Clinical evaluation
A Caucasian woman, initially 19 years-old, had a story of persistent abdominal cramps. Late in 2012 an abdominopelvic ultrasound showed a heterogeneous nodular expansive mass occupying the vesicouterin space, with 5,0 × 3,2 cm in the largest dimension. In December 2012, an abdominopelvic magnetic resonance (MRI) demonstrated similar findings, and additionally peritoneal implants without lymph node enlargement. No primary tumor was suggested after the MRI. The primary diagnostic hypothesis was an immature teratoma of the ovary.
Initial treatment
The patient underwent biochemical investigation, clinical and radiological staging. The biomarkers were characteristic of an epithelial tumor as it follows: CA-125 278.8 UI/mL (Reference Value – RV < 35 UI/mL), Carcinoembriogenic Antigen (CEA) < 0.5 ng/mL (RV < 5 ng/mL), CA19-9 13.7 UI/mL (RV < 39 UI/mL), hCG (human chorionic gonadotrofin) < 0.5 nUI/mL (RV < 2 nUI/mL), alpha fetoprotein (AFP) < 0.6 ng/mL (RV < 13.7 ng/ML).
In January 2013, an exploratory laparotomy was performed, with biopsies of right ovary, sigmoid implant, omentum and left uterine tube. Every biopsy demonstrated a low-grade serous papillary adenocarcinoma of the ovary and peritoneal cytology was positive for malignant cells. In February 2013, a complete cytorreduction was performed with pelvic and retroperitoneal lymphadenectomy. The histopathologic analysis of the right ovary reported a low-grade serous papillary cystadenocarcinoma, with invasion of right uterine tube, uterine corpus, adjacent colon, cecal appendix, omentum, and multiple peritoneal areas. Six of 67 lymph nodes were positive for malignancy. There were areas containing cells with high-grade nuclear index and low mitotic activity, which suggests the presence of scattered high-grade neoplastic areas.
She was admitted to the Medical Oncology Department in March 2013. The therapeutic plan consisted of 6 cycles of adjuvant Carboplatin with a predicted area under the curve (AUC) of 5 with paclitaxel 175 mg/m2 every 21 days. The patient was offered for the dosage of circulating tumor cells (CTC) with complete acceptance. Chemotherapy cycles were completed in August 2013, without any delays or grade 3 or 4 toxicities. Follow-up was started in September 2013. No previously identified pathogenic mutations in BRCA1 and BRCA2 genes were found.
Disease recurrence
Planned follow-up was provided with imaging studies every three months, combined with laboratory tests and CA125 dosage. CTC dosage was paralleled to ambulatory visits and not deemed as part of the follow-up protocol. The images did not show progressive disease (PD) until April/2014, when the CA125 modestly rose (14.3 UI/mL in December 2013 to 34.2 UI/mL in April/2014 and continued to rise until July/2014, with 47.2 UI/mL peak). An abdominopelvic CT was performed, showing disease recurrence.
In August/2014 an optimal secondary cytorreduction was performed. The pathological report showed recurrence of the disease: low grade serous papillary cystadenocarcinoma of the ovary with infiltration of vesical peritoneum, sigmoid colon, obturatory fossa, spleen, stomach, duodenum, liver, gallbladder, and diaphragm, including other peritoneal areas.
After the secondary cytorreduction, a new course of chemotherapy was initiated with Carboplatin AUC 4 day 1 + Gemcitabine 1000 mg/m2 days 1 and 8 + Bevacizumab 15 mg/kg [8] in September/2014. She underwent a difficult course of chemotherapy for 6 cycles, requiring granulocyte colony stimulating factor (G-CSF) support. Maintenance bevacizumab was offered until January/2016, when PD was identified in a nodule in abdominal wall. Third line chemotherapy was initiated with Carboplatin AUC 5 day 1 + Pegylated Liposomal Doxorubicin 30 mg/m2 every 28 days.
Methods
This patient was evaluated according to best practice protocols for Ovarian Cancer. Every treatment was conducted at A.C.Camargo Cancer Center, following every rule for patient safety.
Isolation, counting and characterization of circulating tumor cells
Blood samples were collected from patient after she had signed the informed consent to study with circulating tumor cells in solid tumors, which has been approved by the Local Research Ethics Committee of A.C. Camargo Cancer Hospital, São Paulo, Brazil (protocol number: 1367–10).
Blood (8 mL) was collected in EDTA tubes, stored at room temperature under homogeneization for up to 4 h, and treated by ISET (Isolation by Size of Tumor cells (ISET) (Rarecells Diagnostics, Paris, France) according to manufacture procedure. This methodology uses polycarbonate membrane with 8-μm-diameter cylindrical pores to isolate intact CTCs from blood through direct filtration, based on the concept that of the larger size of tumor cells as compared with leukocytes.
Rapidly, 8 ml blood was diluted on ISET buffer, transferred to the ISET block and filtered. Cells that have more than 8 μm were maintained by negative pressure in the membrane, that was stored at - 20 °C until time of analysis. Cells were considered as CTCs if they have presence of hypercromatic nucleus, irregular shape, high cytoplasm nucleus ratio (>0.8), stain negatively for CD45 and have nuclear size ≥12 μm (Krebs et al., 2012). For cell counting, ISET membrane spots were cut out and used for single immunocytochemistry (DAB+/DakoTM) for exclusion of leucocytes (CD45; 1:100; clone 2B11+ PD7/26, DakoTM), according to the manufacturer’s instructions. Eight spots on the ISET filter were used for counting, corresponding to 8 mL of blood [9, 10].
Circulating tumor cells analysis
The chronologic relationship between dosages of CA125 and CTCs is shown in Fig. 1. At the first evaluation, before receiving any surgical or chemotherapeutic treatment CTC dosage was 7.5 CTCs/mL. In that opportunity, the CA125 was 26.8 UI/mL. After this first dosage, the patient underwent the first treatment course (Carboplatin AUC 5 + Paclitaxel 175 mg/m2). Her following evaluations did not show any prediction of progression for CA125 until ascension in CTC dosage (see Fig. 1) with a peak of 3.6 CTCs/mL in April/2014, and an only modest ascension in CA125 (33.9 UI/mL). In July 2014, CTCs and CA125 were still rising (5.3 CTCs/mL and 45.0 UI/mL respectively), with demonstration of PD of images in August 2014. After the end of the proposed second line chemotherapy and during bevacizumab maintenance CTCs levels were stable.
Follow-up was continued during bevacizumab maintenance, with a novel and slow rise until PD. In March/2016 there was an increase on CA125 without an increase in CTC. Later, on May/2016, CTC levels rised, with a stable level of CA125. This finding is considered as related to the low burden of disease on the abdominal wall and probable stability of peritoneal disease, thus, CTCs may not be of analytic value for superficial progressive disease, such as in this case, when progressive disease was detected only in the abdominal wall.
To evaluate the direct correlation between CTC and CA125 levels, a paired samples T-test was performed considering the value for every data-point using IBM SPSS Statistics 20.0 for Macintosh. This test showed a 0.868 significance when comparing the 12 paired dosages for both variables. The results of this test confirmed the impossibility to draw a definitive conclusion between CTC and CA125 and the possible correlation among both, considering a statistically significant p value of < 0.05.