Objective
We sought to identify effective chemotherapy regimens against uterine serous papillary adenocarcinoma (USPC).
Study Design
Six USPC, half of which overexpress HER-2/neu at 3+ level, were evaluated for growth rate and in vitro sensitivity to 14 single-agent chemotherapies and 5 combinations by ChemoFx (Precision Therapeutics Inc, Pittsburgh, PA).
Results
Cell lines overexpressing HER-2/neu showed higher proliferation when compared to low HER-2/neu-expressing cell lines and a lower half maximum inhibitory concentration (IC 50 ) when exposed to the majority of single-agent chemotherapies. High HER-2/neu expressors were more sensitive to platinum compounds, manifesting a 5.22-fold decrease in carboplatin-IC 50 ( P = .005) and a 5.37-fold decrease in cisplatin-IC 50 ( P = .02). When all cell lines were analyzed as a group, chemotherapy agents tested demonstrated lower IC 50 when used in combination than as individual agents.
Conclusion
USPC overexpressing HER-2/neu display greater in vitro sensitivity to platinum compounds when compared to low HER-2/neu expressors. Higher proliferative capability rather than increased drug resistance may be responsible for the adverse prognosis associated with HER-2/neu overexpression in USPC.
Endometrial cancer is the most common female genital tract malignancy in the United States, with an incidence of 42,160 new cases and 7780 deaths annually. Two types of endometrial carcinoma, namely type I and II tumors, have been described, based on both clinical and histopathological variables. Type I endometrial cancers, which account for the majority (about 80%) of cases, are usually well or moderately differentiated and endometrioid in histology. These neoplasms are frequently diagnosed in younger women, are associated with a history of hyperestrogenism as the main risk factor, and typically have a favorable prognosis with appropriate therapy. In contrast, type II endometrial cancers are poorly differentiated tumors, or tumors with serous papillary or clear cell histology. Although type II tumors account for only a minority of endometrial cancers, most recurrences and deaths occur in this group of patients.
Uterine serous papillary adenocarcinoma (USPC), which accounts for about 10% of endometrial cancers, has a propensity for early intraabdominal and lymphatic spread even at presentation and is characterized by a highly aggressive biologic behavior. Unlike the histologically similar high-grade ovarian cancer, USPC is a chemoresistant disease from onset, with in vivo responses to combined cisplatin-based chemotherapy in the order of 20% and of short duration. Our group has recently reported HER-2/neu overexpression by immunohistochemistry (IHC) and amplification of the c-erbB2 gene by fluorescent in situ hybridization (FISH) in a large percentage of patients harboring USPC. These findings, recently confirmed by other groups including the Gynecologic Oncology Group in a cooperative multicentric study, have identified HER-2/neu overexpression in USPC as an independent variable associated with poor outcome, and one that occurs more frequently in African American women than in Caucasian women.
Although HER-2/neu overexpression has been previously associated with resistance to chemotherapy and poor survival in multiple human malignancies including breast, ovarian, and endometrial carcinoma, to our knowledge, no study has carefully evaluated the in vitro chemosensitivity/chemoresistance of this highly aggressive variant of endometrial cancer. To fill this gap in knowledge, we used ChemoFx (Precision Therapeutics Inc, Pittsburgh, PA) to analyze the in vitro sensitivity of 6 primary USPC cell lines recently established and characterized in our laboratory to 14 standard single-agent chemotherapies and 5 combination chemotherapies. Half the cell lines selected overexpress HER-2/neu at 3+ levels and harbor amplification of the c-erbB2 oncogene by FISH. Surprisingly, although in vivo HER-2/neu overexpression is correlated with a more aggressive disease in patients with USPC, growth-inhibition data suggest that these tumors display greater in vitro chemosensitivity to platinum compounds and higher proliferative capability when compared to HER-2/neu-negative tumors.
Materials and methods
Establishment and HER-2/neu expression of USPC cell lines
Primary USPC tumor cell lines from 6 patients with invasive USPC were obtained from fresh tumor biopsies collected at the time of surgery, under approval of the institutional review board. Tumors were staged according to the International Federation of Gynecologists and Obstetricians operative staging system. Six primary USPC cell lines (USPC ARK-1, USPC ARK-2, USPC ARK-3, USPC ARK-4, USPC ARK-5, and USPC ARK-6) were established after sterile processing of the tumor samples from surgical biopsies as described previously. Source-patient characteristics of these 6 USPC cell lines are described in Table 1 . The amplification of the c-erbB2 gene by FISH, expression levels of HER-2/neu receptor by IHC, and messenger RNA expression levels by quantitative real-time polymerase chain reaction for these primary USPC cell lines have been recently reported and are presented in Table 2 .
| Patient | Age, y | Race | FIGO stage | USPC histopathology | Year of diagnosis |
|---|---|---|---|---|---|
| USPC ARK-1 | 62 | AA | IVA | Pure | 1997 |
| USPC ARK-2 | 63 | AA | IVB | Pure | 1998 |
| USPC ARK-3 | 59 | AA | IVB | Mixed | 2006 |
| USPC ARK-4 | 73 | C | IVB | Pure | 2004 |
| USPC ARK-5 | 73 | AA | IIIC | Pure | 2006 |
| USPC ARK-6 | 62 | C | IB | Mixed | 2005 |
| RT-PCR | |||
|---|---|---|---|
| Sample | IHC | FISH | mRNA copy no. |
| Control | 1 | ||
| USPC ARK-1 | 3+ | 2.5 | 373 |
| USPC ARK-2 | 3+ | 5.2 | 607 |
| USPC ARK-3 | 3+ | 4.7 | 677 |
| USPC ARK-4 | 0 | 1.6 | 7 |
| USPC ARK-5 | 0 | 1.4 | 13 |
| USPC ARK-6 | 1+ | 0.9 | 6 |
Primary USPC growth rate analysis
The growth rate of each of the USPC cell lines was determined by counting the number of live cells 24, 48, 72 and 96 hours after plating. In brief, cell lines were cultured in RPMI 1640 (Mediatech, Manassas, VA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). When cultures had grown to approximately 80% confluence, cells were harvested from the flask using 0.25% trypsin EDTA (Invitrogen, Carlsbad, CA), then counted on a hemacytometer chamber and assessed for viability via trypan blue exclusion. Cell density was adjusted to a concentration of 8000 cells/mL, then cells were seeded into a 384-well microtiter plate (Corning Life Sciences, Lowell, MA) at a density of 320 cells per well. Twenty-two replicate wells were plated per cell line per time point. Cell plates were incubated at 37°C with 5% carbon dioxide for 24, 48, 72, and 96 hours, at which time they were removed from the incubator, fixed with 95% ethanol (Fisher Scientific, Pittsburgh, PA) then stained with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Molecular Probes; Invitrogen, Carlsbad, CA). When DAPI staining was complete, cell plates were scanned on an automated fluorescent imaging system, and the number of cells in each well was counted.
Chemoresponse assay
The chemoresponse assay was performed as previously described. The chemotherapy agents, concentrations, and combination used in the assays are described in Table 3 . Briefly, cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. When cultures had grown to approximately 80% confluence, cells were harvested from the flask using 0.25% trypsin EDTA, then counted on a hemacytometer chamber and assessed for viability via trypan blue exclusion. Cell density was adjusted to a concentration of 8000 cells/mL. Then cells were seeded into a 384-well microtiter plate at a density of 320 cells per well. Cell plates were incubated at 37°C with 5% carbon dioxide overnight. On the next day, serial dilutions of each anticancer agent or combination of 2 agents were prepared in growth medium to create 8, 9, or 10 distinct testing concentrations. In total, 14 single agents were tested as well as 5 combinations of 2 agents. Due to the limited amounts of cells available for testing for USPC-ARK-5 primary cell line, 6 of the 14 single agents were not tested (ie, cyclophosphamide, fluorouracil, ifosfamide, vinblastine, vincristine, and vinorelbine) nor were any of the combinations. Three replicates of each dose of agent or combination were applied to each cell line. The cell plates were incubated for an additional 72 hours after treatment, at which time they were removed from the incubator, fixed with 95% ethanol, then stained with DAPI. When DAPI staining was complete, cell plates were scanned on an automated fluorescent imaging system, and the number of surviving cells in each well was counted, and compared to a control population not exposed to any agent.
| Chemotherapy | Lowest dose | Highest dose | Units | No. of concentrations |
|---|---|---|---|---|
| Carboplatin | 0.98 | 500 | μmol/L | 10 |
| Cisplatin | 0.2 | 50 | μmol/L | 8 |
| Cyclophosphamide | 0.33 | 14 | μmol/L | 9 |
| Docetaxel | 0.1 | 25 | nmol/L | 9 |
| Doxorubicin | 2.34 | 1200 | nmol/L | 9 |
| Etoposide | 2.867 | 200 | μmol/L | 9 |
| Fluorouracil | 0.2 | 50 | μmol/L | 8 |
| Gemcitabine | 0.73 | 50 | nmol/L | 9 |
| Ifosfamide | 0.2 | 100 | μmol/L | 9 |
| Paclitaxel | 0.2 | 100 | nmol/L | 9 |
| Topotecan | 0.78 | 200 | nmol/L | 8 |
| Vinblastine | 0.15 | 10 | nmol/L | 9 |
| Vincristine | 0.98 | 125 | nmol/L | 8 |
| Vinorelbine | 0.42 | 29.41 | nmol/L | 9 |
| Carboplatin and docetaxel | 0.98 | 500 | μmol/L | 9 |
| 0.1 | 25 | nmol/L | ||
| Carboplatin and doxorubicin | 0.98 | 500 | μmol/L | 9 |
| 2.34 | 1200 | nmol/L | ||
| Carboplatin and gemcitabine | 0.98 | 125 | μmol/L | 8 |
| 0.73 | 19.53 | nmol/L | ||
| Carboplatin and paclitaxel | 0.98 | 250 | μmol/L | 9 |
| 0.2 | 50 | nmol/L | ||
| Carboplatin and topotecan | 0.98 | 250 | μmol/L | 9 |
| 0.39 | 100 | nmol/L |
Statistical analysis
For each cell line, the half maximum inhibitory concentration (IC 50 ) for a given drug was determined from the dose-response curve fit to the cell-count data using XLFit program (ID Business Solutions, Parsippany, NJ). The Levenburg-Marquardt algorithm was used to obtain and calculate the IC 50 and IC 50 curves for each drug and drug combination in each cell line using a nonlinear regression-curve fit. To facilitate statistical inference on IC 50 fold changes, IC 50 were transformed to log 10 units. Differences in log 10 (IC 50 ) between high and low HER-2/neu expressors were assessed for significance via unequal-variance t test, while cell line differences in log 10 (IC 50 ) for agents used singly vs in combination were assessed for significance via paired t test. Fold changes in IC 50 were calculated as 10 raised to the power of the differences in log 10 (IC 50 ). Differences were considered significant at P values < .05. All statistical analysis was conducted using software (Excel 2007; Microsoft Corp, Redmond, WA).
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