Cancer Investigation, 32:533–542, 2014 ISSN: 0735-7907 print / 1532-4192 online C 2014 Informa Healthcare USA, Inc. Copyright  DOI: 10.3109/07357907.2014.964409

REVIEW ARTICLE

ErbB Receptors as Prognostic and Therapeutic Drug Targets in Bone and Soft Tissue Sarcomas Hongsheng Wang,1,2∗ Qingbo Yang,1,2∗ Zeze Fu,1∗ Dongqing Zuo,1,2 Yingqi Hua,1# and Zhengdong Cai1# 1

Department of Orthopedics, Shanghai 1st People’s Hospital, Shanghai Jiaotong University, Shanghai, China, 2 Department of Orthopedics, Tenth People’s Hospital of Tongji University, Shanghai, China cluding prognostic relevance and targeted therapy that may be worthy of further exploration.

ErbB receptors have been intensely studied to understand their importance in cancer biology and as therapeutic targets, and many ErbB inhibitors are now used in the clinical setting. A large number of studies have been conducted to examine the expression of ErbB family members in bone and soft tissue sarcomas, including osteosarcomas, synovial sarcomas, Ewing sarcomas, rhabdomyosarcomas, and so on. Nevertheless, the clinical implications of ErbB receptors remain elusive. To illustrate the potential of ErbB family members as prognostic and therapeutic drug targets in bone and soft tissue sarcomas, we summarized the molecular evidence and observations from clinical and basic trials.

OVERVIEW OF ERBB RECEPTORS, LIGANDS, AND TRANSDUCTION PATHWAY The ErbB family of receptor tyrosine kinases (RTKs) consists of the following four members: ErbB1 (also called EGFR, HER1), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4), which play a key role in embryonic development, tissue renewal/repair, and cancer (7). All four members share an overall structure of an extracellular ligand-binding domain, a single hydrophobic transmembrane region, and an intracellular segment that contains a conserved tyrosine kinase domain (8). ErbB1 and ErbB4 are two fully functional receptors of the family, and they can activate similar downstream processes in response to their different ligands (3, 9). In contrast, ErbB2 is unable to bind any known ligands, and ErbB3 lacks intrinsic tyrosine kinase abilities. Therefore, activation of ErbB2 and ErbB3 is dependent upon dimerization with other family members (10, 11). Ligand binding to ErbB receptors results in the formation of receptor homo- and heterodimers that are essential for activation of the tyrosine kinase (9). There are up to 13 recognized ligands of the ErbB family, which include the following: transforming growth factor-α (TGF-α), epidermal growth factor (EGF), heparin-binding (HB)-EGF, amphiregulin, betacellulin, epwigen, epiregulin, and neuregulins 1–6 (12, 13). EGF and TGF-α are the key ErbB1 binding ligands, betacellulin can bind and activate all receptors, and the neuregulins have a preference for ErbB3 and ErbB4 (14, 15). Furthermore, ErbB receptors can become transactivated by G-proteincoupled receptors (GPCR). ADAMs are the key metalloproteases activated by several GPCR agonists to produce a mature EGFR ligand leading to EGFR transactivation (16).

Keywords: ErbB receptors, Bone and soft tissue sarcomas, Prognostic relevance, Targeted therapy

INTRODUCTION In the last two decades, evidence has accumulated indicating that the ErbB receptor tyrosine kinases play important roles in cancer. Abundant clinical and basic studies have suggested that they play key roles in cancer development and progression by activating a multiplicity of intracellular pathways (1, 2). Furthermore, many ErbB-targeted inhibitors have been developed as therapeutic agents against cancers that have been associated with ErbB receptors (3). To date, the most widely used ErbB targeted therapy in clinic is for breast cancer (4) and non-small cell lung cancer (NSCLC) (5), which gives us a promising prognosis by follow-up. Bone and soft tissue sarcomas are a rare group of tumors that are derived from mesenchymal tissues (6). The molecular pathogenesis of these heterogeneous tumors is largely unknown, which explains the lack of targeted therapies. In this review, we will concentrate on the role of ErbB receptors in mesenchymal originated bone and soft tissue sarcomas, in∗ #

Hongsheng Wang, Qingbo Yang, and Zeze Fu are co-first authors; Yingqi Hua and Zhengdong Cai are co-corresponding authors

Correspondence to: Yingqi Hua, Musculoskeletal Oncology Center, Department of Orthopedics, Shanghai 1st People’s Hospital, Shanghai Jiaotong University, Shanghai, 200080, China. e-mail: Hua [email protected] or Zhengdong Cai, Department of Orthopedics, Shanghai 1st People’s Hospital, Shanghai Jiaotong University, Shanghai, 200080, China. e-mail: [email protected] Received 19 February 2014; revised 30 April 2014; accepted 8 September 2014.

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Downstream signaling follows three dominant transduction pathways: PI3K/AKT, RAF/MEK/MAPK, or JAK/STAT (17–19). Despite extensive overlap in the molecules that are recruited by the different active receptors, different ErbBs preferentially modulate certain signaling pathways, owing to the ability of individual ErbBs to bind specific effector proteins (20). For example, STAT5 is a direct binding partner of ErbB1 and ErbB4, and this interaction is required during lactation (21). ErbB3 is activated by neuregulin-1 and -2 and is a strong activator of PI3K kinase activity (20). Interestingly, several studies have shown that MEK inhibition leads to increase AKT activation by relieving a negative feedback on ErbB receptors (22, 23). ROLES ERBB RECEPTORS PLAY IN BONE AND SOFT TISSUE SARCOMAS A large number of studies have been conducted to examine the expression of ErbB family members in bone and soft tissue sarcoma, including osteosarcomas, synovial sarcomas, Ewing sarcomas, rhabdomyosarcomas, and so on (Table 1). However, a conclusion cannot be drawn whether ErbB family plays a role in oncogenesis of sarcomas, and whether overexpression will lead to a worse outcome. Furthermore, the factors that affect ErbB receptors in bone and soft sarcomas are poorly understood. ErbB receptors in osteosarcoma Osteosarcoma is the most common primary malignant tumor arising from bone in children and adolescents. Although the routine use of intensive chemotherapy has significantly improved survival for patients with localized disease, patients with metastatic, refractory, or recurrent disease continue to do poorly (24, 25). In the literature, it appears that the expression of ErbB1 and ErbB2 are prevalently expressed in osteosarcomas, and little is known about ErbB3 and ErbB4. However, the prognostic impact of ErbB still varies significantly within individual studies.

ErbB1 in osteosarcoma Various studies have been conducted to characterize the expression of ErbB1 in osteosarcoma, irrespective of histologic subtype, patient age, or tumor site. The expression of ErbB1 has been reported in 30–90% of tumor tissues (26–28); however, correlations between ErbB1 expression and clinical prognosis have been controversial. The study by Kersting et al. (29) found that all patients with strong ErbB1 expression were alive for up to 12 years after diagnosis (overall survival), irrespective of the degree of neoadjuvant chemotherapy and induced tumor regression, which indicted that expression of ErbB1 in high-grade osteosarcoma showed a positive dose–response relation with favorable clinical outcome. Wen et al. reported that ErbB1 expression was observed in six out of nine (67%) patients with metastasis and four out of five (80%) of those with local recurrence (30). However, they considered that the small number of cases with metastasis and local recurrence does not allow any definitive conclusions regarding a correlation between EGFR expression and clinical outcome. Several other studies have failed to detect any association between ErbB1 expression and clinical findings in osteosarcoma (27, 31). The value of ErbB1 downstream signaling molecules as prognostic factors has been examined in osteosarcoma cases. AKT is phosphoryated and activated by ErbB1 through PI3 kinase, which plays an important role in modulating the mammalian target of rapamycin (mTOR) and is effectively inhibited by PTEN (32). Wu et al. (31) performed a clinical study and constructed a mediation model to explain and confirm the correlation pattern of ErbB1 and the AKT signal pathway for cancer cell proliferation in osteosarcoma patients. Lee et al. (28) found that loss of PTEN in tumors with ErbB1 expression was associated with resistance to antiErbB1 tyrosine kinase inhibitors. However, Freeman et al. (33) did not identify a correlation between ErbB1 expression, or PTEN, and clinical features. ERK1/2 is activated by mitogenic agents and is a component of signal transduction cascades that regulate cell proliferation, survival, and transformation. Do et al. presented data that indicated that ERK

Table 1. The Basic Molecular Characteristics of ErbB Receptors and Their Ligands ErbB Receptors/Ligands ErbB1 (EGFR, HER1) ErbB2 (HER2) ErBB3 (HER3) ErbB4 (HER4) TGF-α EGF HB-EGF AREG BTC EPGN EREG NRG-1 NRG-2 NRG-3 NRG-4

GenBank Accession Number

Chromosome Location

1956

7p12.3-p12.1

2064 2065 2066 7039 1950 1839 374 685 71920 2069 3084 9542 10718 145957

17q21.1 12q13 2q33.3-q34 13 4 5 4 4 4 4 8 5 10 15

Major Molecular Functions Cell growth, proliferation, motility, adhesion, invasion, apoptosis inhibition, metastasis, autophagy, and resistance to chemotherapy Tumorigenicity, metastasis, and resistance to chemotherapy Proliferation and differentiation Mitogenesis and differentiation Proliferation, differentiation, and development Growth, proliferation, and differentiation Proliferation, migration, and adhesion Growth and development Growth and reproduction Unclear Angiogenesis, vascular remodeling, and proliferation Growth, differentiation, and survival Growth, differentiation, and survival Growth, differentiation, and survival Growth, differentiation, and survival

TGF-α, transforming growth factor-α; EGF, epidermal growth factor; AREG, Amphiregulin; BTC, Betacellulin; EPGN, Epigen; EREG, Epiregulin; NRGs, neuregulins.

Cancer Investigation

ErbB Receptors Sarcomas could be used as a prognostic factor in patients in osteosarcoma. They demonstrated that cytoplasmic and membrane expression of ErbB1 showed a statistical correlation with ERK expression, which supports ERK as a downstream signaling molecule of ErbB1 (34). With regard to the JAK/STAT signaling pathway, there are few studies that discuss the relationship with ErbB1 in osteosarcoma. In addition, various ErbB1 mutations have been found in human cancers, with varying clinical implications. Several groups have provided evidence that the presence of mutations or amplifications may correlate with the clinical response to kinase inhibitors of ErbB1. Such alterations include ErbB1 amplification, gain-of-function somatic mutations, protein overexpression, and a point mutation in the tyrosine kinase domain (35). We have not found any known mutations, such as ERBB1vIII, L858R, L861Q, and G719C in osteosarcoma. Studies of ErbB1 gene mutations identify a point mutation at codon 863 in exon 21, E829E, and R831C in exon 21 (36,41) (36, 37). Of all of these mutations, only one mutation (R831C in exon 21) resulted in an amino acid change from arginine to cysteine (28, 33, 34). Furthermore, the methylation status of ERBB1 CpG island was examined in osteosarcomas. Montero et al. found that hypermethylation density was 14% in a human osteosarcoma cell line SAOS2 (38). Considering the positive expression of ErbB1 and the presence of ErbB1 mutations, we consider that patients with osteosarcoma may profit from the tyrosine kinase inhibitor therapy. ErbB2 in osteosarcoma Data concerning the expression of ErbB2 in osteosarcoma are conflicting, with some studies reporting up to 100% positive cases (39) and others reporting only negative cases (40). Furthermore, the prognostic value of ErbB2 remains controversial. Onda et al. (41) and Gorlick et al. (42) first reported that ErbB2 correlates with increased pulmonary metastasis and worse survival. Later, Zhou et al. (43), Scotlandi et al. (44), and Fellenberg et al. (39) had similar findings implicating ErbB2 as an adverse prognostic factor. Based on these results, some phase II clinical trial by the Children’s Oncology Group used ErbB2 as an adjuvant target with conventional chemotherapy. Two clinical trials have been completed; however, no reports from these studies have been published thus far. Experimental investigations (45) have shown that although ErbB2-positive osteosarcoma cell lines are not responsive to Trastuzumab—a monoclonal antibody against ErbB2, ErbB2-specific T cells have profound efficiency in killing osteosarcoma both in vitro and in vivo. A small molecular pan-ErbB tyrosine kinase inhibitor, which hits all four members of the ErbB family kinases also showed good efficacy of proliferation inhibition in vitro against osteosarcoma (46). These studies support the conclusion that ErbB2 could be a potential target for improving treatment of osteosarcoma. However, several other studies could not confirm these conclusions. They suggested that ErbB2 is not important in the development or progression of osteosarcoma, and ErbB2-targeted therapy was unlikely to yield promising results in the treatment of osteosarcoma C 2014 Informa Healthcare USA, Inc. Copyright 

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(40, 47–49). Interestingly, Maitra et al. (40), Kilpatrick et al. (47), and Thomas et al. (49) considered that ErbB2 is either not expressed or expressed at low levels in osteosarcoma cases. Two years later, three of these authors were included in a study by Hughes et al. (50), which demonstrated variable expression of ErbB2 using immunohistochemistry, quantitative real-time PCR, and flow cytometry. Interesting, Scotlandi et al. (44) found that the growthinhibitory effects of Trastuzumab (an ErbB2 antibody) was definitely low in vitro, although ErbB2 expression is found in these cell lines. They considered that the lack of gene amplification may be, at least partly, responsible for the scarce antitumor effects of Trastuzumab. Indeed, Anninga et al. (48) and Willmore-Payne et al. (51) failed to find ErbB2 gene amplification using FISH, and suggested ErbB2-targeted therapy was unlikely to yield promising results in the treatment of osteosarcoma. According to the literature seen earlier, we have considered that ErbB2 may play a role in the tumor biology of osteosarcoma, but the efficacy of anti-ErbB2 agents should be further studied. ErbB receptors in Ewing sarcoma Ewing sarcoma (ES) is the second most common malignant bone tumor, arising in children and adolescent with a peak incidence at 15 years of age. The pathognomonic genetic marker of ES is the recurrent translocation t(11;22)(q24:q12), which encodes an oncogenic fusion protein and transcription factor EWS/FLI (52). In ES, ErbB receptors have been evaluated by immunohistochemistry in a small series of clinical samples or in few cell lines, and its biological relevance in this neoplasm remains to be clarified. Studies about ErbB2 by Kim et al. (53), Ye et al. (54), Thomas et al. (49), and Scotlandi et al. (44) had similar conclusions, i.e., that ErbB2 is not an important prognostic factor in ES. Nevertheless, Zhou et al. (55) showed that downregulation of ErbB2 expression will decrease cell growth, and increase cellular sensitivity to VP-16 and Adriamycin. Sorensen et al. conducted trials to examine ErbB4 expression in metastatic disease compared to primary patient-matched ES biopsies, and found activation of the ERBB4 tyrosine kinase suppresses anoikis (56), or detachment-induced cell death, and induces chemoresistance in ES cell lines in vitro (57). ErbB receptors in chordoma Chordomas are a rare bone tumor, arising from notochordal remnants in the midline skeletal axis, for which the current treatment is limited to surgery and radiotherapy. Notably, there is no effective drug therapy for treating chordomas (58). However, the ongoing elucidation of the molecular mechanisms underlying chordomas has led to new therapeutic hopes (59). There are several reports evaluating ErbB1 expression in chordomas. These immunohistochemistrybased studies reported ErbB1 and phosphorylated ErbB1 were found in 32—100% and 43—100% chordoma patients (60–64), respectively. Nevertheless, EGFR polysomy has been reported in a variable number of chordoma samples (17–52%) by FISH (60, 62, 64). With regard to ErbB2, between 0% and 33% of chordoma samples examined were

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positive (61, 65, 66). Sporadic clinical observations have been corroborated by preliminary data on the possible role of ErbB1 in chordoma pathogenesis. Deniz et al. (67) found that increased levels of the ErbB1 ligand TGF-α in chordomas correlate with an increased incidence of local recurrence, suggesting that the ErbB1 pathway may be active. Shalaby et al. (62) demonstrated that ErbB1 inhibitor tyrosine (AG1487) markedly inhibited proliferation of the chordoma cell line U-CH1 in vitro and diminished ErbB1 phosphorylation, and these data implicated aberrant ErbB1 signaling in the pathogenesis of chordomas. Furthermore, four clinical cases highlighted the antitumor activity of ErbB1 inhibition in advanced chordomas (68–71). These studies support the conclusion that ErbB1 could be a potential target for improving treatment of osteosarcomas. ErbB receptors in synovial sarcoma Synovial sarcoma (SS) is a malignant soft tissue sarcoma that accounts for approximately 5–10% of all soft tissue tumors and tends to occur primarily in children and young adults (72). The role of ErbB1 and ErbB2 as molecular markers, have been evaluated in SS. The first report on expression of ErbB receptors in SS was published in 1985 (73). Subsequent reports have shown that ErbB1 overexpression was detected in 50–90% of synovial sarcoma (74–78), and ErbB2 in 10–40% cases (77, 79). The results of studies differ substantially and frequently depend on the methods used. In most studies using immunohistochemical methods, only membranous staining is considered specific for ErbB2 and cytoplasmic staining is usually dismissed as nonspecific. The study by Nuciforo et al. (79) demonstrated that the expression of ErbB2 in SS by RT-PCR is associated with features of good prognosis. The prognostic relevancy of ErbB receptors in synovial sarcoma still lacks consensus. The study by Thomas et al. (77) demonstrated that ErbB1 and ErbB2 expression may represent an important molecular event in the oncogenesis of synovial sarcoma, albeit at low level. In the study of Tawbi et al. (75), patients with SS that expressed ErbB1, phosphorylated ErbB1, or EGFRvIII did not have any statistically significant difference in time to first recurrence or death when compared with patients that lacked expression. Furthermore, recent studies have explored the prevalence and association of genomic alterations in downstream molecules of the ErbB1 pathway with clinical factors in SS. Mutations of PI3K, KRAS, and BRAF are rare events, while deletion of PTEN was correlated with poor survival in patients with SS (78). The majority of SS harbor a specific chromosomal translocation t(X;18)(p11.2;q11.2), in which the proximal portion of the SYT gene at chromosome 18q11 is fused to the distal portion of one of several duplicated SSX genes (most notably SSX1 and SSX2) at chromosome Xp11 (80). Several studies have explored the contribution of SYT/SSX fusion type to the expression of ErbB receptors. One of the studies has demonstrated that ErbB1 and ErbB2 are expressed in the majority of patients with SYT/SSX1, albeit at low levels (77). It is possible that SYT/SSX1 indirectly mediates the ErbB expression as a transcriptional regulator

because SSX1 and SSX2 are nearly identical in amino acid composition (81). Synovial sarcomas are uniquely composed of the following two morphologically distinct cell types: spindle cells and epithelioid cells. The presence of the two cell types in varying proportions lends to the classification of tumors into the following three histologic subtypes that exist along a continuous spectrum: biphasic, monophasic (monophasic fibrous or rare monophasic epithelial), and poorly differentiated (72). Strong ErbB2 protein expression is seen in the epithelial component of all biphasic synovial sarcomas, and approximately one-third of monophasic tumors, notably those with solid epithelioid areas (82). ErbB receptors in Rhabdomyosarcoma Rhabdomyosarcoma (RMS) is the most common soft-tissue malignant tumor of childhood, which is derived from primitive skeletal muscle tissue (83). RMS is composed of two main subtypes, embryonal RMS (eRMS) and alveolar RMS (aRMS). Whereas eRMS histologically resembles embryonic skeletal muscle, the aRMS subtype is more aggressive and has a poorer prognosis (84, 85). With regard to ErbB receptors expressed in RMS, Ganti et al. (86) reported that expression of ErbB1 was identified in 31/66 (47%) by immunohistochemistry and correlated with the embryonal subtype, while ErbB2 was expressed in 22/66 (33%) and tended to be more frequent in the alveolar subtype. Nordberg et al. (87) found that the ErbB3 transcript is highly expressed in pediatric aRMS in vitro and in vivo. Although much is known of the histopathology and clinical behavior of this neoplasm, the actual causes of carcinogenesis are still unknown. The ErbB family plays an essential role in the malignant phenotype: ErbB1 sustains RMS cell proliferation and growth, ErbB2 regulates myoblast cell transformation and survival, and ErbB3 induces myogenic differentiation (88–90). Additionally, Nanni et al. (91) conducted a study in which activation of ErbB2 coupled with inactivation of the oncosuppressor gene p53 causes RMS in mice and speculated that the interaction between ErbB family genes and the p53 pathway might be involved in the origin of human RMS. TARGETED THERAPY OF ERBB FAMILY IN BONE AND SOFT TISSUE SARCOMA Most efforts have concentrated on ErbB receptors due to their vital roles in tumor etiology and progression. Hence, many ErbB inhibitors have been intensely pursued as therapeutic targets. The following two important types of ErbB inhibitors are in clinical use: humanized monoclonal antibodies (mAbs) directed against the extracellular domain of ErbB1 or ErbB2, and small-molecule tyrosine–kinase inhibitors (TKIs) that compete with ATP in the tyrosine-kinase domain of the receptor (Table 2). Monoclonal antibodies mAbs bind to the ectodomain of the ErbB receptors with high specificity and compete with endogenous ligands to inhibit the ligand-induced ErbB tyrosine kinase activation by blocking the ligand-binding region (92, 93). Cancer Investigation

ErbB Receptors Sarcomas

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Table 2. Prognostic Relevancy of ErbB Receptors in Bone and Soft Tissue Sarcomas Study

Cancer

Method(s)

Kersting (29)

Osteosarcoma

IHC FISH

Scotlandi (46)

Osteosarcoma Ewing sarcoma

IHC

Gorlick (41)

Osteosarcoma

IHC

Ma (43)

Osteosarcoma

IHC

Morris (44)

Osteosarcoma

IHC

Onda (40)

Osteosarcoma

IHC

Zhou (42)

Osteosarcoma

IHC

Fellenberg (38)

Osteosarcoma

IHC

Akatsuka (45)

Osteosarcoma

IHC

Bakhshi (52)

Osteosarcoma

IHC

Willmore-Payne (54) Osteosarcoma

FISH

Somers (53)

Osteosarcoma

TMA

Thomas (51)

IHC

Lee (54)

Osteosarcoma Ewing sarcoma Osteosarcoma

Rt-PRC FISH

Anninga (50)

Osteosarcoma

IHC

Klipatrick (49)

Osteosarcoma

IHC (two antibodies)

Shalaby (67)

Chordoma

IHC

Weinberger (71)

Chordoma

IHC

Tawbi (80)

Synovial sarcoma

IHC

Thomas (82)

Synovial sarcoma

IHC

Teng (83)

Synovial sarcoma

IHC

Barbashina (79)

Synovial sarcoma

IHC

Nociforo (84)

Synovial sarcoma

FISH IHC

Ganti (91)

Rhabdomyosarcoma

IHC

No. of Patients 111 39

ErbB Receptor(s) ErbB1

Postive (%)

Correlation with Prognosis

81% 23%

Related with improved event-free and overall survival. 84 113 ErbB2 32% 16% An adverse role of HER2 expression in the progression of osteosarcoma. 47 ErbB2 42.6% ErbB-2 should be evaluated prospectively as a prognostic indicator. 63 ErbB2 60% A pivotal role of ErbB2 expression as a prognostic factor of osteosarcoma. 73 ErbB2 42.6% ErbB2 expression as a prognostic indicator. 26 ErbB2 42% ErbB2 is a useful prognostic marker in osteosarcoma. 25 ErbB2 45% ErbB2 expression is association with an increased risk of metastasis. 17 ErbB2 100% ErbB2 as prognostic marker for therapy outcome in osteosarcoma. 81 ErbB2 61% ErbB2 expression is correlated with increased survival patients with osteosarcoma. 59 ErbB2 47.1% ErbB2 and p53 combined expression were not related to grade or stage of osteosarcoma. 21 ErbB2 none ErbB2 expression was a relatively infrequent event, and anti-ErbB2 therapy is unlikely to have much value in osteosarcoma 18 ErbB2 11% ErbB2 expression is not important in the development or progression of osteosarcoma. 66 11 ErbB2 47% 27% ErbB2 is not an important prognostic factor. 21 ErbB2 100% 26% ErbB2 is not a poor prognostic factor in low-grade osteosarcoma. 27 ErbB2 4% ErbB2 does not play a role in osteosarcoma, and ErbB2-targeted therapy is unlikely to have much value in osteossarcoma. 41 ErbB2 98%/83% ErbB2 was not correlated with either response chemotherapy or survival. ErbB1 173 69% ErbB1 signaling in the pathogenesis of chordoma. ErbB1 ErbB2 12 100% 58% ErbB1 and ErbB2 play a significant role in the growth of chordomas 48 ErbB1 71% ErbB1 expression did not correlated with disease outcome. 38 ErbB1 ErbB2 55.3% 52.6% ErbB1 and ErbB2 expression may play an important role in the oncogenesis of synovial sarcoma. 30 ErbB1 63.3% Overexpression ErbB1 is significantly more prevalent in patients who were 35 or old. 19 ErbB1 ErbB2 68% 52% ErbB2 overexpression is not association with clinical outcome. 13 ErbB2 31% 23% Increased ErbB2 expression is associated with a more favorable clinical course. 66 ErbB1 ErbB2 47% 33% ErbB1 expression correlates with the embryonal subtype.

IHC, Immunological Histological Chemistry; FISH, Fluorescence In Situ Hybridization; Rt-PCR, reverse transcription-polymerase chain reaction;

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Table 3. Targeted Therarpy of ErbB1 Receptors in Bone and Soft Tissue Sarcomas Study

Type of Cancer

Bone and Soft Tissue Sarcoma

No. of Patients

Age

Trial Phase

Drug

Regimen

Treatment outcome

Trastuzumab and cytotoxic chemotherapy Gefitinib: 150, 300, 400, or 500 mg/m2

Therapeutic benefit of Trastuzumab remains uncertain. Oral Gefitinib is well tolerated in children.

Gefitinib

Gefitinib and Irinotecan

Phase I

Gefitinib

Gefitinib 25, 100, 250 mg/m2

>15

Phase II

Gefitinib

Gefitinib 500 mg/day

≤22

Phase I

Erlotinib

Erlotinib with or without Temozolomide

Gefitinib significantly enhances the bioavailability of oral Irinotecan. Gefitinib did not induce any consistent variation in ErbB1, VEGF, MMP-2, and MMP-9 blood levels. Gefitinib given as a single agent has no therapeutic role in advanced synovial sarcoma. The recommended dose of Erlotinib is 85 mg/m2 /d.

61

Phase II

Lapatinib

Lapatinib 1500 mg/day

96

≤32

Phase II

Trastuzumab

Osteosarcoma Ewing sarcoma Synovial sarcoma Osteosarcoma Ewing sarcoma family of tumor

632

≤22

Phase I

Gefitinib

63

≤22

Phase I

Pediatric tumors

Osteosarcoma Ewing sarcoma Synovial sarcoma

311

14.8

Ray-Coquard (100)

Synovial sarcoma

Advance ErbB1 expressing Synovial sarcoma

46

Jakacki (101)

Pediatric tumors

685

Stacchiotti (103)

Chordoma

Osteosarcoma Rhabdomyosarcoma Soft tissue sarcoma Advanced ErbB1-positive chordoma

Ebb (93)

Osteosarcoma

Metastatic osteosarcoma

Daw (96)

Pediatric tumors

Furmam (97)

Pediatric tumors

Jimeno (99)

18

Trastuzumab, Cetuximab, and Panitumumab are the currently FDA-approved ErbB antibodies, while Pertuzumab, Zalutumumab, and Nimotuzumab have been used in advanced clinical testing. Nevertheless, these targeted agents are never or rarely used in the clinical setting, probably owing to the prognostic relevance of ErbB receptors that are ambiguous in bone and soft-tissue sarcoma.

Trastuzumab Trastuzumab, a humanized monoclonal antibody against ErbB2, is a pivotal component in the treatment of patients with ErbB2-overexpressed or amplified tumors. The Children’s Oncology Group initiated a phase II trial of Trastuzumab in addition to standard chemotherapy for patients with newly diagnosed metastatic osteosarcoma from 2001 to 2010 (94). In this study, only patients with ErbB2 overexpression received concurrent therapy with Trastuzumab given for 34 consecutive weeks. Results showed there was no significant difference in event-free and overall survival between the HER-2-positive group treated with Trastuzumab and the HER-2-negative group treated with cytotoxic chemotherapy alone. It is important to note that, if ErbB2 overexpression is associated with inferior prognosis in osteosarcoma, a similar outcome for ErbB2-negative and HER2-positive patients may suggest benefit from the addition of Trastuzumab.

A modest antitumor activity of Lapatinib in chordoma.

Cetuximab Cetuximab is a chimeric IgG1-isotype mAb that binds to ErbB1 with high affinity and abrogates ligand-induced ErbB1 phosporylation. In numerous clinical studies, the addition of Cetuximab to conventional multidrug chemotherapy or radiotherapy has led to a significant improvement of clinical response rates, progression-free survival, and overall survival. One recent study found Cetuximab-mediated immunotherapy may be considered a novel treatment modality in the management of advanced osteosarcoma (95). In this study, Cetuximab treatment can improve the lysis of ErbB1-expressing, autologous primary osteosarcoma cells by patient-derived NK cells via Cetuximab-mediated antibodydependent cellular cytotoxicity (ADCC).

Tyrosine-kinase inhibitors Some molecule TKIs act in ErbB receptor’s intracellular domain to inhibit enzyme tyrosine kinase, which is responsible for signal transduction cascade and downstream activation of many proteins (92, 96). Several TKIs have been found to have effective antitumor activity and have been approved or are in clinical trials. The following sections discuss different TKIs including Gefitinib, Erlotinib, Lapatinib, and Canertinib in bone and soft tissue sarcomas. Cancer Investigation

ErbB Receptors Sarcomas Gefitinib Gefitinib is an orally active synthetic quinazoline that reversibly and selectively targets ErbB1 and blocks its downstream signaling pathways. A phase I trial of single-agent Gefitinib was initiated in 25 children with recurrent refractory solid tumors, including Ewing sarcoma, osteosarcoma, and synovial sarcoma (97). The conclusion of this trial showed that oral Gefitinib is well tolerated in children, and a combination with cytotoxic chemotherapeutic agents should be explored. Gefinitib enhances the bioavailability of Irinotecan, and no adverse neurologic events were observed in 29 pediatric patients with refractory solid tumors (98). Gefinitib was evaluated in combination with the VEGFR signal inhibitor Cediranib in a phase I trial in patients with advanced solid tumors and demonstrated favorable response in one osteosarcoma patient (99). Additionally, a phase I trial showed Gefitinib did not induce any consistent variation in ErbB1, VEGFR, MMP-2, and MMP-9 blood levels in pediatric patients with solid tumors (100). Nevertheless, one phase II study of Gefitinib had bad results, in which the best response was observed only in 10/46 patients with advanced ErbB1expressing synovial sarcoma (101). Erlotinib Erlotinib is a potent, reversible, and selective inhibitor of the ErbB1 tyrosine kinase, which blocks cell cycle progression in G1 phase. Preclinical and clinical trials suggest that combining Erlotinib with active chemotherapeutic agents leads to incremental improvements in outcome. A phase I and pharmacokinetic study had been completed with few adverse effects for Erlotinib, in combination with Temozolomize in children with refractory solid tumors including osteosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas (102). Lapatinib Lapatinib is a reversible and specific receptor tyrosine kinase inhibitor for both ErbB1 and ErbB2. This inhibition blocks downstream MAPK and PI3K/AKT proliferation and survival signaling pathways both in vitro and in vivo. In human osteosarcoma, Lapatinib alters the malignant phenotype of cells via downregulation of the activity of the ErbB2-PI3K/AKT-FASN axis in vitro (103). A phase II trial on Lapatinib showed a modest antitumor activity in ErbB1-positive advance chordoma patients. Major toxic effects included: anemia, fatigue, rash, hypertension, and thromboembolism [104]. Canertinib Canertinib is an orally available pan-erbB tyrosine kinase inhibitor that, unlike the majority of receptor inhibitor, effectively blocks signal transduction through all four members of the ErbB family. In addition, it covalently binds to these receptors, irreversibility inhibiting them, and thereby provides for prolonged suppression of erbB receptor-mediated signaling. Canertinib can abrogate ErbB receptor phosphorylation and cause osteosarcoma cell growth inhibition and apoptosis (46). C 2014 Informa Healthcare USA, Inc. Copyright 

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CONCLUSIONS AND FUTURE DIRECTIONS In recent decades, ErbB receptors have emerged as key players in the establishment of malignancy, and many basic and clinical trials have indicated that these molecules should have considerable promise in the treatment of various types of tumors. Identification of the molecular markers, which can predict patients more likely to respond to anti-ErbB therapy will help individualize cancer treatment and reduce the associated costs. Nevertheless, the roles played by ErbB receptors in bone and soft tissue sarcomas remain elusive with little consensus opinion achieved. All of the published studies so far are single-institution, retrospective studies, limited in size and are not powered adequately. The controversies of any correlation between the expression of ErbB protein and clinical outcomes have limited the clinical application of anti-ErbB agents. Additionally, a general problem in research on rare tumors like osteosarcoma is the limited number of cases included in these phase I, II, and III clinical trials with anti-ErbB agents. Statistical analysis and the impact of these agents always have to be interpreted with caution. In the future, one of the key goals will be the development of accurate predictors of response to ErbB-targeted therapy. The identification of prognostic factors and the evaluation of anti-ErbB agents in these rare tumors will require the establishment of multiinstitutional and international collaborations. ACKNOWLEDGMENTS This work was supported by NSFC (81202115), the Key Project of Basic Research of Shanghai (11JC1410101), the Shanghai Pujiang Program (12PJ1407100), and the Excellent Young Talent program of Shanghai Municipal Commission of Health and Family Planning (XYQ2013108). DECLARATION OF INTEREST The author(s) indicated no potential conflicts of interest including employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/ registrations, and grants or other funding. The authors alone are responsible for the content and writing of the paper. REFERENCES 1. Hynes NE, Lane HA. ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer 2005;5:341–354. 2. Hynes NE, MacDonald G. ErbB receptors and signaling pathways in cancer. Curr Opin Cell Biol 2009;21:177–184. 3. Tebbutt N, Pedersen MW, Johns TG. Targeting the ERBB family in cancer: couples therapy. Nat Rev Cancer 2013;13:663–673. 4. Verma S, Miles D, Gianni L, Krop IE, Welslau M, Baselga J, Pegram M, Oh DY, Dieras V, Guardino E, Fang L, Lu MW, Olsen S, Blackwell K. Trastuzumab emtansine for HER2-positive advanced breast cancer. N Engl J Med 2012;367:1783–1791. 5. Kim ES, Hirsh V, Mok T, Socinski MA, Gervais R, Wu YL, Li LY, Watkins CL, Sellers MV, Lowe ES, Sun Y, Liao ML, Osterlind K, Reck M, Armour AA, Shepherd FA, Lippman SM, Douillard JY. Gefitinib versus Docetaxel in previously treated non-small-cell lung cancer (INTEREST): a randomised phase III trial. Lancet 2008;372:1809–1818.

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