ERCC1 Expression As a Predictive Marker of Cervical Cancer Treated with Cisplatin-based Chemoradiation

Materials and Methods

A total of 217 patients diagnosed with locally advanced cervical cancer between 2006 and 2012 at the Charité University Hospital, Charité Mitte, Virchow, and Benjamin Franklin campuses were identified depending on retrospective databases. Seventy-two patients were excluded due to lack of documentation, incomplete radiochemotherapy, and prior definitive treatment for cervical cancer or active malignancy at another abdominal site.

In 33 cases out of the remaining 145, the analyses for ERCC1 could not be completed. This was either due to insufficient pathological specimen or when the primary pathological study was performed outside our campuses. The final collective of this study consisted of 112 patients.

Standard staging procedures were clinical examination with or without cystoscopy and sigmoidoscopy. Nodal status was determined by pre-treatment staging laparascopy with systematic transperitoneal pelvic and para-aortic lymph node dissection as described elsewhere (12).

Radiochemotherapy planning. All patients underwent planning computed tomography (CT) scan (CT scanner LightSpeed; GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) with intravenous contrast media (Xenetix 350®; Guerbet GmbH, Sulzbach, Hessen, Germany) at a slice thickness of 3.75 mm. In cases of para-aortic involvement, CT scans were carried out from the diaphragm to the lesser trochanter. In histologically-proven negative para-aortic lymph nodes (LN), the CT scan was performed from the second lumbar vertebra to the trochanter minor. The planning CT was performed while the patient was in the supine position using a knee and foot positioning device, and patients were asked to have a full bladder.

Target volumes and organs at risk (OAR). Target volumes and OAR were delineated in all axial CT slices according to the recommendations of the Radiation Therapy Oncology Group (RTOG) (13) and the International Commission on Radiation Units and Measurements Reports 50 (14). The planned target volumes (PTV) were defined on the basis of the full-bladder CT scan. They were divided into PTV-A and PTV-B (boost) volumes, and the concept of simultaneously-integrated boost (SIB) was applied. The clinical target volume (CTV-A) was defined as the macroscopic tumor, including the cervix and the corpus uteri and external, internal, common iliac, and pre-sacral LNs plus/minus the para-aortic LNs with a 5-mm margin. In patients treated before 2009, a sequential boost to the parametric regions was given with 1.8 Gy single doses to 59.4 Gy total dose to the PTV-B, while PTV-A was treated as described below.

The following OARs were delineated: spinal cord, femoral heads, kidneys, bladder, rectum up to the sigmoidal loop, and the small bowel (SB) as the whole peritoneal cavity except for LNs, muscles, and OAR other than the SB. The delineation of the SB exceeded the upper and lower border of the PTV-A by two slices. Institutional dose constraints were used: SB: V45 <20%; V20 <40%; D mean <30 Gy; rectum: V40<70%; V50 <50%; bladder: V30 <60%; V50 <30%: femoral heads: D mean <40 Gy. No constraints were used for the bone marrow.

In addition to external-beam radiation, patients underwent Ir-192-High dose rate (HDR)-brachytherapy (Gammamed 12i and Brachyvision; Varian Medical Systems, Palo Alto, CA, USA) with a total dose of 25-30 Gy (five fractions at 5-6 Gy each) delivered to the cervix including the macroscopic tumor. Since 2007, PTV for brachytherapy has been defined on the basis of the magnetic resonance imaging (MRI).

Dose prescription and planning parameters. The total dose prescribed for the PTV-A was 50.4 Gy delivered in 28 fractions (five weekly) as single doses of 1.8 Gy each. The corresponding dose prescribed to the PTV-B was 59.36 Gy delivered as single doses of 2.12 Gy each for the SIB concept and 1.8 Gy single dose to a total dose of 50.4 Gy (whole pelvis ± para-aortic LN) and 59.4 Gy to the parametrial region.

Radiation technique. Until 2007, 3D planning was performed; later patients were treated either with conventional 7-field intensity-modulated radiotherapy (IMRT) or helical tomotherapy® (HT) or Rapid Arc®.

Contouring for both IMRT modalities and HT was performed with the Therapy Planning System (TPS) of Eclipse (Varian Medical Systems). The CT datasets with contoured structures were then transferred to the Tomotherapy TPS (TomoTherapy Inc., Madison, WI, USA), enabling inverse treatment planning for photon irradiation at 6 MV with HT.

Conventional linac-based IMRT plans were calculated for photon irradiation at 6 MV using the Eclipse TPS; before 2009, 6-20 MV X-rays were used.

Treatment. Primary treatment consisted of external-beam radiation and simultaneous chemotherapy with cisplatin 40 mg/m² body surface once weekly for five applications. In case of contraindications, carboplatin was used [Area under the curve (AUC) 1.5 weekly] in only three patients (2.7% of cases). Mean treatment time was 45 days. Therapy was completed in 110 patients of our final study group (98.2%).

Follow-up. According to German Guidelines, follow-up examination was carried out four times in the first two years after treatment, then twice a year, and included a gynecological examination, detailed patient history and renal ultrasound scan. Additionally to this standard procedure, all patients underwent at least one curettage three months after completion of chemoradiation to confirm histological response.

Immunohistochemistry for ERCC1. Tumor specimens from 112 patients were evaluated for ERCC1 expression. Monoclonal antibody against ERCC1 (clone 8F1; dilution 1:50; Abcam, Cambridge, UK) was used to detect ERCC1 expression.

ERCC1 staining was performed on a Ventana BENCHMARK® XT Instrument using UltraView DAB kit (Ventana Medical Systems, Tucson, AZ, USA). Briefly, for epitope retrival, slides were exposed to heat with EDTA, then enogenous peroxidase activity was blocked by incubation with 3% H₂O₂ (ERCC1: 30 min EDTA and 4 min H₂O₂). Incubation with primary antibody to ERCC1 was carried out for 32 min at 37°C. Immunoreaction was revealed by incubation with secondary antibody for 8 min, with 3’-3’-diaminobenzidine as the chromogen and Mayer's hematoxylin as the counterstain (Figure 1).

Microscopical analyses. An experienced investigator who was unaware of clinical data evaluated ERCC1 staining under a light microscope. She recorded the percentage of the tumor cells expressing ERCC1. Nuclear immunoreactivity was scored as positive for ERCC1.

The staining intensity was graded on a scale of 0-3: 0, negative staining; 1, mild staining; 2, moderate staining; 3, strong staining. The percentage of positively stained tumor cells was calculated for each specimen, and a proportion score was assigned (0 if 0%, 0.1 if 1-9%, 0.5 if 10-49%, and 1 if 50% or more). This proportion score was multiplied by the staining intensity of nuclei to obtain an overall score, the final semiquantitative H-score. Score 3 was assessed as high expression, score between 1.5 and 2.5 as intermediate, and score between 0 and 1 as low expression. Statistical method. Primary outcome measures were progression-free survival and overall survival. Recurrence was defined as any histologically confirmed disease recurrence. Death from disease was defined as any complications associated with cancer as well as strict disease mortality. Progression-free survival (PFS) was measured from the date of diagnosis to the date of relapse, progression, death, or last follow up. Overall survival (OS) was measured from the date of diagnosis to the date of death, or date of last follow-up. Kaplan–Meier event-free survival curves were used to estimate 2-year rates, and the log-rank (Mantel–Cox) test was used to compare the curves for statistical significance.