ORIGINAL ARTICLE - ENDOCRINE TUMORS
Analysis of IGF and IGFBP as Prognostic Serum Biomarkers for Adrenocortical Carcinoma
Dhaval Patel, MD1, Ryan Ellis, BS2, Brandi Howard, BS1, Myriem Boufragech, PhD1, Sudheer Kumar Gara, PhD1, Lisa Zhang, PhD1, Martha M. Quezado, MD3, Naris Nilubol, MD1, and Electron Kebebew, MD1
1Endocrine Oncology Branch, National Cancer Institute, NIH, Bethesda, MD; 2Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA; 3Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, MD
ABSTRACT
Background. Adrenocortical carcinoma (ACC) lacks diagnostic and prognostic biomarkers to guide treatment. A consistently dysregulated pathway in ACC is the IGF signaling pathway, specifically overexpression of IGF2, IGF-I-receptor, and IGFBP2. The objective of this study was to perform a comprehensive analysis of serum IGF and IGFBP levels and to determine their utility as diagnostic and prognostic biomarkers in ACC.
Methods. Preoperative serum samples from 53 patients who underwent surgery for adrenocortical adenomas, 3 patients who underwent initial surgery for ACC, 16 patients who underwent reoperative surgery for ACC, and 5 healthy volunteer controls were analyzed. The serum concentration of IGF1, IGF2, IGFBP1, IGFBP2, and IG- FBP3 was determined by enzyme-linked immunosorbent assay.
Results. No difference in the levels of IGF2 (p = . 231) and IGFBP2 (p = . 511) was observed between patients with ACC, benign adrenocortical tumors, and healthy volunteers. IGF1, IGFBP1, and IGFBP3 levels were not detected. High IGFBP2 levels were associated with better overall survival (OS) (p = . 001) and showed a trend toward better abdomi- nal progression-free (APFS) survival (p = . 070) in patients with ACC. A subanalysis of patients undergoing reoperation for recurrent ACC showed better OS with high levels of IGFBP2 (p = . 003) and a trend toward better APFS (p = . 107). There was no significant difference in IGF2 and IGFBP2 levels by extent of disease.
Conclusions. IGF2 and IGFBP2 are not elevated in the serum of patients with ACC compared with patients with benign neoplasms and healthy volunteers. Elevated serum IGFBP2 is associated with better survival in patients with ACC and those undergoing reoperative surgery for recurrent ACC.
Adrenocortical carcinoma (ACC) is a rare malignancy with an estimated incidence of 0.7 to 2.0 cases per million people per year.1 Each year in the United States, 0.2 % of all cancer deaths are related to ACC, and the prognosis continues to be dismal as evidenced by the 5-year survival of 32-45 %.2,3 Surgical resection is the mainstay of treatment, followed by adjuvant mitotane, with combination chemotherapy with etoposide, doxoru- bicin, and cisplatin reserved for patients with recurrence or metastasis. However, we have no curative treatment for locally advanced or metastatic ACC, and survival continues to be poor for this rare malignancy.1,4 The poor prognosis and dearth of therapeutic options has led investigators to search for novel pharmacologic targets, and prognostic biomarkers, that identify patients who should receive adjuvant therapy to reduce their risk of recurrent disease and determine who would benefit from reoperative surgery for recurrent ACC. With the dis- covery of the association of overexpression of the IGF2 gene in the majority of sporadic ACC and Beckwith- Wiedemann Syndrome (BWS), several investigators have focused on the role of the insulinlike growth factor (IGF) system in ACC to further define its effects on tumori- genesis, potential therapeutic utility for targeted therapy, and as a potential biomarker.5-9
The IGF family comprises IGF1, IGF2, and 6 insulin- like growth factor binding proteins (IGFBP1-6). 10-12 IGF1 and IGF2 are short polypeptides that regulate cell prolif- eration, differentiation, and apoptosis.13 Circulating levels
@ Society of Surgical Oncology 2014
D. Patel, MD e-mail: divot999@gmail.com
of IGF1 have been previously associated with an increased risk of breast, prostate, colon, and lung cancer.14 IGF2 has been implicated in cancer initiation and progression. 13,15 Previous in vitro experimental work by Logie and col- leagues showed IGF2 to act in an autocrine manner causing enhanced cell proliferation in the ACC H295R cell line.16 Boulle and colleagues further demonstrated that increased levels of tissue IGF2 are associated with ACC.9
IGF1 and IGF2 mediate their autocrine and paracrine effects primarily through activation of the insulin-like growth factor receptor 1 (IGF1R).17 The activation of IGF1R, a tyrosine kinase receptor, results in increased cellular proliferation, dedifferentiation, and cell survival.18 The IGFBPs serve in a counter-regulatory role by binding IGF1 and IGF2, which effectively reduces circulating IGF1 and IGF2 levels and IGF1R activation.19 Overexpression of tissue IGFBP2 has been previously shown in patients with ACC as well as elevated circulating levels of IGFBP2 in patients with metastatic ACC, indicating that serum levels of IGFBP2 could potentially be a diagnostic and prognostic biomarker in ACC.9,20
The aim of this study was to perform a comprehensive analysis of serum IGF and IGFBP levels in patients with adrenocortical tumors and to determine their utility as potential biomarkers for diagnosis, prognosis, and thera- peutic targets for ACC.
MATERIALS AND METHODS
The study was approved by the Office of Human Research Protections, U.S. Department of Health and Human Services, and Institutional Review Board at the National Institutes of Health. All research participants provided written informed consent.
Serum Samples
IGF1, IGF2, IGFBP1, IGFBP2, and IGFBP3 protein levels were measured in serum samples from patients with
adrenocortical tumors and healthy volunteer controls. Fast- ing serum samples from patients with adrenocortical tumors were obtained the day of surgery for initial operation, recurrent abdominal disease, or metastasectomy. Samples were stored at -80 ℃. Demographic, clinical, and patho- logic information and tissue samples were collected under an Institutional Review Board (IRB) approved protocol. Clin- ical characteristics of the study cohort are summarized in Table 1. Tumors were classified as ACC if the Weiss criteria was ≥3 and/or evidence of tumor recurrence or distant metastasis (Table 2). Tumors were classified as benign if the Weiss criteria was <3 and there was no evidence of recur- rence or metastasis during follow-up.
Enzyme-Linked Immunosorbent Assay (ELISA)
Serum levels of IGF1, IGF2, IGFBP1, IGFBP2, and IGFBP3 were measured with a commercially available ELISA kit (Cusabio; Wuhan, Hubei Province, China) fol- lowing the manufacturer instructions. All samples were measured in duplicate with a standard curve for each of the protein levels tested. The lowest detection level for IGF1 was 7.8 ng/ml; IGF2 was 62.5 pg/ml; IGFBP1 was 3.12 ng/ml; IGFBP2 was 15.6 pg/ml; and IGFBP3 was 0.78 ng/ml.
Statistical Analysis
A One-way ANOVA and the Mann-Whitney U test were used to compare protein levels between groups. The primary outcome variables analyzed were abdominal pro- gression-free survival (APFS) and overall survival (OS). APFS was defined as time to abdominal recurrence post- operatively for those patients who underwent repeated abdominal surgery (16 of 19 patients had stable pulmonary metastasis at the time of surgery). OS was defined as time from preoperative blood samples drawn to disease-related mortality or last follow-up, which was censored for those patients who underwent repeated abdominal surgery. The association between IGF2 and IGFBP2 protein level and
| Adrenocortical carcinoma | Benign adrenocortical tumor | Healthy controls | |
|---|---|---|---|
| No. patients | 19 | 53 | 5 |
| Age (average ± SD) | 51.5 ± 10.0 | 47.4 ± 18.2 | 52.4 ± 13.1 |
| Sex (female/male) | 11/8 | 32/21 | 3/2 |
| Syndromeª | |||
| Cushing's | 5 | 27 | – |
| Subclinical Cushing's | 0 | 2 | – |
| Conn's | 0 | 21 | – |
| Nonfunctioning | 14 | 3 | – |
a Functional status at initial presentation
| Patient ID | Age at diagnosis | Age at operation | Sex | Operation No. | TNMb | Ki67 (%) | Weiss score | Distant mets | Chemotherapyd |
|---|---|---|---|---|---|---|---|---|---|
| 1 | 59 | 69 | F | 2 | – | >20 | – | Yes | EDP + mitotane |
| 2 | 44 | 48 | F | 2 | – | >20 | – | Yes | EDP + mitotane |
| 3 | 59 | 65 | M | 2 | – | >20 | – | Yes | EDP + mitotane |
| 4 | 39 | 55 | F | 2 | – | – | – | Yes | Tariquidar, doxorubicin, vincristine, etoposide + mitotane |
| 5 | 49 | 53 | M | 2 | – | >20 | – | Yes | Streptozocin + mitotane |
| 6 | 32 | 35 | F | 2 | – | >20 | – | Yes | None |
| 7 | 49 | 52 | F | 1 | IV | <5 | 5/9 | Yes | Cisplatin |
| 8 | 52 | 55 | F | 2 | – | >20 | – | Yes | IMC-A12 + mitotane |
| 9 | 49 | 51 | M | 2 | – | e – | – | No | None |
| 10 | 68 | 73 | F | 3 | – | >20 | – | No | Mitotane |
| 11 | 34 | 38 | M | 2 | – | >20 | – | Yes | Mitotane |
| 12 | 63 | 70 | F | – | – | >20 | – | Yes | Tariquidar, doxorubicin, vincristine, etoposide + mitotane |
| 13 | 58 | 62 | F | – | – | >20 | – | Yes | OSI-906, EDP, streptozocin + mitotane |
| 14 | 57 | 62 | F | 3 | – | >20 | – | No | Mitotane |
| 15 | 55 | 57 | M | 2 | – | >20 | – | Yes | EDP + mitotane |
| 16 | 52 | 57 | F | 2 | – | >20 | – | Yes | Mitotane |
| 17 | 39 | 41 | M | 1 | IV | – | – | Yes | EDP, cetuximab + mitotane |
| 18 | 50 | 54 | M | 1 | IV | <5 | 5/9 | Yes | AT-101 + mitotane |
| 19 | 63 | 64 | M | 2 | – | >20 | – | Yes | Mitotane |
a Number of abdominal operations
b TNM staging at initial operation (if the initial operation was performed at the NIH)
” Distant metastatic disease at presentation to the NIH and time of the operation
d Chemotherapy prior to operation (including both neoadjuvant and adjuvant)
e Information not available
f Patient underwent pulmonary metastasectomy
survival was assessed using the Kaplan-Meier method with statistical differences determined by log-rank test. The relationship between IGF2 and IGFBP2 was assessed using a Spearman correlation. A p value of < . 05 was considered statistically significant. All calculations were performed using GraphPad Software 5.0 (La Jolla, CA, USA).
RESULTS
IGF2 and IGFBP2 protein levels were detected in the serum of patients with adrenocortical tumors and healthy volunteer controls. IGF2 (p = . 231) and IGFBP2 (p = . 511) levels in serum were not significantly different between patients with ACC (combining patients with initial and reoperative surgery), benign adrenocortical tumors, and healthy control volunteers (Fig. 1). There was no sig- nificant difference in IGF2 (p = . 135) and IGFBP2 (p = . 808) levels in patients with benign adrenocortical tumors and malignant adrenocortical tumors when healthy control volunteers were excluded. IGF1, IGFBP1, and
IGFBP3 protein levels were not detectable in any of the serum samples from patients with adrenocortical tumors and healthy volunteer controls at lower limit of assay detection (7.8, 3.12, and 0.78 ng/ml for IGF1, IGFBP1, and IGFBP3, respectively).
Elevated serum levels of IGFBP2 were associated with improved OS (p = . 001, median follow-up, 423 days) and showed a trend toward better APFS (p = . 070, median time to recurrence, 194 days) in patients with ACC when these patients underwent either initial or reoperative abdominal surgery (Fig. 2). A subanalysis of patients undergoing reoperative abdominal surgery for recurrent ACC disease showed high serum levels of IGFBP2 were significantly associated with improved OS (p = . 003, median follow-up, 475 days) and showed a trend toward better APFS (p = . 107, median time to recurrence, 195 days) in patients with recurrent ACC (Fig. 3). There was no significant difference in OS or APFS based on levels of IGF2. There was no significant association in IGF2 and IGFBP2 protein levels by tumor burden and
A
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Benign (n=53)
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Survival Stratified by Median IGFBP2
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Progression Free Interval by IGFBP2
100
100
- High IGFBP2 (n=9)
- High IGFBP2 (n=9)
-Low IGFBP2 (n=8)
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Survival Stratified by Median IGFBP2
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Progression Free Interval by IGFBP2
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100
- High IGFBP2 (n=8)
- High IGFBP2 (n=8)
-Low IGFBP2 (n=6)
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-Low IGFBP2 (n=6)
80
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Percent survival
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20
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standard uptake value (SUV) of recurrent abdominal dis- ease. There was no difference in OS or APFS when patients were stratified by a history of chemotherapy.
DISCUSSION
In this study we show IGF2 and IGFBP2 serum levels were not significantly different between patients with benign and malignant adrenocortical tumors, and the other IGF and IGFBPs were undetectable. A higher level of circulating IGFBP2, however, was associated with improved OS and a trend toward better APFS in patients who underwent initial and/or reoperative abdominal sur- gery for ACC. Furthermore, a subanalysis of the data showed that elevated IGFBP2 levels in patients undergoing reoperative surgery for recurrent ACC was associated with improved OS and a trend toward better APFS.
Previous studies have shown that IGF2 is overexpressed in sporadic ACC, that overexpression leads to proliferation in vitro, and that blocking IGF1R with antibodies leads to decreased growth.5,6,16,21 Gao and colleagues showed the antibody, MEDI-573, targeting both IGF1 and IGF2, has a strong effect on decreasing tumor growth in vitro and in vivo.22 Thus, targeting the IGF system could be effective in ACC given the overexpression of IGF2 in most tumors and the expected elevated circulating levels of IGF2. However, our data do not demonstrate that IGF2 is uni- formly elevated in patients with ACC, indicating that nonselective use of this pharmacologic strategy may explain the lack of significant activity of targeting IGFR1 with monoclonal antibody, especially in patients with recurrent ACC.23-25 With no difference in circulating IGF2 levels between patients with malignant and benign adre- nocortical tumors, IGF2 neutralizing antibodies are unlikely to have uniform benefit in all patients.
The levels of IGFBP2 in patients with malignant, benign adrenocortical tumors, and in healthy volunteers were not significantly different in this study. Previously, Boulle and colleagues had shown plasma levels of IGFBP2 to be higher in patients with metastatic ACC compared with patients with localized ACC and healthy volunteers.20 Our study did not confirm this finding of high IGFBP2 levels in the serum of patients with metastatic ACC compared with patients with localized ACC and healthy volunteers. We believe this discrepancy may be explained by differences in cohort selection between the 2 studies. The study by Boulle and colleagues compared patients with localized ACC undergoing their initial operation compared with a majority of patients undergoing reoperation for metastatic ACC. In this study, the majority of patients were undergoing reop- erative surgery and had metastatic disease at the time of presentation. All 3 of the patients with localized disease in
this cohort were undergoing reoperative surgery, which may be an indication of biologically aggressive disease.
Furthermore, the Boulle study included patients actively receiving mitotane or mitotane and etoposide, doxorubicin, and cisplatin (EDP) chemotherapy, while the present cohort mostly comprised patients with a history of che- motherapy not currently receiving chemotherapy. The administration of chemotherapy is a possible confounder in both studies, as chemotherapy may lead to varying levels of IGFBP2. An association between chemotherapeutic resistance and expression levels of IGFBP2 was recently shown in patients with acute myeloid leukemia and thus could be a confounder in our data.26 However, given the known intracellular elevation of IGFBP2 in ACC, the previous observation of elevated IGFBP2 in the serum may also be due to higher tumor cell death in the setting of concurrent chemotherapy.9 Since the IGF system is com- plex, further studies will need to be undertaken to elucidate how chemotherapy affects the IGF and IGFBP serum and tumor levels in patients with ACC. IGFBP2 may still serve as a valuable prognostic marker, especially when consid- ering reoperative surgery.
The current management for recurrent ACC is limited. The prognosis of ACC is heavily based on initial surgical resection and the tumor biology of each individual patient. The management of recurrence is based on clinical judgement.27 A recent study by Erdogan and colleagues showed that prognostic factors for reoperative surgery included time to first recurrence and R0 resection.28 Dy and colleagues29 performed a retrospective review of patients undergoing reoperation for recurrent ACC and also found that a disease-free interval of greater than 6 months and R0 resection to be associated with improved survival. This study has identified IGFBP2 as a potential biomarker in guiding surgical decision making in recurrent ACC. Kap- lan-Meier analysis revealed that an elevated level of IGFBP2 was associated with a better OS and a trend toward a better APFS in patients undergoing reoperation for recurrent ACC. The APFS was not statistically signif- icant, and this may be explained by the clinical timing of image acquisition and the fact that progression-free sur- vival is an imperfect surrogate marker of OS.30 We also used APFS and some patients with ACC-related mortality died of heavy burden of disease in the lung and bones. Nonetheless, our data suggest that patients with high levels of IGFBP2 may benefit most from reoperative abdominal surgery for recurrent ACC.
Although this current study only assessed IGF2 and IG- FBP2 in the serum, there may be implications to the current research of IGF system as a potential chemotherapeutic target. Currently, the clinical management of ACC relies on surgical resection, with or without adjuvant mitotane, and chemotherapy for locally advanced and metastatic
ACC.31,32 Given the dismal prognosis of patients with ACC and evidence of IGF2 overexpression, substantial research resources have been focused on modulation of the IGF pathways for ACC treatment.5,6 IGF1 and IGF2 both acti- vate IGF1R, which results in downstream activation of the MAPK and AKT pathways leading to cellular proliferation and survival.33 The lack of difference of serum IGF2 levels implies that therapies targeting the IGF system may ulti- mately not be clinically beneficial. One of the limitations of this implication, however, is that ACC has been reported to overexpress IGF1R. Weber and colleagues showed 3 of 4 ACC tissue samples overexpress IGF1R.17 IGF1R overex- pression may indicate that serum IGF2 levels may not be significant to observable clinical response with monoclonal antibodies such as OSI-906, an IGF1R antagonist. Patients may benefit from tissue biopsy to determine IGF1R expression levels to determine if they will receive a response to therapy targeting the IGF system. The current results may dampen the enthusiasm for treating patients by targeting the IGF system, unless further study is done on the overex- pression of IGF1R. However, the authors are equally hopeful to be proven wrong and await the results of the current multicenter randomized double-blinded phase 3 trial evaluating OSI-906, a small molecule inhibitor of IGF1R.
There are a few limitations to the current study. The IGF system is extremely complicated, with 14 different proteins interacting and competing to bind with IGF1 and IGF2.34 The measurement and the effects of IGF2 are complicated by the multiple interactions of IGF2 has with the 6 secreted IGFBPs. These interactions complicate any interpretation of serum IGF and IGFBP. Since IGF1R also can be alterna- tively spliced, form a hybrid receptor with HER2, and insulin receptor A or B, interpretation of associations of IGF2 levels and IGF1R may be too simplistic, but none of the other IGFBPs measured were detectable in our study.34,35 The lack of difference in IGF2 and IGFBP2 in patients with benign and malignant tumors may not reflect the tumor microenvironment interaction or levels of these proteins in the tumor. Similarly, the lack of difference may not reflect tissue levels, specifically of IGFBP2. This data is very important as serum levels of IGFBP2 may be inde- pendent of tumor levels and derive from other tissues. Although IGFBP2 levels may be independent of tumor tis- sue, the serum levels may still potentially influence overall survival by blocking the effects of IGF signaling pathway. Therefore, future studies assessing and correlating the serum and tissue levels of IGFBP2 would be beneficial. Since the cohort analyzed consisted of patients undergoing surgical treatment at different time points in the course of their ill- ness, correlations with tumor stage, Weiss score, and tumor size were not feasible. Furthermore, the duration and type of chemotherapy received by each patient was not uniform, which could be a potential confounder.
In summary, we found no significant difference between IGF2 and IGFBP2 levels in patients with ACC and benign adrenocortical tumors. Elevated serum IGFBP2 was a prognostic marker for better OS and showed a trend toward better APFS in patients with ACC. Furthermore, our data indicate that targeting IGF2 with neutralizing antibodies will likely not be effective, and targeting IGF1R should be undertaken after a tissue sample confirms overexpression of the IGF1R receptor with elevated levels of IGF2.
ACKNOWLEDGMENTS We thank the staff and patients at the NIH for providing the serum samples used in these studies. We are also very thankful to Tito Fojo, MD, at the NIH, for referring his patients to the Endocrine Oncology Branch for surgery. This research was sup- ported by the intramural research program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health.
DISCLOSURE The authors have nothing to disclose.
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