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Gemcitabine-Based Chemotherapy In Adrenocortical Carcinoma: A Multicentric Study On Efficacy and Predictive Factors.
Judith E.K. Henning, Timo Deutschbein, Barbara Altieri, Sonja Steinhauer, Stefan Kircher, Silviu Sbiera, Vanessa Wild, Wiebke Schlötelburg, Matthias Kroiss, Paola Perotti, Andreas Rosenwald, Alfredo Berruti, Martin Fassnacht, Cristina L. Ronchi
The Journal of Clinical Endocrinology & Metabolism Endocrine Society
Submitted: July 20, 2017
Accepted: September 14, 2017
First Online: September 19, 2017
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Gemcitabine in adrenocortical carcinoma
Gemcitabine-Based Chemotherapy In Adrenocortical Carcinoma: A Multicentric Study On Efficacy and Predictive Factors.
Judith E.K. Henning1, Timo Deutschbein1, Barbara Altieri1,2, Sonja Steinhauer1, Stefan Kircher3,4, Silviu Sbiera1, Vanessa Wild3, Wiebke Schlötelburg5, Matthias Kroiss4, Paola Perotti6, Andreas Rosenwald3,4, Alfredo Berruti7, Martin Fassnacht1,4, Cristina L. Ronchi1
1Division of Endocrinology and Diabetes, Dpt. of Internal Medicine I, University Hospital, University of Wuerzburg, Germany. 2Division of Endocrinology and Metabolic Diseases, Catholic University of the Sacred Heart, Rome, Italy. 3 Institute of Pathology, University of Wuerzburg, Germany. 4Comprehensive Cancer Center Mainfranken, University of Wuerzburg, Germany. 3Institute for diagnostic and interventional Radiology, University Hospital of Wuerzburg, Germany. Division of Internal Medicine I, University of Turin, San Luigi Hospital, Turin, Italy.
Medical Oncology, Dpt. of Medical and Surgical Specialties, Radiological Sciences, and Public Health, University of Brescia, Spedali Civili Hospital, Brescia, Italy
Received 20 July 2017. Accepted 14 September 2017.
Context: Adrenocortical carcinoma (ACC) is rare and confers an unfavorable prognosis in advanced stages. Beyond combination chemotherapy with cisplatin, etoposide, doxorubicin and mitotane, second/third line regimens are not well-established. Gemcitabine (GEM)-based chemotherapy was suggested in a phase 2 clinical trial with 28 patients. In other solid tumors, hENT1 and/or RRM1 expression have been associated with resistance to GEM.
Objective: To assess the efficacy of GEM-based chemotherapy in ACC in a real world setting and the predictive role of molecular parameters.
Design: Retrospective multicentric study.
Setting: Referral centers of university hospitals.
Patients and materials: 145 patients with advanced ACC were treated with GEM-based chemotherapy (132 with concomitant capecitabine). FFPE tumor material was available for 70 patients for immunohistochemistry.
Outcome measures: Main outcome measures were progression-free survival (PFS) and objective response to GEM-based chemotherapy. Secondary objective was the predictive role of hENT1 and RRM1.
Results: Median PFS in the patient population was 12 weeks (range: 1-94). Partial response or stable disease was achieved in 4.9 and 25.0% of cases with a median duration of 26.8 weeks. Treatment was generally well tolerated with adverse events grade 3 or 4 occurring in 11.0% of cases. No significant impact of hENT1 and/or RRM1 expression was observed on response to GEM-based chemotherapy.
Conclusions: GEM-based chemotherapy is a well-tolerated, but modestly active regimen in ACC patients with advanced disease. No reliable molecular predictive factors could be identified. Due to scarce alternative therapeutic options, GEM-based chemotherapy remains an important option for salvage treatment in advanced ACC.
We retrospectively evaluated efficacy of gemcitabine-based chemotherapy in 145 patients with adrenocortical carcinoma and elucidated the potential predictive role of hENT1 and RRM1 expression. .
1. Introduction
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Adrenocortical carcinomas (ACC) are rare and highly malignant tumors with a generally poor prognosis. Initial tumor stage is the single most important prognostic factor with overall 5-year survival of less than 15% in presence of metastasis (1-4). Systemic treatments in advanced disease are very limited and usually include mitotane, either alone or in combination with chemotherapy (5, 6). According to the results of the first randomized controlled phase III clinical trial (FIRM-ACT), combination chemotherapy with etoposide, doxorubicin, cisplatin and mitotane (EDP-M) represents the current first line treatment in advanced ACC (7). In a phase II clinical trial, 28 patients with advanced ACC were treated with gemcitabine (2’,2’- difluorodeoxycytidine; GEM) (8), which has been proposed as second-line option in combination with fluoropyrimidines (capecitabin or 5-fluorouracil) (6). In this pilot study, the rate of non- progressing patients after 4 months of treatment was 46%, with a median time to progression of 5.3 months (including complete and partial responses in one patient each). Since then, GEM- based chemotherapy has found widespread use in view of the absence of established second- /third line treatments. So far, however, the promising findings of the phase II trial have not been confirmed in any other series (9). Moreover, to date no reliable predictive biomarkers are available, impeding the identification of potentially responsive patients.
The predictive role of molecules involved in the metabolism of GEM has already been investigated in various solid tumors. For instance, the human equilibrative nucleoside transporter type 1 (hENT1) mediates cellular entry of nucleosides (10), including some cytotoxic nucleoside analogs, such as cytarabin, fludarabin, and GEM (11, 12). hENT1 deficiency impairs efficacy of nucleotide drugs both in vitro (12) and in vivo, as repeatedly demonstrated in different solid cancer types such as pancreatic cancer (13-15), biliary tract cancer (16, 17), and bladder cancer (18). Another promising biomarker is the subunit of ribonucleotide reductase (RRM1), which catalyzes the rate-limiting step in the production of deoxyribonucleotides. The latter are essential for DNA synthesis and repair, thereby representing another important cellular determinant of GEM-efficacy. Previous studies demonstrated that low RRM1 expression levels are associated with a better response to GEM-containing treatment regimens in different tumor types (19, 20) and to adjuvant treatment with mitotane in ACC (21).
Thus, the major goal of the present retrospective multicentric study was to assess the efficacy and safety of GEM-based chemotherapy in a large series of patients with advanced ACC. Moreover, we investigated the expression of both hENT1 and RRM1 in adrenocortical tissues and their prognostic and predictive role.
2. Material and Methods
The collection of clinical data and biomaterial for this retrospective study was approved by the local ethics committee in accordance with the principles of the Declaration of Helsinki. Written informed consent was obtained from all patients.
A. Patients and Treatment Regimen
Inclusion criteria in the study were: age of at least 18 years, histologic diagnosis of ACC, and treatment with gemcitabine according to the clinical practice. Thus, we identified a total of 155 patients with advanced ACC who had been treated with GEM-based chemotherapy (at least one administration of i.v. gemcitabine) at our referral centers between January 2004 and December 2016 (Germany n=124, Italy n=31) and were not part of the published series (8). Among them, 10 patients were lost to follow-up and were excluded from further analysis. Thus, a final series of 145 patients with advanced ACC were included in the present study. The baseline clinical parameters, such as initial tumor size, tumor stage at the time of diagnosis according to the
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European Network for the Study of Adrenal Tumors (ENSAT) classification (3), hormonal secretion pattern, Weiss score, Ki67 proliferation index, presence and number of distant metastases, and previous treatments are given in Table 1. All data were collected through the ENSAT Registry (www.ens@t.org/registry). Detailed follow-up data during GEM-based treatment were collected from patients’ records. In brief, the standard treatment scheme consisted of 800 mg/m2 i.v. gemcitabine on days 1 and 8, repeatedly administered in 21 day-cycles, as an infusion over 30 min. The median number of GEM-cycles was 4 (range: 1-31). Altogether, 132 patients (91.0% of cases) received a combination with oral capecitabine (1500 mg/daily) (8), 7 a combination with oral erlotinib (100 mg/daily, (9)), and 4 a combination with other drugs (i.e. vinorelbin, carmustin or 5-Fluorouracil). Moreover, 114 patients (78.6% of cases) were concomitantly treated with mitotane with a target plasma concentration of 14-20 mg/L. Exact mitotane concentration values were available for 89 patients, of which 42 reached the “target plasma levels” of ≥14 mg/L (47.2%). Only one patient received gemcitabine as monotherapy. Finally, GEM-based chemotherapy was administered as first-line treatment in 12 patients (8.3%), and as second-line treatment in 83 patients (with former treatment failure of platinum-based chemotherapy or streptozotocin in 81 and 2 cases, respectively). The remaining 50 patients were treated with GEM-based chemotherapy as third- to fifth-line therapy, with a history of failed platinum-based chemotherapy, streptozotocin, sunitinib (multi-targeted receptor tyrosin kinase) (22), linsitinib (IGFR and IR inhibitor) (23), trofosfamid (24) or thalidomide (Table 1).
B. Evaluation of response and toxicity
All 145 patients underwent standardized follow-up visits with a staging interval usually every 8- 12 weeks. These evaluations included physical examination, biochemical workup (with routine chemistry and steroid hormones), and multislice imaging (usually contrast-enhanced computed tomography (CT)) of chest and abdomen. Treatment was discontinued in case of unacceptable toxicity, patients’ refusal, or evidence of disease progression.
Efficacy of GEM-based chemotherapy was retrospectively assessed by progression-free survival (PFS, for definition see below) and best overall objective response. For this evaluation, according to our clinical practice, all radiological images were reviewed by the local expert radiologists and discussed in our multidisciplinary tumor board meetings to determine a final consensus response (progressive disease, stable disease, partial or complete response). Clinical benefit was defined as stable disease or treatment response for a minimum of 4 months.
Potential treatment related adverse effects were regularly registered during the follow up visits and retrospectively summarized from the patients’ medical records. Toxicities were then graded using the Common Terminology Criteria for Adverse Events (CTCAE), version 4.03.
C. Formalin-Fixed Paraffin Embedded Tissue Samples and Immunohistochemistry
The series of formalin-fixed paraffin embedded (FFPE) specimens included 303 ACC samples (52 standard slides and 251 assembled into seven tissue microarrays) from 262 patients, 51 adrenocortical adenomas and 18 normal adrenal glands (including 3 adrenal hyperplasia) (Supplementary Table 1). 87 of the ACC samples were from 70 patients out of the 145 treated with GEM-based chemotherapy. In 17 cases we had available FFPE material from subsequent surgeries in the same patients (primary tumors and local recurrences and/or distant metastasis). They showed baseline clinical characteristics (median age: 43 years; median tumor size: 12.5 cm; median Weiss score: 6; median proliferation index ki67: 20%) similar to the other 75 patients without available FFPE material. The remaining 216 FFPE tumor specimens came from 192 patients (117F&75M, median age at diagnosis: 50 years, range: 17-81) who had not been treated with GEM-based chemotherapy (Supplementary Table 1). In this series of patients, 88
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had an ENSAT tumor stage of 1-2 (48.9% of known), 53 of 3 (29.4%) and 38 of 4 (21.1%), the median Weiss score was 5 (range: 2-9) and the median ki67 was 10 (range: 1-80). The entire series was used to investigate the hENT1 and RRM1 expression by immunohistochemistry.
The TMA and the full sections were deparaffinized and immunohistochemical detection was performed, using an indirect immunoperoxidase technique after high temperature antigen retrieval in 10 mM citric acid monohydrate buffer (pH 6.5) in a pressure cooker for 13 min. Blocking of unspecific protein-antibody interactions was performed with 20% human AB serum in PBS for 1h at room temperature. Primary antibody for hENT1 was a polyclonal anti-rabbit antibody against SLC29A1 (Sigma Aldrich Cat# HPA012383, epitope PrEST, dilution 1:30). Primary antibody for RRM1 was a polyclonal anti-rabbit antibody (AbCam Cat# ab81085, epitope C-terminal; dilution 1:100). As negative control, the N-Universal Negative Control Anti- Rabbit (Dako Cat# IS600) was used. Signal amplification was achieved by En-Vision Advance System Labeled Polymer-HRP Anti-Rabbit (Dako) for 40 min and developed for 10 min with DAB Substrate Kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. Nuclei were counterstained with Mayer’s hematoxilin for 2 min. For positive controls, sections of colon and testis cancer (for hENT1) or hepatocellular and prostate cancer (for RRM1) were chosen, while cells of the tumor stroma served as internal negative control.
All slides were analyzed independently by two investigators blinded to clinical information (J.E.K.H. and C.L.R.), with evaluation of membranous staining for hENT1 and of cytoplasmic staining for RRM1. Membranous hENT1 staining intensity was classified as negative (0) or positive (1). Staining RRM1 intensity in the cytoplasm was graded as negative (0), low (1), medium (2), or strong (3). The percentage of positive tumor cells was calculated for each specimen and scored 0 if 0% were positive, 0.1 if 1-19% were positive, 0.5 if 20-49% were positive and 1 if 50% or more were positive. A semi-quantitative H-score was then calculated by multiplying the staining intensity grading score with the proportion score as described previously (25). In case of discrepant results, staining intensities were jointly assessed by both investigators, forming the final score by consensus. Inter-observer agreement was investigated via Pearson’s correlation coefficients: 0.66 (95%CI 0.59-0.71) for hENT1 and 0.62 (95%CI 0.56-0.68) for RRM1. Both hENT1 and RRM1 were homogeneously distributed with a percentage of positive cells higher than 50% in 86 and 76% of samples, respectively.
D. Statistical analysis
The Fisher’s exact or the Chi-square tests were used to investigate dichotomic variables, while continuous variables were investigated with a two-sided t test or non-parametric test. A non- parametric Kruskal-Wallis test, followed by Dunn’s test, was used for comparison among several groups for non-normal distributed variables. Correlations and 95% confidence intervals (95%CI) between different parameters were evaluated by linear regression analysis.
PFS was defined as the time from the date primary tumor resection or start of GEM-therapy (as indicated) to the first radiological evidence of disease progression or death. Similarly, disease-specific survival was defined as the time from the date of primary surgery or start of GEM-therapy (as indicated) to disease-specific death or last follow-up. Survival curves were obtained with Kaplan-Meier estimates and the differences between survival curves were assessed by the log-rank (Mantel-Cox) test. For the calculation of hazard ratios (HR), two ACC-groups with negative/low or positive/high hENT1 and RRM1 protein expression, respectively, were considered. A multivariate regression analysis was performed via a Cox proportional hazard regression model, aiming to identify factors that might independently influence survival.
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Statistical analyses were made using GraphPad Prism (version 6.0, La Jolla, CA, USA) and SPSS Software (PASW Version 21.0, SPSS Inc., Chicago, IL, USA). P values <0.05 were considered as statistically significant.
3. Results
A. Efficacy and tolerability of GEM-based chemotherapy
Detailed characteristics of the 145 patient’s cohort (F:M=85:60, mean+SD age at diagnosis: 45.4±13.4 years) are given in Table 1. The median PFS was 12 weeks (90 days), ranging from 1 to 94 weeks (7-658 days) (Figure 1A). 116 patients died during the study or follow-up and all but one died from progressive ACC. The median disease-specific survival after initiation of GEM-treatment was 40 weeks (range 1-280) (Figure 1A). Four patients did not receive the second cycle of GEM due to death from ACC unrelated to treatment after 7 to 21 days within the first cycle (n=3) or due to clinically evident disease progress confirmed by premature imaging (n=1).
Considering the best objective response to therapy (n=144, Table 1), 7 patients showed a partial response (4.9%) and 36 patients had stable disease (25.0%) with a median duration of 26.8 weeks (range: 4-94) (Figure 1B). The remaining 102 patients (70.8%) experienced progressive disease. In total, 30 patients (20.8%) experienced a clinical benefit from treatment for at least 4 months, with a median duration of 33.6 weeks (235 days), ranging from 17-94 weeks (120-658 days). All these 30 patients had concomitant capecitabine and 27/30 had concomitant mitotane.
No significant differences were observed between patients being treated with GEM-based chemotherapy as first-/second-line treatment or in a later line of treatment (P=0.55). However, patients with concomitant mitotane that reached the target concentration of above 14 mg/L (n=42) had a longer PFS than those that did not (n=47) or that did not receive mitotane (n=21) (HR for progress=0.56, 95%CI=0.35-0.77, P=0.0026) (Supplementary Figure 1A). Similar results were obtained in terms of objective response (Supplementary Figure 1B) Finally, a longer PFS during treatment was observed in patients receiving capecitabine in combination with gemcitabine in comparison with the other ones (HR for progress=0.55, 95%CI=0.19-0.84, P=0.026).
Data on toxicity were available for 109/145 patients (75.2%). In general, gemcitabine was well tolerated with severe adverse events (CTCAE grade 3-4) being reported in 11.0% of cases (Supplementary Table 2). The most frequent adverse effects were asthenia (25.7%), edemas (11.0%), nausea or vomiting (10.0%), thoracic or abdominal or generalized pain (10%), fever (7.3%), dyspnea (7.6%), reduced appetite (7.4%), numbness (6.4%), and diarrhea (6.4%). Other rare adverse events included mucositis, hand-foot syndrome, dyspnea, and headache (all below 3%). Hematologic events namely transitory anemia, neutropenia, and thrombocytopenia were observed in 34.8%, 20.2% and 11.9% of cases, respectively. Nine patients needed to discontinue GEM due to adverse events.
No significant differences in terms of drug tolerability were observed between patients treated with or without concomitant capecitabin and/or mitotane.
B. hENT1 and RRM1 as prognostic and predictive factors
Acknowledging the limited number of patients with clinical benefit, we next aimed to identify potential predictive factors in the subgroup of 70 patients with available tumor samples. Compared to the entire group of 145 patients, this subgroup showed a similar treatment regimen and a superimposable PFS during treatment (with a median of 13 weeks (88 days)).
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Representative examples for hENT1 and RRM1 immunostaining in normal and neoplastic adrenocortical FFPE samples are shown in Figure 2. For this evaluation we used FFPE material coming from 65 primary tumors, 3 local recurrences, and 2 distant metastases. Moreover, we had available tissues from multiple surgeries for 17 patients, where we could investigate the hENT1 and RRM1 staining over the time, without observing any relevant differences in the single cases. Despite positive results in other tumor entities, we did not observe any significant relationship between hENT1 staining and the PFS during treatment (HR for progress=0.99, 95%CI=0.57- 1.70, P=0.97, Figure 3A). Similarly, no difference in terms of best objective response to GEM- based therapy was found in patients with positive or negative hENT1 (progressive disease in 33/45 vs 16/25, respectively, P=0.67 by Chi-square test).
Examination of RRM1 revealed that RRM1 expression also did not influence PFS during treatment (HR=0.84, 95%CI=0.46-1.46, P=0.52, Figure 3B). Patients with high RRM1 expression had a similar best objective response to GEM-based therapy in comparison with those with low RRM1 levels (progressive disease in 34/50 vs 16/20, respectively, P=0.43 by Chi- square test). Furthermore, we investigated the association between the combination of hENT1 and RRM1 immunostaining and the PFS during GEM-based therapy without identifying any significant correlation (P=0.99, Figure 3C).
Finally, we analyzed the role of hENT1 and RRM1 in a large cohort of additional 216 ACC samples (total=303 samples from 262 patients) and 51 adenomas and 18 normal adrenal glands (Supplementary Table 1). For details on staining characteristics see Appendix. Interestingly, hENT1m staining was positive in significantly fewer ACC (92/303, 30%) than adenomas (36/51, 71%) or normal adrenal glands (11/18, 61%, P=0.005 by Chi-square test on absolute values, Supplementary Figure 2). Considering all the ACC samples, hENT1 staining was only slightly inferior in tissues coming from distant metastasis (n=30, 88% negative) or local recurrences (n=33, 70% negative) or primary tumors (n=240, 67% negative, P=0.058 by Chi-square test). Such differences were not observed for RRM1 expression. For the correlation analyses between immunostaining and general clinical outcome, only ACC patients with samples from primary tumors were considered (n=240 out of 262). Interestingly, those patients with negative hENT1 staining had a significantly shorter disease-specific survival (median 30 vs 89 months, HR for specific death=1.71, 95%CI=1.18-2.29-0.84, P=0.0033) and PFS after primary surgery (median 8 vs 13 months, HR for progress=1.44, 95%CI=1.08-1.94, P=0.0172) (Figure 4A-B). The impact of hENT1 on disease-specific survival remained significant in a multivariate analysis, including ENSAT tumor stage, resection status, and ki67 proliferation index (HR=0.53, 95%CI=0.33-0.85, P=0.009). On the other hand, no significant relationship between RRM1 staining levels and the general clinical outcome (either disease-specific survival or PFS after primary surgery) was observed.
4. Discussion
The present work not only represents the largest study on GEM-based chemotherapy in patients with advanced ACC (n=145), but also after the FIRM-ACT trial the second largest study on pharmacological treatment in ACC (7, 23). We could clearly demonstrate that this pharmacological approach is moderately active in the clinical practice, with a disease control rate (disease stabilization or response to therapy) of about 30% of patients and a clinical benefit (disease stabilization or response to therapy for at least 4 months) in about 20%. This is actually inferior to the rate of 46% previously described in the prospective phase 2 trial (n=28) (8), but this is not a rare situation in subsequent real-world studies. Furthermore, it seems to be inferior to
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the rates reported for EDP-M schema (disease control in 58.2% of cases), but similar to those shown for streptozotocin-M regimen (disease control in 31.4% of cases) by the FIRM-ACT trial (7). Finally, the response rates of GEM-based therapy result superior to the rates demonstrated for linsitinib (23) (Supplementary Table 3). Even if several influencing factors, such as different staging intervals, the retrospective nature of the present study and the modalities and duration of concomitant mitotane, prevent a direct comparison, we believe that it might be helpful for putting these results in clinical context. Concerning the toxicity, we could show a pattern superimposable to what previously published (8), with severe drug-related adverse events in only 11% of patients, further confirming that GEM-based chemotherapy is generally very well tolerated. This and the fact that promising “targeted” drug alternatives are missing, suggest that GEM-based chemotherapy remains a possible therapeutic option as salvage treatment in advanced ACC. Our data indicate (but cannot prove) that GEM should be combined with capecitabine as this brings a slightly better efficacy without additional toxicity. Furthermore, the sub-analysis on mitotane-cotreated patients suggests that this ACC-specific drug might be also a good combination partner if drug levels above 14mg/l can be achieved.
Moreover, we provide here the first extensive evaluation of hENT1 and RRM1 protein expression in a large series of normal and neoplastic adrenocortical tissues. In particular, we evaluated their prognostic potential for general clinical outcome in ACC and their predictive role for the response to GEM-based chemotherapy. First, we demonstrated that a hENT1 membranous immunostaining, which is the most relevant indicator for protein expression of hENT1 (a nucleoside transporter), is significantly less frequent in ACC than in ACA and normal adrenal glands. Furthermore, we could show that hENT1 staining slightly decreases with the progression of the disease and is significantly associated with prolonged disease-specific and progression-free survival in ACC patients, thus potentially representing a new molecular prognostic factor. These finding are in accordance with previous studies on some other cancer types (15, 26, 27), but in contrast with others (28). Our current results may be of particular interest because the downregulation of hENT1 itself has been shown to be related to an altered epithelial mesenchymal transition (EMT), probably independently of its role as a drug transporter (29, 30). Thus, hENT1 may also serve as an independent prognostic biomarker. Up to now, only a limited number of such molecular markers have been proposed in addition to ki67 proliferation marker (31-35). Accordingly, our current findings may by itself be of particular relevance.
We also separately considered 70 ACC patients who had been treated with GEM-based chemotherapy, thereby aiming to investigate the predictive potential of hENT1/RRM1 expression for the sensitivity to therapy. Here, we did not observe any significant impact between hENT1 or RRM1 staining results and the PFS during treatment or the objective response to therapy. These findings are in contrast with previously reported data for hENT1 in other cancer types, such as pancreatic cancer (13-15), biliary tract cancer (16) or bladder cancer (18). However, other large studies on advanced pancreatic carcinomas also did not detect a relevant role of hENT1 immunostaining for predicting the sensitivity to GEM-based chemotherapy (36, 37). Similarly, RRM1 has been recognized as promising predictive biomarker in some studies (38), but not in all (39, 40). Even a combination of these two biomarkers did not allow us for a reliable prediction of response to GEM-based chemotherapy in ACC, what is in contrast with previously published results for pancreatic cancer (41) and cholangiocarcinoma (42). These discrepancies may be due to several factors, including the different methods used to evaluate the immunostaining (e.g. no standardized scoring protocol and/or differences in the
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antibodies, as already outlined elsewhere (43)) and the variability of molecular and clinical factors that may have influenced both the sensitivity to and the treatment efficacy of GEM-based chemotherapy itself. In fact, other molecular mechanisms, such as the thymidylate synthase (TYMS), the ribonucleotide reductase large subunit 2 (RRM2) or the activating enzyme deoxycytidine kinase (dCK), might be at least theoretically involved in the chemoresistance to GEM and may therefore behave synergistically with hENT1 and/or RRM1 (44, 45). Furthermore, concomitant or parallel treatments (e.g. alternative drugs such as mitotane and/or capecitabine), may have influenced the time to progression during treatment. Another limitation of this analysis is that both molecular markers were assessed mostly on tumor specimens from primary tumors and not on tumor biopsies before GEM-based therapy. However, we believe that our data demonstrate that hENT1 and RRM1 expression either alone or in combination cannot serve as robust predictive biomarkers of GEM-based therapy in ACC.
5. Conclusion
GEM-based chemotherapy is moderately active but generally well-tolerated in ACC patients with advanced disease. Furthermore, no reliable predictive molecular factors are available so far. Thus, we suggest GEM-based chemotherapy, preferentially in association with mitotane, as a salvage treatment of selected patients with advanced ACC.
6. Appendix
hENT1 and RRM1 immunostaining in adrenocortical samples
For hENT1, the staining in the cytoplasma was highly homogenous, as illustrated by a percentage of positive cells >50% in 86% of all samples (median 75%, range 20-100%). For RRM1, cytoplasmatic staining was also relatively homogenous, with a percentage of positive cells >50% in 76% of samples (median 70%, range 15-100%).
7. Acknowledgments
The authors are grateful to Martina Zink for excellent technical support and to Michaela Haaf for coordinating the ENSAT ACC Registry in Wuerzburg.
Corresponding author: Cristina L. Ronchi (MD, PhD), Division of Endocrinology and Diabetes, Department of Internal Medicine I, University Hospital of Wuerzburg, Oberduerrbacher-Str 6, 97080 Wuerzburg (Germany), Tel. 0049-0931-20139704, E-mail: Ronchi C@ukw.de
Funding: This work was supported by grants from the Deutsche Forschungsgemeinschaft DFG (grant numbers KR4371/1-1 to M.K., FA466/4-1 to M.F., and CRC/Transregio 205/1 „The Adrenal: Central Relay in Health and Disease“ to M.K. and M.F.).
Funding
This work was supported by grants from the Deutsche Forschungsgemeinschaft DFG (grant numbers KR4371/1-1 to M.K. and FA466/4-1 to M.F.).
Author contributions
J.E.K. Henning: performed most of the immunostaining, served as first operator for the evaluation of the staining, collected the clinical data for the german center, interpreted the relationship between clinical and molecular data, actively contributed to write the manuscript. T. Deutschbein & M. Kroiss: contributed in the collection and interpretation of the clinical data, as well as in writing the manuscript. B. Altieri: contributed to preparing tables and figures as well
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as in writing the manuscript. S. Steinhauer: contributed to performing the immunostaining. V. Wild, S. Kircher, A. Rosenwald: provided paraffin embedded blocks or slides from adrenocortical tissues. S. Sbiera: contributed to the staining evaluation. A. Berruti & P. Perotti: collected and provided the clinical data for Italian centers. M. Fassnacht & C.L. Ronchi: conceived the design of the study, contributed in the collection and interpretation of the clinical data, C.L.R. additionally served as second operator for the evaluation of the staining, performed the statistical analysis, prepared tables and figures, and wrote the manuscript draft. All authors actively revised the manuscript and approved the final version.
Disclosure Summary. The author reports no conflicts of interest in this work.
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6. Berruti A, Baudin E, Gelderblom H, Haak HR, Porpiglia F, Fassnacht M, Pentheroudakis G, Group EGW 2012 Adrenal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 23 Suppl 7:vii131-138
7. Fassnacht M, Terzolo M, Allolio B, Baudin E, Haak H, Berruti A, Welin S, Schade- Brittinger C, Lacroix A, Jarzab B, Sorbye H, Torpy DJ, Stepan V, Schteingart DE, Arlt W, Kroiss M, Leboulleux S, Sperone P, Sundin A, Hermsen I, Hahner S, Willenberg HS, Tabarin A, Quinkler M, de la Fouchardiere C, Schlumberger M, Mantero F, Weismann D, Beuschlein F, Gelderblom H, Wilmink H, Sender M, Edgerly M, Kenn W, Fojo T, Muller HH, Skogseid B, Group F-AS 2012 Combination chemotherapy in advanced adrenocortical carcinoma. The New England journal of medicine 366:2189-2197
8. Sperone P, Ferrero A, Daffara F, Priola A, Zaggia B, Volante M, Santini D, Vincenzi B, Badalamenti G, Intrivici C, Del Buono S, De Francia S, Kalomirakis E, Ratti R, Angeli A, Dogliotti L, Papotti M, Terzolo M, Berruti A 2010 Gemcitabine plus
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metronomic 5-fluorouracil or capecitabine as a second-/third-line chemotherapy in advanced adrenocortical carcinoma: a multicenter phase II study. Endocrine-related cancer 17:445-453 9. Quinkler M, Hahner S, Wortmann S, Johanssen S, Adam P, Ritter C, Strasburger C, Allolio B, Fassnacht M 2008 Treatment of advanced adrenocortical carcinoma with erlotinib plus gemcitabine. The Journal of clinical endocrinology and metabolism 93:2057-2062
10. Young JD, Yao SY, Sun L, Cass CE, Baldwin SA 2008 Human equilibrative nucleoside transporter (ENT) family of nucleoside and nucleobase transporter proteins. Xenobiotica; the fate of foreign compounds in biological systems 38:995-1021
11. Pastor-Anglada M, Perez-Torras S 2015 Nucleoside transporter proteins as biomarkers of drug responsiveness and drug targets. Frontiers in pharmacology 6:13
12. Mackey JR, Mani RS, Selner M, Mowles D, Young JD, Belt JA, Crawford CR, Cass CE 1998 Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines. Cancer research 58:4349-4357
13. Spratlin J, Sangha R, Glubrecht D, Dabbagh L, Young JD, Dumontet C, Cass C, Lai R, Mackey JR 2004 The absence of human equilibrative nucleoside transporter 1 is associated with reduced survival in patients with gemcitabine-treated pancreas adenocarcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research 10:6956- 6961
14. Farrell JJ, Elsaleh H, Garcia M, Lai R, Ammar A, Regine WF, Abrams R, Benson AB, Macdonald J, Cass CE, Dicker AP, Mackey JR 2009 Human equilibrative nucleoside transporter 1 levels predict response to gemcitabine in patients with pancreatic cancer. Gastroenterology 136:187-195
15. Greenhalf W, Ghaneh P, Neoptolemos JP, Palmer DH, Cox TF, Lamb RF, Garner E, Campbell F, Mackey JR, Costello E, Moore MJ, Valle JW, McDonald AC, Carter R, Tebbutt NC, Goldstein D, Shannon J, Dervenis C, Glimelius B, Deakin M, Charnley RM, Lacaine F, Scarfe AG, Middleton MR, Anthoney A, Halloran CM, Mayerle J, Olah A, Jackson R, Rawcliffe CL, Scarpa A, Bassi C, Buchler MW, European Study Group for Pancreatic C 2014 Pancreatic cancer hENT1 expression and survival from gemcitabine in patients from the ESPAC-3 trial. Journal of the National Cancer Institute 106:djt347
16. Santini D, Schiavon G, Vincenzi B, Cass CE, Vasile E, Manazza AD, Catalano V, Baldi GG, Lai R, Rizzo S, Giacobino A, Chiusa L, Caraglia M, Russo A, Mackey J, Falcone A, Tonini G 2011 Human equilibrative nucleoside transporter 1 (hENT1) levels predict response to gemcitabine in patients with biliary tract cancer (BTC). Curr Cancer Drug Targets 11:123-129 17. Murata A, Amano R, Yamada N, Kimura K, Yashiro M, Nakata B, Hirakawa K 2013 Prognostic predictive values of gemcitabine sensitivity-related gene products for unresectable or recurrent biliary tract cancer treated with gemcitabine alone. World J Surg Oncol 11:117
18. Matsumura N, Nakamura Y, Kohjimoto Y, Inagaki T, Nanpo Y, Yasuoka H, Ohashi Y, Hara I 2011 The prognostic significance of human equilibrative nucleoside transporter 1 expression in patients with metastatic bladder cancer treated with gemcitabine-cisplatin-based combination chemotherapy. BJU Int 108:E110-116
19. Jordheim LP, Seve P, Tredan O, Dumontet C 2011 The ribonucleotide reductase large subunit (RRM1) as a predictive factor in patients with cancer. The Lancet. Oncology 12:693-702
20. Akita H, Zheng Z, Takeda Y, Kim C, Kittaka N, Kobayashi S, Marubashi S, Takemasa I, Nagano H, Dono K, Nakamori S, Monden M, Mori M, Doki Y, Bepler G 2009
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Significance of RRM1 and ERCC1 expression in resectable pancreatic adenocarcinoma. Oncogene 28:2903-2909
21. Volante M, Terzolo M, Fassnacht M, Rapa I, Germano A, Sbiera S, Daffara F, Sperone P, Scagliotti G, Allolio B, Papotti M, Berruti A 2012 Ribonucleotide reductase large subunit (RRM1) gene expression may predict efficacy of adjuvant mitotane in adrenocortical cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 18:3452-3461
22. Kroiss M, Quinkler M, Johanssen S, van Erp NP, Lankheet N, Pollinger A, Laubner K, Strasburger CJ, Hahner S, Muller HH, Allolio B, Fassnacht M 2012 Sunitinib in refractory adrenocortical carcinoma: a phase II, single-arm, open-label trial. The Journal of clinical endocrinology and metabolism 97:3495-3503
23. Fassnacht M, Berruti A, Baudin E, Demeure MJ, Gilbert J, Haak H, Kroiss M, Quinn DI, Hesseltine E, Ronchi CL, Terzolo M, Choueiri TK, Poondru S, Fleege T, Rorig R, Chen J, Stephens AW, Worden F, Hammer GD 2015 Linsitinib (OSI-906) versus placebo for patients with locally advanced or metastatic adrenocortical carcinoma: a double-blind, randomised, phase 3 study. Lancet Oncol 16:426-435
24. Kroiss M, Deutschbein T, Schlotelburg W, Ronchi CL, Neu B, Muller HH, Quinkler M, Hahner S, Heidemeier A, Fassnacht M, German Adrenocortical Carcinoma Study G 2016 Salvage Treatment of Adrenocortical Carcinoma with Trofosfamide. Horm Cancer 7:211-218
25. Ronchi CL, Sbiera S, Kraus L, Wortmann S, Johanssen S, Adam P, Willenberg HS, Hahner S, Allolio B, Fassnacht M 2009 Expression of excision repair cross complementing group 1 and prognosis in adrenocortical carcinoma patients treated with platinum-based chemotherapy. Endocrine-related cancer 16:907-918
26. Kim R, Tan A, Lai KK, Jiang J, Wang Y, Rybicki LA, Liu X 2011 Prognostic roles of human equilibrative transporter 1 (hENT-1) and ribonucleoside reductase subunit M1 (RRM1) in resected pancreatic cancer. Cancer 117:3126-3134
27. Fisher SB, Fisher KE, Patel SH, Lim MG, Kooby DA, El-Rayes BF, Staley CA, 3rd, Adsay NV, Farris AB, 3rd, Maithel SK 2013 Excision repair cross-complementing gene-1, ribonucleotide reductase subunit M1, ribonucleotide reductase subunit M2, and human equilibrative nucleoside transporter-1 expression and prognostic value in biliary tract malignancy. Cancer 119:454-462
28. Santini D, Perrone G, Vincenzi B, Lai R, Cass C, Alloni R, Rabitti C, Antinori A, Vecchio F, Morini S, Magistrelli P, Coppola R, Mackey JR, Tonini G 2008 Human equilibrative nucleoside transporter 1 (hENT1) protein is associated with short survival in resected ampullary cancer. Ann Oncol 19:724-728
29. Guillen-Gomez E, Pinilla-Macua I, Perez-Torras S, Choi DS, Arce Y, Ballarin JA, Pastor-Anglada M, Diaz-Encarnacion MM 2012 New role of the human equilibrative nucleoside transporter 1 (hENT1) in epithelial-to-mesenchymal transition in renal tubular cells. J Cell Physiol 227:1521-1528
30. Lee Y, Koay EJ, Zhang W, Qin L, Kirui DK, Hussain F, Shen H, Ferrari M 2014 Human equilibrative nucleoside transporter-1 knockdown tunes cellular mechanics through epithelial-mesenchymal transition in pancreatic cancer cells. PLoS One 9:e107973
31. Fenske W, Volker HU, Adam P, Hahner S, Johanssen S, Wortmann S, Schmidt M, Morcos M, Muller-Hermelink HK, Allolio B, Fassnacht M 2009 Glucose transporter GLUT1 expression is an stage-independent predictor of clinical outcome in adrenocortical carcinoma. Endocrine-related cancer 16:919-928
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32. Ronchi CL, Sbiera S, Leich E, Tissier F, Steinhauer S, Deutschbein T, Fassnacht M, Allolio B 2012 Low SGK1 expression in human adrenocortical tumors is associated with ACTH- independent glucocorticoid secretion and poor prognosis. The Journal of clinical endocrinology and metabolism 97:E2251-2260
33. Ronchi CL, Sbiera S, Altieri B, Steinhauer S, Wild V, Bekteshi M, Kroiss M, Fassnacht M, Allolio B 2015 Notch1 pathway in adrenocortical carcinomas: correlations with clinical outcome. Endocrine-related cancer 22:531-543
34. Beuschlein F, Weigel J, Saeger W, Kroiss M, Wild V, Daffara F, Libe R, Ardito A, Al Ghuzlan A, Quinkler M, Osswald A, Ronchi CL, de Krijger R, Feelders RA, Waldmann J, Willenberg HS, Deutschbein T, Stell A, Reincke M, Papotti M, Baudin E, Tissier F, Haak HR, Loli P, Terzolo M, Allolio B, Muller HH, Fassnacht M 2015 Major prognostic role of Ki67 in localized adrenocortical carcinoma after complete resection. The Journal of clinical endocrinology and metabolism 100:841-849
35. de Reynies A, Assie G, Rickman DS, Tissier F, Groussin L, Rene-Corail F, Dousset B, Bertagna X, Clauser E, Bertherat J 2009 Gene expression profiling reveals a new classification of adrenocortical tumors and identifies molecular predictors of malignancy and survival. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 27:1108-1115
36. Poplin E, Wasan H, Rolfe L, Raponi M, Ikdahl T, Bondarenko I, Davidenko I, Bondar V, Garin A, Boeck S, Ormanns S, Heinemann V, Bassi C, Evans TR, Andersson R, Hahn H, Picozzi V, Dicker A, Mann E, Voong C, Kaur P, Isaacson J, Allen A 2013 Randomized, multicenter, phase II study of CO-101 versus gemcitabine in patients with metastatic pancreatic ductal adenocarcinoma: including a prospective evaluation of the role of hENT1 in gemcitabine or CO-101 sensitivity. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 31:4453-4461
37. Sinn M, Riess H, Sinn BV, Stieler JM, Pelzer U, Striefler JK, Oettle H, Bahra M, Denkert C, Blaker H, Lohneis P 2015 Human equilibrative nucleoside transporter 1 expression analysed by the clone SP 120 rabbit antibody is not predictive in patients with pancreatic cancer treated with adjuvant gemcitabine - Results from the CONKO-001 trial. European journal of cancer 51:1546-1554
38. Dong X, Hao Y, Wei Y, Yin Q, Du J, Zhao X 2014 Response to first-line chemotherapy in patients with non-small cell lung cancer according to RRM1 expression. PLOS One 9:e92320 39. He YW, Zhao ML, Yang XY, Zeng J, Deng QH, He JX 2015 Prognostic value of ERCC1, RRM1, and TS proteins in patients with resected non-small cell lung cancer. Cancer chemotherapy and pharmacology 75:861-867
40. Bepler G, Williams C, Schell MJ, Chen W, Zheng Z, Simon G, Gadgeel S, Zhao X, Schreiber F, Brahmer J, Chiappori A, Tanvetyanon T, Pinder-Schenck M, Gray J, Haura E, Antonia S, Fischer JR 2013 Randomized international phase III trial of ERCC1 and RRM1 expression-based chemotherapy versus gemcitabine/carboplatin in advanced non-small-cell lung cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 31:2404-2412
41. Nakagawa N, Murakami Y, Uemura K, Sudo T, Hashimoto Y, Kondo N, Sueda T 2013 Combined analysis of intratumoral human equilibrative nucleoside transporter 1 (hENT1) and ribonucleotide reductase regulatory subunit M1 (RRM1) expression is a powerful predictor of survival in patients with pancreatic carcinoma treated with adjuvant gemcitabine-based chemotherapy after operative resection. Surgery 153:565-575
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42. Sasaki H, Murakami Y, Uemura K, Sudo T, Hashimoto Y, Kondo N, Sueda T 2014 Concurrent analysis of human equilibrative nucleoside transporter 1 and ribonucleotide reductase subunit 1 expression increases predictive value for prognosis in cholangiocarcinoma patients treated with adjuvant gemcitabine-based chemotherapy. Br J Cancer 111:1275-1284
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Figure 1. Efficacy of gemcitabine-based chemotherapy in 145 patients with adrenocortical carcinoma. A) Progression-free and disease-specific survival expressed by Kaplan-Meyer curves B) Duration of disease response in 43 patients with partial response (dark grey, n=7) or stable disease (light grey, n=36): median duration 6.7 months (range: 0.8-23.5).
Figure 2. Representative examples of hENT1 (A-D) and RRM1 (E-H) immunostaining A) normal adrenal gland (cytoplasmatic staining H-score=2, membranous staining highly positive); B) adrenocortical adenoma (cytoplasmatic staining H-score=2, membranous staining positive); C) adrenocortical carcinoma - primary tumor (cytoplasmatic staining H-score=2, membranous staining negative); D) adrenocortical carcinoma - metastasis (cytoplasmatic staining H-score=1, membranous staining negative). E) normal adrenal gland (cytoplasmatic staining H-score=2); F) adrenocortical adenoma (cytoplasmatic staining H-score=2); G) adrenocortical carcinoma - primary tumor (cytoplasmatic staining H-score=2); H) adrenocortical carcinoma - local recurrence (cytoplasmatic staining H-score=1).
Figure 3. Relationship between potential predictive biomarkers and sensitivity to gemcitabine-based chemotherapy. Analysis performed in a subgroup of 70 patients with advanced adrenocortical carcinomas, as illustrated by the progression-free survival during treatment (Kaplan-Meyer curves and log-rank test). A) hENT1 membranous immunostaining; B) RRM1 cytoplasmatic immunostaining; C) Combination between hENT1 and RRM1 immunostaining.
| Age - years (median, range) | 44 (20-79) |
| Sex (F/M) | 69/45 |
| Initial hormone secretion: | |
| Glucocorticoids - n (% of known) | 41 (41) |
| Androgens - (% of known) | 8 (8) |
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| Glucocorticoids+androgens - (% of known) | 23 (23) |
| Endocrine inactive - n (% of known) | 28 (28) |
| Unknown - n (%) | 45 (31) |
| Size of the primary tumor - cm - median (range) | 13 (1.8-26.0) |
| Initial ENSAT tumor stage: | |
| I-II - n (% of known) | 58 (42.3) |
| III - n (% of known) | 26 (19.0) |
| IV - n (% of known) | 53 (38.7) |
| Unknown - n (%) | 8 (5.5) |
| Resection status of the primary surgery: | |
| R0 - n (% of known) | 61 (48.0) |
| RX - n (%of known) | 16 (12.6) |
| R1/R2 - n (%of known) | 41 (32.3) |
| Unknown - n (%) | 18 (12.4) |
| No surgery - n (%) | 9 (7.1) |
| Histopathological workup of the primary tumor: | |
| Proliferation index (ki67) % - median (range) | 20 (1-85) |
| Unknown - n (%) | 11 (7.5) |
| Weiss score - median (range) | 6 (2-9) |
| Unknown - n (%)* | 51 (32.4) |
| Local therapeutic approaches before GEM-based therapy **: | |
| One additional surgery - n (%) | 32 (22.1) |
| Multiple additional surgeries -n | 37 (23.8) |
| Radiotherapy (tumor bed or metastasis) - n | 58 (40.0) |
| Other local therapies *** - n | 17 (11.7) |
| Chemotherapies before GEM-based therapy: | |
| Platinum-based chemotherapy - n (%) | 128 (88.3) |
| Streptozotozin - n (%) | 44 (30.3) |
| Other chemotherapies - n (%) | 13 (9.0) |
| None - n (%) | 12 (8.3) |
| Treatment sequence of GEM-based therapy: | |
| First-line - n (%) | 12 (8.3) |
| Second-line - n (%) | 83 (57.2) |
| Third-line or further - n (%) | 50 (34.5) |
| Combination treatment | |
| Concomitant capecitabine - yes (%) | 132 (91.0) |
| Concomitant mitotane - yes (%) | 114 (78.6) |
| Best objective response to therapy ****: | |
| Progressive disease - n (%) | 102 (70.8) |
| Stable disease - n (%) | 36 (25.0) |
| Partial/Complete remission - (%) | 7 (4.9) |
| Unknown - n (%) | 1 (0.7) |
| Clinical benefit (partial response or stable disease for at least 4 months - n (%) | 30 (20.8%) |
Notes: * Weiss score ≥3 but not exactly known. ** more than one treatment per patient possible *** iodometomidate or radiofrequency ablation. **** evaluated locally by expert radiologist and multidisciplinary team Abbreviations: n: number; F: female; M: male; RO=complete resection; RX=uncertain resection; R1=microscopic incomplete resection; R2=macroscopic incomplete resection; GEM: gemcitabine.
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| Normal adrenal gland | Adrenocortical adenoma | Adrenocortical carcinoma (primary tumor) | Adrenocortical carcinoma (local recurrence/metastasis) |
|---|---|---|---|
| A | B | C | D |
| E | F | G | H |
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