic in situ hybridization and FISH in pathology. Methods Mol Biol 204:343-351, 2002
19. Suit H, Spiro I, Mankin HJ, et al: Radiation in management of patients with dermatofibrosarcoma protuberans. J Clin Oncol 14:2365-2369, 1996
20. Ballo MT, Zagars GK, Pisters P, et al: The role of radiation therapy in the management of dermatofibrosarcoma protuberans. Int J Radiat Oncol Biol Phys 40:823-827, 1998
21. Wan PT, Garnett MJ, Roe SM, et al: Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 116:855-867, 2004
22. Heinrich MC, Corless CL, Duensing A, et al: PDGFRA activating mutations in gastrointestinal stromal tumors. Science 299:708-710, 2003
23. Lynch TJ, Bell DW, Sordella R, et al: Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350:2129-2139, 2004
24. Segatto O, Lonardo F, Pierce JH, et al: The role of autophosphorylation in modulation of erbB-2 transforming function. New Biol 2:187-195, 1990(Grant A. McArthur, George)
Dana-Farber Cancer Institute and Harvard Medical School, Sarcoma Center
Department of Pathology, Brigham & Women's Hospital, Boston, MA
Universiteit Ziekenhuis Gasthuisberg dienst oncology
Cytogenetics and Molecular Genetics of Human Malignancies, Department of Human Genetics, Catholic University of Leuven, Leuven, Belgium
Departments of Medicine and Pathology, Oregon Health Sciences University Cancer Institute and Portland Veterans Affairs Medical Center, Portland, OR
Clinical Research Oncology, Novartis Pharma AG, Basel, Switzerland
ABSTRACT
PATIENTS AND METHODS: We analyzed the objective radiologic and clinical response to imatinib at 400 mg twice daily in eight patients with locally advanced DFSP and two patients with metastatic disease.
RESULTS: Each of eight patients with locally advanced DFSP had evidence of t(17;22) and showed a clinical response to imatinib. Four of these patients had complete clinical responses. The two patients with metastatic disease had fibrosarcomatous histology and karyotypes that were substantially more complex than those typically associated with localized DFSP. One patient with metastatic DFSP and an associated t(17;22) had a partial response to imatinib but experienced disease progression after 7 months of therapy. In contrast, the other patient with metastatic disease had a tumor lacking t(17;22), and there was no clinical response to imatinib. Unexpectedly, there was minimal platelet-derived growth factor receptor–beta phosphorylation in the untreated DFSP, despite the documented presence of a PDGFB autocrine mechanism.
CONCLUSION: Imatinib has clinical activity against both localized and metastatic DFSP with t(17;22). However, fibrosarcomatous variants of DFSP lacking t(17;22) may not respond to imatinib.
INTRODUCTION
More than 90% of DFSP feature a translocation involving distinct regions of chromosomes 17 and 22.6 Most commonly, the t(17;22) breakpoint region is tandemly repeated within a supernumerary ring chromosome.7 The translocation breakpoint generally involves the second exon of the platelet-derived growth factor-B (PDGFB) gene on chromosome 22, which is fused with the strongly expressed collagen 1 alpha 1 (COL1A1) gene on chromosome 17. This distinctive translocation mechanism results in transcriptional upregulation of the PDGFB gene, in the form of a COL1A1-PDGFB fusion oncogene. The associated COL1A1-PDGF-B fusion protein8-10 is post-translationally processed to yield mature and fully functional PDGFB.11 Therefore, the t(17;22) results in PDGFB-mediated activation of platelet-derived growth factor receptor–beta (PDGFRB) by autocrine and paracrine production of a functional ligand for PDGFRB. Because PDGFRB is the major PDGFR isoform expressed on DFSP,12,13 the t(17;22) most likely activates PDGFRB rather than PDGFR-alpha (PDGFRA).11
The identification of deregulated expression of PDGFB as a result of the t(17;22) led to the hypothesis that inhibitors of PDGFRB, such as imatinib, might have activity in DFSP. After supportive preclinical studies,11,12 there have been several reports of clinical activity of imatinib in DFSP.14-16 Here we report a series of 10 patients with DFSP who received imatinib for locally advanced or metastatic DFSP. Strikingly, all eight patients with locally advanced disease responded to imatinib. Unexpectedly, these dramatic clinical responses were seen even in DFSPs expressing relatively low amounts of activated PDGFRB.
PATIENTS AND METHODS
Patients were treated with 800 mg of imatinib daily (400 mg twice daily) with dose reductions for grade 3 toxicity or for recurrent grade 2 toxicity after an initial 1-week treatment break. The initial dose reduction was to 600 mg, with a second dose reduction to 400 mg allowed for further grade 3 toxicity or recurrent grade 2 toxicity after a 1-week treatment break. Full details of the clinical trial will be published separately.
Study investigations were performed after approval by a local human investigations committee and for United States centers in accord with an assurance filed with and approved by the United States Department of Health and Human Services. Informed consent was obtained from each patient.
Eligibility
Patients with primary or metastatic DFSP, Eastern Cooperative Oncology Group performance status of 0 to 2, and adequate end organ function were eligible for treatment. Prior systemic therapy for DFSP was allowed.
Efficacy Assessments
Objective responses to imatinib were evaluated using traditional Southwest Oncology Group criteria (pre-Response Evaluation Criteria In Solid Tumors). Partial response was defined as a ≥ 50% decrease from baseline in the sum of products of perpendicular diameters of all measurable lesions. Complete response was defined as complete disappearance of all measurable and nonmeasurable but assessable disease.
All patients were evaluated with either tumor imaging with computed tomography or magnetic resonance imaging scans (patients 9 and 10), clinical photography in the case of locally advanced tumors with superficially visible disease (patients 1, 2, 4, and 6), or clinical examination by palpation with measurements confirmed by ultrasound (patients 3, 5, 7, and 8). Measurable disease in the skin was defined as lesions with at least one diameter ≥ 0.5 cm that could be assessed by medical photography or two diameters ≥ 2 cm that could be assessed by palpation with measurements confirmed by ultrasound. Data presented include follow-up to March 1, 2004.
Western Blot Analyses
Frozen tumor specimens were homogenized in ice-cold cell lysis buffer using a Tissue Tearor unit (Biospec, Bartlesville, OK). The lysates were then rocked at 4°C for 30 minutes, cleared by centrifugation, and quantitated using the BioRad protein assay (Hercules, CA). Electrophoresis was performed using 30 μg of DFSP cell lysate per lane, and with inclusion of control lanes containing PDGFA- and PDGFB-stimulated NIH3T3 cells, and a PDGFRA mutant gastrointestinal stromal tumor, as positive controls for phospho PDGFRB and phospho PDGFRA, respectively. The gels were blotted to polyvinylidenedifluoride membranes, then stained for phospho PDGFRs, using a rabbit phosphoPDGFRA Y754 antibody (which binds to both phosphoPDGFRA and phosphoPDGFRB) from Santa Cruz Biotechnology Inc (SC-12911; Santa Cruz Biotechnology Inc, Santa Cruz, CA). Detection was performed with enhanced chemiluminescence, and protein expression was quantitated using a FUJI LAS1000-plus chemiluminescence imaging system (Fuji; Stamford, CT).
Karyotyping
DFSP karyotyping was performed after 3 to 6 days in tissue culture, and the metaphase cells were stained and analyzed by Giemsa-trypsin banding according to standard methods.17
Fluorescence In Situ Hybridization
Fluorescence in situ hybridization (FISH) was performed by labeling bacterial artificial chromosomes (BACs) centromeric (RP11-1149B8 and RP11-348I17) and telomeric (RP11-101B10 and RP11-434E5) to the PDGFB locus with biotin and digoxigenin, respectively.18 FISH evaluations of cytogenetic DFSP metaphase material were performed by standard methods, using either the DFSP BAC probes or using painting probes for chromosomes 17 and 22. FISH evaluations of paraffin sections were performed after pretreating 4-μm sections by microwaving and digestion with Digest All-III (Zymed, South San Francisco, CA), then applying the DFSP BAC probes and codenaturing the probe and section in a polymerase chain reaction machine. Detection of the biotinylated and digoxigenin-labeled probes was performed with streptavidin Alexa 594 (Molecular Probes, Eugene, OR) and fluorescein isothiocyanate antidigoxigenin (Roche, Indianapolis, IN), respectively.
RESULTS
All patients were commenced on imatinib at 800 mg daily (400 mg bid). In this patient population, 800 mg daily was well tolerated, with only one patient requiring dose reduction to 600 mg daily. Four patients received tumor biopsies before and approximately 28 days after commencement of imatinib. Responses were evaluated using traditional Southwest Oncology Group criteria (pre-Response Evaluation Criteria in Solid Tumors). Notably, all eight patients with locally advanced DFSP had partial (four patients) or complete (four patients) responses to imatinib (Table 2). Two patients (patients 7 and 8) who proceeded to surgical resection after initial clinical response had pathologically confirmed complete responses. In four further cases (patients 1, 2, 3, and 5), the patients were rendered disease-free by surgical resection after partial response to imatinib. Of the two patients with metastatic disease and complex cytogenetics, patient 10, whose DFSP lacked the t(17;22), had no clinical response to imatinib by either conventional imaging or fluorodeoxyglucose positron emission tomography scan at day 19 of imatinib therapy. However, follow-up on this patient was quite short because of the patient's death on day 32. By contrast, patient 9, whose metastatic DFSP featured the t(17;22) and other cytogenetic aberrations, had a robust partial response (Table 2 and Fig 2). The clinical responses to imatinib were sustained in all four patients treated with imatinib for greater than 6 months. However, two of these patients subsequently experienced disease progression. Patient 1, with locally advanced DFSP on the cheek, responded for just less than 2 years before the tumor progressed, at which point the patient was rendered disease-free after resection using Mohs surgical technique. Patient 9 with metastatic disease experienced disease progression after 7 months. Patients rendered free of disease by surgical resection had no evidence of progression at 4, 18, 12, and 1 months after the resection.
Several patients had matched pretreatment and on-therapy tumor biopsy specimens. Biopsy specimens obtained during imatinib therapy had reduced cellularity and the development of hyaline changes (Fig 3). PDGFB FISH analysis showed persistence of the t(17;22) in the spindle-cell component of the resected stable disease. However, the t(17;22) was found in only 20% of the spindle cells, suggesting that most of the residual spindle cells were either reactive or were DFSP cells lacking t(17;22).
Tumor tissue was examined for expression and activation of PDGFRA and PDGFRB by immunoblot using antisera to the active phosphorylated forms of the PDGF-receptor. Interestingly, PDGFR activation was low in five DFSPs evaluated before imatinib therapy (two examples are shown in Fig 4). Indeed, PDGFR activation was at least 10-fold lower than in gastrointestinal stromal tumors (GISTs) expressing PDGFR with an intrinsic gain-of-function mutation (Fig 4). The weak baseline activation in the DFSP precluded accurate quantification of the reduction in activated PDGFRB inhibition in response to imatinib.
DISCUSSION
The high response rate to imatinib therapy in DFSP supports the hypothesis that DFSP cells are dependent on aberrant activation of PDGFRB for cellular proliferation and survival. Our clinical results are consistent with studies of DFSP cell culture and animal models, in which t(17;22)-mediated PDGFRB autocrine activation is effectively inhibited by imatinib. Unexpectedly, the level of PDGFRB activation as assessed by receptor autophosphorylation was substantially lower than PDGFR activation in a GIST with intrinsic PDGFRA oncogenic mutation. Therefore, although mutation of a serine/threonine kinase such as BRAF in melanoma21 or mutation or amplification of a receptor tyrosine kinase (RTK) such as PDGFRA in GIST tumors,22 epidermal growth factor receptor in epithelial tumors,23 or ERB-B2 in breast cancer24 leads to strong activation of the involved oncogenic RTKs, it seems that autocrine/paracrine oncogenic mechanisms—as seen in DFSP—can be associated with substantially lower levels of RTK activation. These novel observations indicate that neither high levels of RTK activation nor RTK overexpression are required for clinical response to therapeutic inhibition of receptor signaling. Rather, inhibition of low-level RTK activation can be clinically effective, providing that the tumor cells are dependent on that signaling mechanism.
Our findings demonstrating low activation of PDGFRB in DFSP tumors contrast with those of Sjblom et al,12 who readily detected phosphorylation of PDGFRB in cultured cells derived from DFSP tumors. We propose two possible explanations for this apparent discrepancy: first, the culture conditions used by Sjblom et al included the use of fetal calf serum, a source of exogenous PDGFs. It is possible that these culture conditions induced greater phosphorylation of PDGFRB than we observed in tumor biopsy specimens. Second; Sjblom et al used immunoprecipitation rather than direct immunoblotting to detect phosphorylation of PDGFRB. It is likely that immunoprecipitation is more sensitive than immunoblotting at detecting phosphorylated PDGFRB. Nonetheless, there is a clear and substantial difference between receptor phosphorylation in GISTs or PDGFA/B-stimulated NIH3T3 cells compared with the DFSP tumors (Fig 4). Moreover, in our experience, only weak activation of PDGFRB is detectable using immunoprecipitation of protein lysates purified from fresh DFSP tumors (J.A.F., unpublished data).
We treated two patients with metastatic disease; both patients had fibrosarcomatous histology associated with complex karyotypes. In one case (patient 10), both the locally recurrent and metastatic lesions lacked t(17;22), suggesting that this DFSP-like tumor was not necessarily dependent on signaling through PDGFRs. Consistent with this prediction, the second metastatic DFSP (patient 9) had an associated t(17;22) and had a partial clinical response to imatinib, although the DFSP progressed after 7 months of therapy. Four other patients with metastatic DFSP have been reported in the literature with clinical responses observed in all of these cases,14-16 although one patient had only a transient response of some but not all lesions.14 Notably, this patient had fibrosarcomatous histology with an associated complex karyotype but no evidence of t(17;22).14 Only one of the three other patients whose metastatic DFSP had a significant clinical response to imatinib had cytogenetic evaluation of their tumor; this case had evidence of PDGFB rearrangement by FISH. However, neither a ring chromosome or t(17;22) were present, and the PDGFB rearrangement involved an unidentified translocation partner. The limited clinical experience in metastatic DFSP suggests that imatinib therapy has a role in the management of advanced disease. Given the ineffectiveness of cytotoxic chemotherapy for this disease, we conclude that a trial of imatinib therapy is clinically indicated in patients with metastatic disease. Further studies may be helpful in determining the usefulness of cytogenetics and/or PDGFB FISH in predicting the likelihood of clinical response of metastatic DFSP to imatinib therapy.
Authors' Disclosures of Potential Conflicts of Interest
NOTES
Presented in part at the 39th Annual Meeting of the American Society of Clinical Oncology, Chicago, IL, May 31-June 3, 2003.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
1. Gloster HM Jr: Dermatofibrosarcoma protuberans. J Am Acad Dermatol 35:355-374, 1996
2. Rutgers EJ, Kroon BB, Albus-Lutter CE, et al: Dermatofibrosarcoma protuberans: Treatment and prognosis. Eur J Surg Oncol 18:241-248, 1992
3. Mark RJ, Bailet JW, Tran LM, et al: Dermatofibrosarcoma protuberans of the head and neck: A report of 16 cases. Arch Otolaryngol Head Neck Surg 119:891-896, 1993
4. Bowne WB, Antonescu CR, Leung DH, et al: Dermatofibrosarcoma protuberans: A clinicopathologic analysis of patients treated and followed at a single institution. Cancer 88:2711-2720, 2000
5. Gloster HM Jr, Harris KR, Roenigk RK: A comparison between Mohs micrographic surgery and wide surgical excision for the treatment of dermatofibrosarcoma protuberans. J Am Acad Dermatol 35:82-87, 1996
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