A 60-year-old Caucasian woman presented with a chief complaint of abdominal and back pain. There was no history of smoking or excess alcohol consumption. Her family history was unremarkable. She had no relevant previous medical or surgical history. On admission, physical examination revealed jaundice. Radiographic examination showed a mass, measuring 3.0 cm in diameter, within the head of the pancreas. The computed tomographic imaging of the chest and abdomen did not show any evidence of extra-pancreatic disease.
The patient underwent pylorus-preserving pancreaticoduodenectomy. Examination of the surgical specimen showed a 3.0 cm sized mass, invading the duodenal wall and common bile duct. On light microscopy, the neoplasm was composed entirely of small to medium-sized cells possessing scant cytoplasm and oval nuclei with a finely granular chromatin pattern (). The neoplasm showed extensive necrosis and a high frequency of mitotic figures (). Metastasis was identified in 10 of 23 sampled lymph nodes. Venous/lymphatic vessel and perineural invasion was also present. Complete review of all histologic sections did not reveal evidence of a component of ductal adenocarcinoma. Scattered pancreatic intraepithelial neoplasia-1 (PanIN-1) and 2 lesions were present in the background pancreas. The surgical resection margins were negative for neoplastic cells. The final pathological diagnosis was SCC of the pancreas with lymph node metastases.
Figure 1 Photomicrographs of primary small cell carcinoma of the pancreas (patient’s original tumor) (a) and heterotransplanted tumor tissue in a nude mouse inoculated with A99 (b) demonstrating similar histopathologic characteristics. Necrosis (arrows (more ...)
Six weeks after surgical resection, the patient received chemotherapy consisting of cisplatin (60 mg/m2) given intravenously on day 1 and etoposide (120 mg/m2) intravenously on days 1–3, repeated every 3 weeks for three cycles. Three months after initial chemotherapy, a computed tomographic scan showed multiple liver metastases. The chemotherapy regimen was switched to obatoclax mesylate (GX15-070MS, 12.5 mg/m2), a Bcl-2 antagonist, given intravenously on days 1 and 3 in combination with topotecan (1.25 mg/m2) intravenously on days 1–5 but this second-line chemotherapy was ceased during the first cycle due to neutropenia. The patient’s clinical course continued to deteriorate and she expired 13 months from the time of her surgery.
Prior to death, she consented to an autopsy for research purposes. At autopsy, multiple liver metastases, pleural studding of the lungs and peritoneum, and local recurrence were grossly identified. The local recurrence and multiple metastases were sampled and tumor tissues sliced into 1.0 × 1.0 × 0.2 cm sections for overnight fixation in 10% buffered-formalin and for snap-freezing in liquid nitrogen. Histological examination confirmed the gross impression of recurrent SCC.
Autopsy Specimen Collection and Maintenance of Xenografts and Tumor Cell Line
One liver metastasis was obtained in sterile fashion, and 2 to 3 mm3
samples were placed into sterile RPMI media (Invitrogen, Carlsbad, CA) for transport to the animal facility for xenografting into athymic nude mice that were maintained in a laminar-flow hood under pathogen-free conditions. Xenografting was performed as described by Hahn et al 7
. Tumor tissues were minced with a sterile blade into 1 mm3
pieces in Matrigel (Becton Dickinson, Bedford, MA), and were implanted beneath the skin fold of the back bilaterally while the mice were under isoflourane inhalation anesthesia. The animals were followed weekly for growth of xenografts.
When the xenografts reached ~10 mm in size, the tumors were excised and a portion of each was placed in 10% formalin for paraffin embedding or snap frozen in preparation for subsequent genetic analyses. A final portion was placed in DMEM medium with 20% fetal bovine serum (FBS), minced with sterile blades and digested with collagenase, type IV (Invitrogen), and hyaluronidase (Sigma-Aldrich, St. Louis, MO) for 30 minutes. The digested cells were centrifuged 10 minutes at 1,000 rpm and resuspended into DMEM culture media supplemented with 20% FBS, penicillin 100 U/mL, streptomycin 100 μg/mL, gentamicin 50 μg/mL and amphotericin B 2.5 μg/mL at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. The supernatant with solid remnants was removed 24 hours later and replaced with fresh medium. Cells were passaged at 50% to 100% confluence and medium was exchanged every 2 days. Cell cultures were routinely monitored for mycoplasma contamination, and no mycoplasma growth has been detected.
Colony Formation in Soft Agar
Six-well plates were filled with semisolid medium containing DMEM, 1% Bacto agar and 20% FBS and 50,000 cells in a semisolid medium (DMEM with 1% Bacto agar and 10% FBS) were added per well. Additional medium (DMEM with 20% FBS, penicillin, streptomycin, gentamicin and amphotericin B) was added to the wells to keep the agar moist, and 2.5 mL of fresh medium were added to each well every 3 days thereafter. After incubation for 21 days at 37°C, to assess colony formation, the medium was removed, and 3 mL of 0.005% Crystal Violet (Sigma-Aldrich) staining solution was added to each well. After incubation at 37°C for 3 hours, removal of the staining solution and washing twice with phosphate-buffered saline (PBS), colonies were visualized by trans-UV illumination and counted using the analysis software Quantity One (Bio-Rad, Hercules, CA).
Tumorigenicity in Nude Mice
A99 cells at passage 15 were grown to 80% confluence and trypsinized, and >90% cell viability was confirmed by trypan blue exclusion. Three 5-weeks-age athymic nude mice were subcutaneously(s.c.) inoculated into the back bilaterally. 0.5 × 107 or 1.7 × 107 of A99 cells of two different passages in 200 μL of PBS were used for each injection. The mice were observed weekly for the development of xenografts. At 8 weeks after injection, the s.c. xenografted tumors reached a diameter of 13–22 mm. The mice were sacrificed and the s.c. tumors formed from A99 cells were excised and a portion of each was placed in 10% formalin for paraffin embedding or snap frozen in liquid nitrogen.
The tissue samples of the patient’s original tumor specimen, first passage xenografts and the s.c. tumor formed from A99 cells were formalin-fixed and paraffin-embedded. Paraffin blocks were cut into sections 4 μm thick for the immunolabeling by the avidin-biotin complex method, with all labeling processes, from deparaffinization to counterstaining with hematoxylin, being performed automatically with the Ventana Discovery staining system (Ventana Medical Systems, Tucson, AZ). Antibodies used for immunohistochemical labeling are summarized in .
Antibodies used in the immunohistochemical study
DNA Extraction and Sequencing for KRAS, p16 and TP53
Genomic DNA was extracted from A99, its corresponding xenograft and the original metastatic tissue from which it was derived at autopsy using standard phenol-chlorophorm-isoamyl alcohol method or DNeasy Blood & Tissue Kits (Qiagen, Valencia, CA). Polymerase chain reactions (PCR) of KRAS
exons 1 and 2, p16
exons 1 and 2 and TP53
exons 5 to 9 were performed as described 8
. PCR products were sequenced by use of a universal M13F primer that was incorporated into the forward primer of each primer pair (Beckman Coulter Genomics, Danvers, MA). Sequence data were analyzed with Sequencher 4.10.1 software (Gene Codes, Ann Arbor, MI).
The cell culture was harvested with exposure to 10 ng/mL Colcemid for 4 hours, incubated in cancer hypotonic solution at 37°C for 30 minutes, and fixed in 3:1 methanol:glacial acetic acid. Trypsin-Leishman staining procedure was used for G-band karyotyping. Chromosomal abnormalities were described using ISCN 2009 nomenclature 9