Topoisomerase a 2 and thymidylate synthase expression in adrenocortical cancer
1Elisa Roca, 1 Alfredo Berruti, 2Silviu Sbiera, 3Ida Rapa, 1Ester Oneda, 4Paola Sperone, 2Cristina L. Ronchi, 1Laura Ferrari, 1Salvatore Grisanti, 5Antonina Germano, 5Barbara Zaggia, 4Giorgio Vittorio Scagliotti, 2Martin Fassnacht, 3Marco Volante, 5Massimo Terzolo, ‘Mauro Papotti.
Department of Medical and Surgical Specialties, Radiological Sciences, and Public Health, University of Brescia, Oncology Unit, ASST Spedali Civili di Brescia, Brescia, Italy.
2Department of Internal Medicine I, Division of Endocrinology and Diabetes, University Hospital, University of Wuerzburg, Wuerzburg, Germany
3Department of Oncology, University of Turin, Pathology Unit, San Luigi Gonzaga Hospital, Orbassano, Italy
4Department of Oncology, University of Turin, Medical Oncology, San Luigi Gonzaga Hospital, Orbassano, Italy
5Department of Clinical and Biological Sciences, University of Turin, Internal Medicine 1. San Luigi Gonzaga Hospital, Orbassano, Italy.
Department of Oncology, University of Turin; Pathology Unit, City of Health and Science Hospital, Turin, Italy.
Correspondence to:
Professor Alfredo Berruti
Oncologia Medica ASST Spedali Civili Piazzale Spedali Civili 1, 25123 Brescia - Italy Tel 030 3995410 Email: alfredo.berruti@unibs.it
Short title
TOP2a and TS expression in adrenocortical carcinoma
Keywords: adrenocortical carcinoma, topoisomerase alpha 2, thymidylate synthase, prognostic and predictive factors
Words count:
· Abstract (maximum 250 words): 250
· Manuscript (maximum 5000 words): 2471
· Figures (maximum 10): 6 figures (1, 2a, 2b, 2c, 2d, 3a, 3b) and 2 tables
· References: (maximum 60 words): 41
1 Abstract
2
Topoisomerase II alpha (TOP2A) and Thymidylate Synthase (TS) are known prognostic parameters 3 4 in several tumors and also predictors of efficacy of anthracyclines, topoisomerase inhibitors and
5 fluoropirimidines, respectively.
6 7 8 9 Expression of TOP2A and TS mRNA was assessed in 98 patients with adrenocortical carcinoma (ACC) and protein expression was assessed by immunohistochemistry in a subset of 39 tumors. Ninety-two patients were radically resected for stage II-III disease and 38 of them received adjuvant mitotane. Twenty-six patients with metastatic disease received the EDP-M (Etoposide, 10 Doxorubicin, Adriamycin, Cisplatin plus mitotane). TOP2A and TS expression in ACC tissue was 11 directly correlated with the clinical data. Both markers were not associated with either disease free
12 survival (DFS) or overall survival (OS) in multivariate analyses and failed to be associated to mitotane efficacy. Disease response or stabilization to EDP-M treatment was observed in 12/17 (71%) and 1/9 (11%) patients with high and low TOP2A expressing tumors (p=0.0039) and 9/13 (69%) and 4/13 (31%) patients with high and low TS expressing ACC, respectively (p=0.049). High TOP2A expression was significantly associated with longer time to progression (TTP) after EDP- M. TOP2A and TS proteins assessed by immunohistochemistry significantly correlated with mRNA expression. Immunohistochemical TOP2A expression was associated with a non-significant better response and longer TTP after EDP-M.
TOP2A and TS were neither prognostic nor predictive of mitotane efficacy in ACC patients.
The predictive role of TOP2A expression of EDP-M activity suggests a significant contribution of adriamycin and etoposide for the efficacy of the EDP scheme.
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24 25
Introduction
Adrenocortical carcinoma (ACC) is a rare and aggressive malignant tumor [Fassnacht M, et al. 26 2011; Terzolo M, et al. 2013]. Surgery is the mainstay of therapy. Although complete surgical 27 28 29 30 31 32 33 34 removal of ACC is the only potentially curative approach, most of the radically resected patients are destined to relapse, often with metastases [Bellantone R, et al. 1997; Icard P, et al. 2001]. Mitotane is the reference systemic therapy for ACC [Hahner S, & Fassnacht M, 2005]. On the basis of the results of a large retrospective multicentric study carried out at several referral centers in Italy and Germany [Terzolo M, et al. 2007], the drug is recommended to be administered in adjuvant setting in radically resected ACC with high risk of recurrence [Berruti A, et al. 2010; Berruti A, et al. 2012]. The recent finding that mitotane serum levels are prognostic in patients receiving the drug as adjuvant therapy provides further evidence in favor of the efficacy of this drug in this setting 35 [Terzolo M, et al. 2013]. However, few prognostic factors are currently available to identify the 36 37 patient risk of relapse and death of disease. Currently only disease stage, completeness of initial resection, and proliferation index are widely accepted prognostic factors [Berruti A, et al. 2010; 38 Volante M, et al. 2008; Beuschlein F, et al 2015]. In the patients with metastatic disease at 39 diagnosis or showing distant recurrence after surgery, chemotherapy plus mitotane is the best 40 treatment strategy. The results of a multinational prospective randomized clinical study have 41 established the combination chemotherapy with the topoisomerase II inhibitor Etoposide, the 42 anthracycline Doxorubicin, Cisplatin plus mitotane (EDP-M) as the reference regimen for this rare disease [Fassnacht M, et al. 2012] Adjuvant mitotane therapy and EDP-M however, are toxic and their overall efficacies limited. Therefore, the establishment of predictive factors that identify a subset of patients, in whom a certain treatment would be more effective, would be highly desirable. We have previously evaluated the expression of ribonucleotide reductase large subunit 1 (RRM1) and ERCC1 genes in a multicenter (German and Italian) cohort of ACC patients and provided the
43 44 45 46 47 48 first evidence in vitro and in vivo that RRM1 gene expression levels are functionally associated to
49 mitotane sensitivity and predict response to mitotane treatment in adjuvant setting [Volante M, et al. 2012].
In the same series we now assessed the expression of two other biomarkers with potential prognostic impact: the topoisomerase II alpha (TOP2A), an enzyme responsible for transcription, replication and chromosome condensation and segregation during cell division [Kellner U1, et al. 2002]; and the thymidylate synthase (TS), an enzyme involved in nucleotide metabolism [Lv YT, et al. 2013]. TOP2A is also a well known predictor of efficacy of anthracyclines and topoisomerase inhibitors [Wang J, et al. 2012] and TS is predictive of the efficacy of fluoropyrimidines [Formentini A, et al. 2004]. In the present study, we explored the prognostic significance of these markers in ACC as well as their predictive role for efficacy of adjuvant mitotane and of EDP-M in patients with advanced disease.
Materials and Methods
Patients.
Ninety-eight patients with radically resected ACC between 1989 and 2007 at the San Luigi Hospital of Orbassano, University of Turin, Italy (51 patients) and 35 centers in Germany coordinated by the German ACC Registry (47 patients) were included according to the following eligibility criteria: 1) age of 18 years or older; 2) histologically confirmed diagnosis of ACC after central pathologic revision (MV, MP); 3) complete resection of primary ACC, 4) availability of follow-up information, 5) availability of representative paraffin-embedded tissue block(s). All patients fulfilling these criteria were included in the study. Ninety-two patients were already considered in the previously published series that have tested ERCC1 and RRM1 [Volante M, et al. 2012]. In the present paper we also considered six patients from the Italian archive that were excluded in the published series because they were metastatic at diagnosis. These patients were radically resected on primary tumor but not on metastatic disease. The diagnosis of ACC was based on the pathological Weiss score [Weiss LM, et al. 1989]. Variables recorded included age, sex, hormone 4
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secretion, ENSAT stage at diagnosis [Fassnacht M, et al. 2009], initial therapeutic options including primary surgery, disease-free survival (DFS) for patients initially radically resected, defined by the time elapsing from diagnosis to either disease relapse or patient death, overall survival (OS), calculated from diagnosis till death, Weiss score, mitotic count, sites of metastases at the time of progression. ACC relapse was defined as the appearance of local recurrence or metastatic disease at imaging techniques during follow-up. Adjuvant mitotane was offered to patients considered at high risk of relapse, in presence of the following criteria: 1) stage III ACC; 2) high mitotic index. When a post-operative adjunctive measure was deemed necessary, a monitored mitotane treatment aiming at plasma concentrations between 14 and 20 mg/l [Baudin E, et al. 2001; Ceppi P, et al. 2008] was 84 employed. In the absence of intolerability to mitotane, treatment was scheduled for at least 2 years, or till ACC recurrence. Follow-up protocols were similar among the different centers including imaging (CT or MRI) of both chest and abdomen at baseline and thereafter every 3-6 months until disease progression or end of the study period. At each visit, the patients underwent physical examination, routine laboratory evaluation and hormonal work-up. Monitoring of mitotane concentrations was done in treated patients. For recurrent disease, radical surgery was performed if complete resection seemed feasible. In case of not resectable disease, patients received mitotane alone or chemotherapy plus mitotane according to disease aggressiveness, tumor bulk and previous mitotane exposure. Twenty-six patients, 4 metastatic at diagnosis and 22 with disease recurrence after primary surgery, received the combination of EDP-M. Patients gave informed consent for collecting tissue and clinical data and the study was approved by the ethics committees of both centres.
RNA isolation from paraffin embedded tissues and quantitative real time PCR.
Representative tumour areas were dissected under stereomicroscopic assistance from 10 um sections of paraffin-embedded tissue in RNAse-free conditions. RNA isolation was performed by commercially available paraffin material RNA extraction kits according to manufacturer’s
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instructions (High Pure RNA Paraffin Kit; Roche Applied Science, Milano, Italy). Complementary DNA was transcribed using 500ug/ml oligo dT (Roche Applied Science, Penzberg, Germany) and 500M-MLV RT (200U/ul) (Invitrogen, Carlsbad, California) according to standard protocols. Relative cDNA quantification of TS and TOP2A and of internal reference gene (beta-actin) was done in duplicate using a fluorescence-based real-time detection method (ABI PRISM 7900 Sequence Detection System-Taqman; Applied Biosystems, Life Technologies). Primers and probes sequences for TS and TOP2A and cycling conditions have already been published elsewhere [21,22]. The relative gene expression levels were expressed as ratios (differences between the Ct values) between 2 absolute measurements (genes of interest/internal reference gene).
Then, the AACt values were calculated subtracting ACt values of each case to the value of the case with the lowest expression, and converting the ratio by the 2-44C formula; cases were considered of low or high expression according to the median expression level obtained. A mixture containing Human Total RNA (Stratagene, La Jolla, CA) was used as control calibrator on each plate.
Immunohistochemical evaluation
116 Five um thick paraffin sections serial to those used for conventional hematoxylin and eosin staining were obtained for immunohistochemical reactions. The following antibodies were employed: TS (1/100, rabbit monoclonal, clone ERP4545) and TOP2A (diluted 1/100, rabbit monoclonal, clone EP1102y) (Abcam, Cambridge, UK). Immunohistochemistry was performed in automated Ventana BenchMark Ultra instrument (Ventana, Roche, Tucson, AZ). Immunoreactivity was scored using the H-score which is generated from the following equation: H-score= >Pi (i + 1), where “2” represents the intensity of staining (0-3+), and “Pi” stands for the percentage of stained tumor cells (0% to 100%).
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Correlations between the expressions of TOP2A and TS genes, as well as between protein and - mRNA expression were tested using the Spearman coefficient. Differences of categorical variables were analyzed using the chi2 test. DFS and OS survival curves were computed using the Kaplan- Meier method and compared using the log-rank test. Hazard ratios (HR) for disease progression and 130 patient death were estimated using the Cox proportional hazard model. Multivariate analyses were carried out adjusting for patient age, sex, Weiss score, ACC stage, mitotic count, and cortisol secretion. Cox models were also used to assess the presence of heterogeneity in the effect of marker expression in the different patient subgroups, defined by the covariates, by including in the model 134 the appropriate treatment/covariate interaction term. Statistical analyses were carried out by using the SPSS for windows software (version 17).
Results
Patients
Patient characteristics are depicted in Table 1. Both Italian and German cohorts were comparable in 140 terms of age, sex proportion, presence of clinical syndromes, and tumor characteristics (data for 92 patients previously published, [Volante M, et al. 2012]. Adjuvant mitotane therapy was administered to 38 patients (38.8%): 18 Italian and 20 German patients, respectively; the remaining 54 patients (55.1%) with non-metastatic disease did not receive any postoperative treatment. The median follow-up was 66.3 months in all patients, being 80 months and 62.8 months in the Italian and German cohorts, respectively. Among patients with stage II-III disease, 64 (65.3%) developed disease recurrence, 29 of the Italian series (67.4%) and 35 of the German series (74.5%). Among them, 30 were treated with chemotherapy; streptozotocyn in 8 patients, EDP (Etoposide, Doxorubicin, Cisplatin) in 22 patients. Both streptozotocyn and EDP were administered in association with mitotane. Overall 51 patients (52.0%) died of ACC progression: 28 (54.9%) in the Italian series and 23 (48.9%) in the German series, respectively.
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126 127 128 129
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Relationship between tissue marker expression and patient and tumor characteristics
As outlined in Table 2, both TOP2A and TS gene expression, dichotomized at the median value, did not show any significant relationship with histopathologic features, except for the significant direct correlation between TS expression levels and Weiss score. TOP2A and TS expressions were significantly directly correlated (p=0.006) and both markers showed a direct relationship with 157 RRM1 (p=0.026 and p=0.011 for TOP2A and TS, respectively) but not with ERCC1 (p=0.54 and p=0.61 for TOP2A and TS, respectively). In the 39 patients in which TOP2A and TS protein 159 expression were assessed by immunohistochemistry, a significant relationship was found between the gene and the relevant protein expression of both enzymes (Spearman R 0.52 and 0.46 for TOP2A and TS, respectively) (figure 1).
Relationship between marker expression and disease free and overall survival
In univariate analysis, TS gene expression was not associated with DFS [Hazard Ratio (HR): 0.96, 95% Confidence Interval (CI): 0.60-1.53, p=0.87] and OS (0.86, 95% CI: 0.49-1.50, p=0.60). High TOP2A expression levels were associated with higher risk of disease recurrence but just failing to 167 attain statistical significance (HR: 1.49, 95% CI: 0.93-2.37, p =0.09) and was significantly 168 associated with a higher risk of death (HR: 1.78, 95% CI: 1.02-3.19, p<0.05). In multivariate analysis, however, TOP2A expression failed to be a significant independent prognostic parameter either in terms of DFS (HR: 1.18, 95% CI: 0.71-1.98, p=0.51) or OS (HR: 1.61, 95% CI: 0.87-2.98, p=0.13).
Predictive role of TOP2A and TS expression for the efficacy of adjuvant mitotane
As previously reported, in this series mitotane-treated patients had a longer median DFS than untreated patients (22.5 months [95% CI, 1.8-43.1] versus 13.2 months [95% CI, 6.2-20.2], HR, 0.70 [95% CI, 0.43-1.16; P= 0.17]), and longer median OS (154 months [95%CI, 65.1-242.9] versus 53 months [95% CI, 22.6-83.4], HR 0.63 [95% CI, 0.34-1.16; P=0.14]).
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169 170 171 172 173 174 175 176 177
The prognostic effect of mitotane administration was assessed stratifying the patients according to marker expression (dichotomised at the median value). As shown in Figure 2, no different effect of mitotane treatment was observed in terms of DFS in patients with high versus low TOP2A and TS expression.
Predictive role of marker expression of EDP + mitotane activity
184 Among the 26 ACC patients who received first line EDP-M for metastatic disease, four (15%) achieved a partial response (PR), nine (35%) stable disease (SD) and 13 (50%) experienced progressive disease (PD) after 2 or 3 cycles.
The clinical benefit (SD or PR) of EDP-M was directly associated with TOP2A expression: 12 SD/PR out of 17 (71%) patients in high TOP2A group as opposed to 1/9 (11%) in low TOP2A group (p=0.0039) (Figure 3a). Stratifying patients according to TS expression, a SD/PR was observed in 9/13 (69%) patients with high gene expression and 4/13 (31%) patients with low TS expression, respectively (p=0.049) (Figure 3b). EDP-M administration was associated with longer time to progression (TTP) in patients with high TOP2A as opposed to those with low TOP2A (p=0.038) (Figure 4a), while no difference in terms of TTP after EDP-M was observed stratifying patients according to TS (data not shown). Fifteen EDP-M treated patients had TOP2A protein expression assessed by immunohistochemistry. TOP2A expression >30% of stained cells (the median value) was associated with a greater chance to attain SD/PR [5/9 (55.5%) vs 1/6 (16.6%), p=0.22] and longer TTP (figure 4b) than TOP2A ≤ 30%. These differences failed to attain the statistical significance. Among the 12 EDP-M patients, whose tumors had TS expression assessed by immunohistochemistry, TS expression above the median value (>30% of stained cells) was associated with a non significant greater SD/PR rate [3/6 (50.0%) vs 1/6 (16.6%), p=0.54] but TTP was similar to that of patients with TS ≤ 30% (data not shown).
Neither ERCC1 nor RRM1 correlated with a clinical benefit of the therapy: SD/PR in 8/15 (53%) versus 5/11 (45%) in high and low ERCC1 expressing tumors (p=0.69) (Figure 3c); SD/PR in 7/16
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(44%) versus 6/10 (60%) in high and low RRM1 expressing tumors, respectively (p=0.42) (Figure 3d). No difference in term of TTP was observed stratifying patients according to both RRM1 and ERCC1 expression (data not shown).
Discussion
TOP2A and TS have been shown to play a prognostic role in several cancers, such as colorectal, 212 esophageal, breast, prostate, pancreatic, lung, and other cancers [Peters GJ, et al. 1994; Lenz HJ, et al. 1998; Suda Y, et al. 1999; Suzuki M, et al. 1999; Edler D, et al. 2000; Harpole DH Jr., et al. 2001; Nakopoulou L, et al. 2001; Di Leo A, et al. 2003; Gonen Met al. 2003; O’Connor JK, et al. 2006; Shvero J, et al. 2008; Faggad A, et al. 2009; Van der Zee JA, et al. 2012; Lee SW, et al. 2013; Yan H, et al. 2015]. At the best of our knowledge, the prognostic role of these two markers has never been explored in ACC.
In this study, we have measured the mRNA expressions of TOP2A and TS in a relatively large series of 98 ACC patients, considering the extreme rarity of this tumor.
The results showed that TS expression is not prognostic in ACC while TOP2A expression in univariate analysis showed a significant correlation with a worse OS although failing to be significantly associated with DFS. In multivariate analysis, after adjusting for prognostic factors including mitotic index, TOP2A expression failed to be a significant independent prognostic parameter. TOP2A is a marker of cell proliferation [Zhang H, et al. 2014] and the direct correlation of this enzyme expression with the mitotic count (although not significant) could account for its failure to be an independent prognostic parameter.
As mentioned in the introduction, both TOP2A and TS are also well known predictors of chemotherapy efficacy. In our series, high TOP2A expression in primary ACC was significantly associated with responsiveness to EDP-M and increased TTP in metastatic patients.
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All these data refer to gene expression of both enzymes. In order to explore whether TOP2A and TS expressed at the protein level provide the same information, the expression of both enzymes by immunohistochemistry was performed in the Italian patients having residual paraffin material available. A significant correlation was found between PCR and immunohistochemistry both for TOP2A and TS expression. In addition, the predictive role of TOP2A of efficacy of EDP-M in advanced ACC patients was confirmed also at immunohistochemistry although without attaining the statistical significance due to the low number of patients considered. Taken together these data suggest that TOP2A and TS protein expression may have the same prognostic and predictive role as mRNA expression levels in ACC.
TOP2A is a notorious predictor of efficacy to anthracycline and topoisomerase inhibitors in several tumors, but not to cisplatin. However, cisplatin is the reference cytotoxic drug in ACC and it is actually unknown whether EDP is more efficacious than cisplatin alone since no randomized studies have been conducted. In our opinion, these data suggest the importance of adriamycin and etoposide in the EDP scheme, particularly if TOP2A is highly expressed.
Also TS showed a direct relationship with the disease response of EDP-M, although with a lesser magnitude than TOP2A, but not with TTP. Since fluoropyrimidines are not included in the EDP-M regimen, the direct relationship of TS with TOP2A may explain the association of this marker expression with EDP-M activity.
In this small series, both RRM1 and ERCC1 failed to be associated with EDP-M activity, this is an expected finding for RRM1 since this marker was repeatedly found to be predictive of gemcitabine efficacy, but not for ERCC1 that is a predictive factor of cisplatin efficacy in non small cell lung cancer [Ronchi CL, et al. 2009]. In ACC patients, tissue ERCC1 assessed by immunohistochemistry was associated with efficacy of cisplatin containing regimens in one study [Baudin E, et al. 2011] but not confirmed in another series [Sbiera S, et al. 2015].
In this paper we also explored the potential role of TOP2A and TS for mitotane efficacy in adjuvant setting. Although very recently sterol-O-acyl-transferase 1 (SOAT1) was identified as a key 11
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intracellular target of mitotane [Sbiera S, et al. 2015], the identification of a possible association between mitotane efficacy with a biological parameter in an explorative analysis, would be very useful to understand the mechanisms of sensitivity and resistance of ACC to this drug.
Both TOP2A and TS failed to have a predictive role for adjuvant mitotane efficacy; however the difference in the DFS curves of adjuvant mitotane versus follow-up only in low versus high TS expressing ACC, although not significant, does not exclude the potential role of this marker of mitotane efficacy.
Conclusions
The predictive role of TOP2A expression with the efficacy of EDP-M is new and of potential clinical relevance. EDP is a toxic regimen and this observation, if confirmed, suggests the potential importance of adding adriamycin and etoposide to cisplatin in highly expressing TOP2A tumors, while these 2 drugs might be unhelpful in low TOP2A expressing ACC.
Declaration of interest:
The authors have no conflict of interest to declare.
Funding:
This study was supported by grants from AIRC, Milan (no. IG/14820/2013 to MP), Fondazione Camillo Golgi, Brescia, and donations of “gli Amici di Carlo” in memory of Carlo Ridon, “gli Amici di Andrea” in memory of Andrea Gadeschi, Pertot Valentino and Stolfa Maria Teresa in memory of Pertot Cristiana, Serena Ambrogini in memory of Guido Cioni.
Author contributions:
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Elisa Roca, Alfredo Berruti, Massimo Terzolo, Marco Volante contributed to study concept and design and to writing of the report
Silviu Sbiera, Paola Sperone, Barbara Zaggia Cristina Ronchi were investigators and contributed to acquisition of data and identifications of eligible patients
Ida Rapa, and Antonina Germano performed immunohistochemistry analysis, the RNA isolation from paraffin embedded tissues and quantitative real time PCR.
Laura Ferrari and Salvatore Grisanti evaluated, analysed, and interpreted the data
Martin Fassnacht and Giorgio Vittorio Scagliotti -performed a critical revision of the manuscript for important intellectual content
Mauro Papotti -contributed to the study concept and design, performed a critical supervision of the study and revised the manuscript.
All authors reviewed and edited the report.
References
· Baudin E, Leboulleux S, Al Ghuzlan A, Chougnet C, Young J, Deandreis D, Dumont F, Dechamps F, Caramella C, Chanson P, et al. Therapeutic management of advanced adrenocortical carcinoma: what do we know in 2011? 2011 Horm Cancer. 6 363-71
281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303
· Baudin E, Pellegriti G, Bonnay M, Penfornis A, Laplanche A, Vassal G, Schlumberger M. Impact of monitoring plasma 1,1-dichlorodiphenildichloroethane (o,p’DDD) levels on the treatment of patients with adrenocortical carcinoma. 2001 Cancer 92 1385-1392
· Bellantone R1, Ferrante A, Boscherini M, Lombardi CP, Crucitti P, Crucitti F, Favia G, Borrelli D, Boffi L, Capussotti L, et al. 1997 Role of reoperation in recurrence of adrenal cortical carcinoma: results from 188 cases collected in the Italian National Registry for adrenal cortical carcinoma. Surgery 122 1212-1218
· Berruti A, Baudin E, Gelderblom H, Haak HR, Porpiglia F, Fassnacht M, Pentheroudakis G ESMO Guidelines Working Group. Adrenal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. 2012 Ann Oncol 23 (suppl 7):vii131-vii138
· Berruti A, Fassnacht M, Baudin E et al. Adjuvant therapy in patients with adrenocortical carcinoma: a position of an international panel. 2010 J Clin Oncol 28(23) e401-2; author reply e403
· Beuschlein F, Weigel J, Saeger W, Kroiss M, Wild V, Daffara F, Libé R, Ardito A, Al Ghuzlan A, Quinkler M, et al. Major prognostic role of Ki67 in localized adrenocortical carcinoma after complete resection. 2015 J Clin Endocrinol Metab. 100 841-9
· Kellner U1, Sehested M, Jensen PB, Gieseler F, Rudolph P. Culprit and victim — DNA topoisomerase II. 2002 Lancet Oncol 3(4) 235-43
· Ceppi P, Longo M, Volante M, Novello S, Cappia S, Bacillo E, Selvaggi G, Saviozzi S, Calogero R, Papotti M, Scagliotti GV. Excision repair cross complementing-1 and topoisomerase IIalpha gene expression in small-cell lung cancer patients treated with platinum and etoposide: a retrospective study. J Thorac Oncol. 2008 Jun;3(6):583-9.
304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325
· Ceppi P, Volante M, Ferrero A, Righi L, Rapa I, Rosas R, Berruti A, Dogliotti L, Scagliotti GV, Papotti M. Thymidylate synthase expression in gastroenteropancreatic and pulmonary neuroendocrine tumors. 2008 Clin Cancer Res. 14 1059-64
· Di Leo A, Isola J. Topoisomerase IIa as a Marker Predicting the Efficacy of Anthracyclines in Breast Cancer: Are We at the End of the Beginning? 2003 Clin Breast Cancer. 4 179-186
· Edler D, Glimelius B, Hallström M, Jakobsen A, Johnston PG, Magnusson I, Ragnhammar P, Blomgren. Thymidylate synthase expression: an independent prognostic factor for local recurrence, distant metastasis, disease-free and overall survival in rectal cancer. 2000 Clin Cancer Res. 6 1378-84
· Faggad A, Darb-Esfahani S, Wirtz R, Sinn B, Sehouli J, Könsgen D, Lage H, Weichert W, Noske A, Budczies J et al. Topoisomerase IIa mRNA and protein expression in ovarian carcinoma: correlation with clinicopathological factors and prognosis. 2009 Mod Pathol 22 579-588
· Fassnacht M, Johanssen S, Quinkler M, Bucsky P, Willenberg HS, Beuschlein F, Terzolo M, Mueller HH, Hahner S, Allolio B; German Adrenocortical Carcinoma Registry Group; European Network for the Study of Adrenal Tumors. Limited prognostic value of the 2004 International Union Against Cancer staging classification for adrenocortical carcinoma: proposal for a Revised TNM Classification. 2009 Cancer 115 243-250
· Fassnacht M, Libé R, Kroiss M 2011 Adrenocortical carcinoma: a clinician’s update. Nat Rev Endocrinol 7 323-335
· Fassnacht M, Terzolo M, Allolio B, Baudin E, Haak H, Berruti A, Welin S, Schade- Brittinger C, Lacroix A, Jarzab B, et al. FIRM-ACT Study Group. 2012 Combination chemotherapy in advanced adrenocortical carcinoma. N Engl J Med. 366 2189-97
326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348
· Formentini A, Henne-Bruns D, Kornmann M. Thymidylate synthase expression and prognosis of patients with gastrointestinal cancers receiving adjuvant chemotherapy: a review. 2004 Langenbecks Arch Surg. 389 405-13
· Gonen M, Hummer A, Zervoudakis A, Sullivan D, Fong Y, Banerjee D, Klimstra D, Cordon-Cardo C, Bertino J, Kemeny N. Thymidylate synthase expression in hepatic tumors is a predictor of survival and progression in patients with resectable metastatic colorectal cancer. 2003 J Clin Oncol/21 406-12
· Hahner S, Fassnacht M 2005 Mitotane for adrenocortical carcinoma treatment. Curr Opin Investig Drugs 6 386 -394
· Harpole DH Jr., Moore MB, Herndon JE 2nd, Aloia T, D’Amico TA, Sporn T, Parr A, Linoila I, Allegra C. The prognostic value of molecular marker analysis in patients treated with trimodality therapy for esophageal cancer. 2001 Clin Cancer Res. 7 562-9
. Icard P, Goudet P, Charpenay C, Andreassian B, Carnaille B, Chapuis Y, Cougard P, Henry JF, Proye C Adrenocortical carcinomas: surgical trends and results of a 253-patient series from the French Association of Endocrine Surgeons study group. 2001 Word J Surg 25 891- 897
· Lee SW, Chen TJ, Lin LC, Li CF, Chen LT, Hsing CH, Hsu HP, Tsai CJ, Huang HY, Shiue YL. Overexpression of thymidylate synthetase confers an independent prognostic indicator in nasopharyngeal carcinoma. 2013 Exp Mol Pathol Aug 95(1) 83-90
· Lenz HJ, Hayashi K, Salonga D, Danenberg KD, Danenberg PV, Metzger R, Banerjee D, Bertino JR, Groshen S, Leichman LP, Leichman CG. p53 point mutations and thymidylate synthase messenger RNA levels in disseminated colorectal cancer: An analysis of response and survival. 1998 Clin Cancer Res/4 1243-50
349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371
· Lv YT, Du PJ, Wang QY, Tan Y, Sun ZB, Su ZL, Kang CM. A novel approach to cloning and expression of human thymidylate synthase. 2013 Asian Pac J Cancer Prev. 14 7523- 7527
· Nakopoulou L, Zervas A, Lazaris AC, Constantinides C, Stravodimos C, Davaris P, Dimopoulos C. Predictive value of topoisomerase Iia immunostaining in urothelial bladder carcinoma. 2001 J Clin Pathol. 54 309-313
· O’Connor JK, Hazard LJ, Avent JM, Lee RJ, Fischbach J, Gaffney DK. Topoisomerase II alpha expression correlates with diminished disease free survival in invasive breast cancer. 2006 Int J Radiat Oncol Biol Phys. 65 1411-1415
· Peters GJ, van der Wilt CL, van Groeningen CJ, Smid K, Meijer S, Pinedo HM. Thymidylate synthase inhibition after administration of fluorouracil with or without leucovorin in colon cancer patients: Implications for treatment with fluorouracil. 1994 J Clin Oncol/12 2035-42
· Ronchi CL, Sbiera S, Kraus L, Wortmann S, Johanssen S, Adam P, Willenberg HS, Hahner S, Allolio B, Fassnacht M. Expression of excision repair cross complementing group 1 and prognosis in adrenocortical carcinoma patients treated with platinum-based chemotherapy. 2009 Endocr Relat Cancer 16 907-918
· Sbiera S, Leich E, Liebisch G, Sbiera I, Schirbel A, Wiemer L, Matysik S, Eckhardt C, Gardill F, Gehl A, et al. Mitotane_Inhibits Sterol-O-Acyl Transferase 1 Triggering Lipid- Mediated Endoplasmic Reticulum Stress and Apoptosis in Adrenocortical Carcinoma Cells. 2015 Endocrinology. 156(11) 3895-908
. Shvero J, Koren R, Shvili I, Yaniv E, Sadov R, Hadar T. Expression of Human DNA topoisomerase ii-a in squamous cell carcinoma of the larynx and its correlation with clinicopathologic variables. 2008 Am J Clin Pathol 130 934-939
372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395
· Suda Y, Kuwashima Y, Tanaka Y, Uchida K, Akazawa S. Immunohistochemical detection of thymidylate synthase in advanced gastric cancer: A prognostic indicator in patients undergoing gastrectomy followed by adjuvant chemotherapy with 5-fluoropyrimidines. 1999 Anticancer Res/19/805-10
· Suzuki M, Tsukagoshi S, Saga Y, Ohwada M, Sato I. Enhanced expression of thymidylate synthase may be of prognostic importance in advanced cervical cancer. 1999 Oncology/57/50-4
· Terzolo M, Angeli A, Fassnacht M, Daffara F, Tauchmanova L, Conton PA, Rossetto R, Buci L, Sperone P, Grossrubatscher E, et al. 2007 Adjuvant mitotane treatment for adrenocortical carcinoma. N Engl J Med 356 2372- 2380
· Terzolo M1, Baudin AE, Ardito A, Kroiss M, Leboulleux S, Daffara F, Perotti P, Feelders RA, de Vries JH, Zaggia B et al. 2013 Mitotane levels predict the outcome of patients with adrenocortical carcinoma treated adjuvantly following radical resection. Eur J Endocrinol 169(3) 263-270
· Terzolo M, Daffara F, Ardito A, Zaggia B, Basile V, Ferrari L, Berruti A 2014 Management of adrenal cancer: a 2013 update. J Endocrinol Invest 37(3) 207-17
· Van der Zee JA, van Eijck CH, Hop WC, van Dekken H, Dicheva BM, Seynhaeve AL, Koning GA, Eggermont AM, Ten Hagen TL. Expression and prognostic significance of thymidylate synthase (TS) in pancreatic head and periampullary cancer. 2012 Eur J Surg Oncol 38(11) 1058-64
· Volante M, Berruti A, Papotti M. Pathological and molecular features of adrenocortical carcinoma: an update. 2008 J. Clin Pathol 61 787-793
396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417
· Volante M, Terzolo M, Fassnacht M, Rapa I, Germano A, Sbiera S, Daffara F, Sperone P, Scagliotti G, Allolio B, et al. Ribonucleotide reductase large subunit (RRM1) gene expression may predict efficacy of adjuvant mitotane in adrenocortical cancer. 2012 Clin Cancer Res. 18(12) 3452-61
· Wang J, Xu B, Yuan P, Zhang P, Li Q, Ma F, Fan Y. TOP2A amplification in breast cancer is a predictive marker of anthracycline-based neoadjuvant chemotherapy efficacy. 2012 Breast Cancer Res Treat. 135 531-7
· Weiss LM, Medeiros LJ, Vickery Al Jr. Pathologic features of prognostic significance in adrenocortical carcinoma. 1989 Am J Surg Pathol. 13 202-202.
· Yan H, Guo X, Wang H, Zhu T. Predictive value of thymidylate synthase for the prognosis and survival of lung adenocarcinoma patients. 2015 Oncol Lett. 9(1) 252-256.
· Zhang H, Li J, Zhang Y, Sun M, Zhao P, Zhang G, Jin C, Sun L, He M, Wang B, Zhang X.ERCC1 mRNA expression is associated with the clinical outcome of non-small cell lung cancer treated with platinum-based chemotherapy. 2014 Genet Mol Res 4 13(4) 10215-22
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Figure legends
Figure 1: Relationship between mRNA and immohistochemical expression of TOP2A a) and TS b) Relationship between TOP2A and TS at protein level c).
Figure 2 - Predictive role of TOP2A and TS expression for the efficacy of adjuvant mitotane. Marker expression is dichotomised at the median value.
Figure 3 - Correlation between markers expression and Clinical Benefit (Partial Response and Disease Stabilization of EDP-M scheme a) -TOP2A, b) - TS, c) - ERCC1, d) - RRM1.
Figure 4 - Relationship between TOP2A expression and time to progression (TTP) in patients treated with EDP-M.
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| Characteristics | N (%) |
|---|---|
| No. | 98 |
| Age, y | |
| Median (range) | 47 (18-85) |
| Sex, no. (%) | |
| Male | 40 (40.8%) |
| Female | 58 (59.2%) |
| ENSAT ACC stage | |
| I | 11 (11.2%) |
| II | 49 (50.0%) |
| III | 32 (32.7%) |
| IV | 6 (6.1%) |
| Secreting tumor, no. (%) | |
| Non-secreting tumor | 51 (52.0%) |
| Cortisol +/- Androgens | 30 (30.6%) |
| Androgens | 8 (8.2%) |
| Estradiol | 3 (3.1%) |
| Mineralcorticoids | 4 (4.1%) |
| Not Available | 2 (2.0%) |
| Weiss score | |
| Median (range) | 5 (3-9) |
| Adjuvant mitotane, no. (%) | |
| Yes | 38 (38.8%) |
| No | 54 (55.1%) |
| Patients with recurring ACC, no. (%) | 64 (65.3%) |
| Patients who died of ACC progression, no. (%) | 51 (52.0%) |
| Patient and tumor Characteristics | TOP2A Low | TOP2A High | TS Low | TS High | ||
|---|---|---|---|---|---|---|
| Sex | ||||||
| Female | 33 (67.35%) | 25 (51.02%) | p=0.10 | 30 (62.50%) | 27 (55.10%) | p=0.46 |
| Male | 16 (32.65%) | 24 (48.98%) | 18 (37.50%) | 22 (44.90%) | ||
| Age | ||||||
| ≤47 | 25 (51.02%) | 25 (51.02%) | p=1 | 26 (54.17%) | 24 (48.98%) | p=0.61 |
| >47 | 24 (48.98%) | 24 (48.98%) | 22 (45.83%) | 25 (51.02%) | ||
| Disease Stage | ||||||
| I-II | 32 (65.31%) | 28 (57.14%) | p=0.41 | 32 (66.67%) | 27 (55.10%) | p=0.24 |
| III-IV | 17 (34.69%) | 21 (42.86%) | 16 (33.33%) | 22 (44.90%) | ||
| Clinical Syndrome | ||||||
| No | 28 (57.14%) | 25 (51.02%) | p=0.54 | 25 (52.08%) | 27 (55.10%) | p=0.76 |
| Yes | 21 (42.86%) | 24 (48.98%) | 23 (47.92%) | 22 (44.90%) | ||
| Cortisol excessism | ||||||
| No | 34 (69.39%) | 34 (69.39%) | p=1 | 33 (68.75%) | 34 (69.39%) | p=0.94 |
| Yes | 15 (30.61%) | 15 (30.61%) | 15 (31.25%) | 15 (30.61%) | ||
| Weiss Score | ||||||
| ≤5 | 27 (56.25%) | 20 (43.48%) | p=0.21 | 28 (60.87%) | 18 (38.30%) | p=0.03 |
| >5 | 21 (43.75%) | 26 (56.52%) | 18 (39.13%) | 29 (61.70%) | ||
| Mitoses | ||||||
| <2 | 30/51 (62.5%) | 21/51 (46.7%) | p=0.12 | |||
| >2 | 18/42 (37.5%) | 24/42 (53.3%) | ||||
| ERCC1 | ||||||
| Low | 27 (55.10%) | 24 (48.98%) | p=0.54 | 24 (50.00%) | 27 (55.10%) | p=0.61 |
| High | 22 (44.90%) | 25 (51.02%) | 24 (50.00%) | 22 (44.90%) | ||
| RRM1 | ||||||
| Low | 30 (61.22%) | 19 (38.78%) | p=0.026 | 18 (37.50%) | 31 (63.27%) | p=0.011 |
| High | 19 (38.78%) | 30 (61.22%) | 30 (62.50%) | 18 (36.73%) | ||
| TOP2A | ||||||
| Low | 31 (64.58%) | 18 (36.73%) | p=0.006 | |||
| High | 17 (35.42%) | 31 (63.27%) | ||||
| TS | ||||||
| Low | 31 (63.27%) | 17 (35.42%) | p=0.006 | |||
| High | 18 (36.73%) | 31 (64.58%) |
15
TOP2A mRNA (ΔΔΟ t)
4
200
TS mRNA (AAC t)
a
b
TS IHC (H-score)
C
3
150
10
2
100-
5
1
50
0
0
0
0
50
100
150
200
0
50
100
150
200
0
50
100
150
TS IHC (H-score)
TOP2A IHC (H-score)
TOP2A IHC (H-score)
| Spearman's R | 0.46 | Spearman's R | 0.52 | Spearman's R | 0.45 |
| 95% CI | 0.10 - 0.72 | 95% CI | 0.22 - 0.73 | 95% CI | 0.09 - 0.70 |
| P (two-tailed) | 0.013 | P (two-tailed) | 0.001 | P (two-tailed) | 0.014 |
Figure 1
Disease Free Survival
| TOP2A high | p=0.79 |
| TOP2A low | p=0.20 |
| TS high | p=0.26 |
| TS low | P=0.23 |
0.0 0.25 0,50 0,75 1,0 1,25 1,5 1,75 2,0
Hazard Ratio
Figure 2
100%
90%
80%
70%
60%
50%
PD
40%
SD/PR
30%
20%
10%
0%
p=0.0039
Low TOP2A
High TOP2A
Figure 3a
100%
90%
80%
70%
60%
50%
PD
40%
SD/PR
30%
20%
10%
0%
p=0.049
Low TS
High TS
Figure 3b
100%
90%
80%
70%
60%
50%
PD
40%
SD/PR
30%
20%
10%
0%
p=0.69
Low ERCC1
High ERCC1
Figure 3c
100%
90%
80%
70%
60%
50%
PD
40%
SD/RP
30%
20%
10%
0%
Low RRM1
High RRM1
p=0.42
Cumulative Proportion Progression Free Surviving
1,0
0,9
0,8
TOP 2A mRNA low
- - TOP 2A mRNA high
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0,0
0
5
10
15
20
25
30
35
40
Months
Figure 4a
1,0
Cumulative Proportion Pregression Free Surviving
0,9
0,8
0,7
IHC TOP2A negative (≤30%)
- - IHC TOP2A positive (>30%)
0,6
0,5
0,4
0,3
0,2
0,1
0,0
0
5
10
15
20
25
30
35
Months
Figure 4b