HER2/neu as a potential target for immunotherapy in gynecological carcinosarcomas

Abstract

Carcinosarcomas of the female genital tract are rare tumors with aggressive clinical behavior. Trastuzumab, a humanized monoclonal antibody, acts by binding to HER2/neu extracellular domain and exhibits therapeutic efficacy in HER2/neu over-expressing cancers. Two uterine carcinosarcomas (UMMT-ARK-1, UMMT-ARK-2) and two ovarian carcinosarcomas (OMMT-ARK-1, OMMT-ARK-2) were established as primary tumor cell lines in vitro and evaluated for HER2/neu expression by immunohistochemistry (IHC), fluorescent-in-situ-hybridization-analysis (FISH), quantitative-real-time-polymerase-chain-reaction and for membrane-bound-complement-regulatory-proteins (mCRP) CD46, CD55 and CD59 by flow-cytometry. Sensitivity to trastuzumab-dependent-cell-mediated-cytotoxicity (ADCC) and complement-dependent-cytotoxicity (CDC) was studied in 5-hour-chromium-release-assays. HER2/neu expression was demonstrated in OMMT-ARK-1 and OMMT-ARK-2. OMMT-ARK-2 demonstrated amplification of the c-erbB2 gene by FISH and a high sensitivity to ADCC (mean killing 45.6%; range: 32.3–72.6%). Lower level of killing was detected against the FISH-negative OMMT-ARK-1 cell line (mean 26.5%; range: 21.0–31.8%). CD46, CD55 and CD59 mCRP were expressed at high levels in all primary MMT cell lines and all these tumors were found highly resistant to CDC with or without trastuzumab. Addition of untreated and heat-inactivated-plasma did not significantly decrease ADCC against OMMT-ARK-2 cell line suggesting that while the cell line is highly resistant to complement, irrelevant IgG do not significantly alter the ability of trastuzumab to mediate ADCC. Our results suggest that HER2/neu may represent a novel target for the immunotherapy of a subset of human carcinosarcomas refractory to salvage chemotherapy.

INTRODUCTION

Carcinosarcomas, also known as Malignant Mixed Müllerian Tumors, of the female genital tract are rare tumors. Uterine carcinosarcomas (UMMT) account for only 2–5% of all uterine cancers¹ and have an estimated recurrence rate of 40–60%². Ovarian carcinosarcomas (OMMT) occur much less frequently and account for less than 1–2% of all ovarian malignancies^3–5. According to an analysis of the Surveillance, Epidemiology, and End Results (SEER) program data, the age adjusted rate of uterine and ovarian carcinosarcomas is 0.6/100,000 and 0.19/100,000, respectively (Surveillance, Epidemiology, and End Results (SEER) Program; http://seer.cancer.gov/data/index.html; date of access 5.16.11).

Carcinosarcomas are histologically comprised of malignant epithelial and stromal components that are classified based on the homologous (tissues physiologically native to the primary site) or heterologous (mesenchymal components that are physiologically foreign to the primary site) nature of their stromal elements. The pathogenesis of carcinosarcomas remains under debate but most research supports the origin of both elements from a common epithelial cell line, rather than two independent progenitors⁶. Compared with other gynecological cancers, carcinosarcomas display an aggressive clinical behavior resulting in poorer survival^7–12. Approximately 35% of patients with uterine carcinosarcomas and 90% with ovarian carcinosarcomas are found to have tumor extending beyond the uterus and ovaries, respectively, at the time of diagnosis and most of these patients will develop recurrent disease^{9, 13}.

Although surgical debulking is the mainstay of treatment, there continues to be a need to identify effective adjuvant therapies given the high rate of tumor recurrence and poor survival in human MMT^{13, 14}. For all these reasons, targeted immunotherapy is increasingly being evaluated as a method for achieving clinically significant levels of tumor cell killing while minimizing damage to healthy tissues.

Human Epidermal Growth Factor Receptor 2 (HER2) (also known as ErbB2, c-erbB2 or HER2/neu) is a 185 kDa protein that belongs to the HER family of receptors which includes four structurally related members - HER1 (ErbB1, also known as EGFR), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). HER2/neu is a type 1 transmembrane glycoprotein composed of three distinct regions: an N-terminal extracellular domain (ECD), a single α-helix transmembrane domain, and an intracellular tyrosine kinase domain^{15, 16}. HER2/neu plays important roles in cell growth, survival, and differentiation in a complex manner^{15, 16}. The major signaling pathways mediated by HER2/neu involve mitogen activated protein kinase (MAPK) pathway and phosphatidylinositol 3-kinase (PI3K) pathway. As a key gene for cell survival, HER2/neu gene amplification and protein overexpression lead to malignant transformation^{15, 16}.

Trastuzumab (Herceptin; Genentech, San Francisco, CA) is a humanized monoclonal antibody that acts by binding to the extracellular domain of HER2/neu and exhibits therapeutic efficacy in HER2/neu over-expressing metastatic and early-stage breast cancers^{17, 18}. Trastuzumab has been shown to induce apoptosis in cancer cells by recruiting host immune cells (natural killer cells) and complement proteins and setting off an antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cell-cytotoxicity (CDC) process¹⁹. It also acts through inhibition of receptor dimerization and angiogenesis, leading to reduced cell growth and microvessel formation in vivo, and reduced endothelial cell migration in vitro¹⁶. Although HER2/neu overexpression has been previously reported in multiple human tumors including MMT of the female genital tract^20–28, its potential as an immunotherapy target in ovarian and/or uterine MMT primary cell lines refractory to standard treatment modalities has not been previously investigated.

The in vivo efficacy of cancer immunotherapy with complement-activating antibodies such as trastuzumab may be limited by overexpression of one or more membrane-bound complement regulatory proteins (mCRPs: CD46, CD55, CD59) on the surface of tumor cells as well as the presence of high concentrations of human IgG in the human plasma^29–31. In this report we have carefully studied the expression of HER2/neu and that of CD46, CD55 and CD59 mCRPs in multiple primary ovarian and uterine MMT cell lines, and evaluated for the first time the in vitro potential of trastuzumab as a novel immunotherapeutic agent against these biologically aggressive and chemotherapy-resistant tumors.

MATERIALS AND METHODS

Establishment of Cancer Cell Lines

Study approval was obtained from the institutional review board, and all patients signed an informed consent form according to institutional guidelines. A total of two primary uterine carcinosarcoma cell lines (UMMT-ARK-1 and UMMT-ARK-2) and two primary ovarian carcinosarcoma cell lines (OMMT-ARK-1 and OMMT-ARK-2) were established after the sterile processing of samples from surgical biopsy specimens, as previously described²². Both UMMT were established from biopsies of the uterus of chemotherapy naïve patients at the time of the primary staging surgery, while both OMMT were obtained from the biopsy of metastatic sites of disease in patients harboring recurrent, chemotherapy-resistant disease. In both OMMT cases, the high in vivo resistance to multiple chemotherapy agents was confirmed in vitro by MTT chemotherapy resistance assays against multiple cytotoxic agents (data not shown). Patient characteristics are described in Table 1. Briefly, tumor tissue was mechanically minced to portions no larger than 1 to 3 mm³ in an enzyme solution made of 0.14% collagenase type I (Sigma) and 0.01% DNase (Sigma, 2000 KU/mg) in RPMI 1640, and incubated in the same solution in a magnetic stirring apparatus for an hour at room temperature. Enzymatically dissociated cells were then washed twice in RPMI 1640 with 10% fetal bovine serum and maintained in RPMI supplemented with 10% fetal bovine serum, 200 µg/ml of penicillin and 200 µg/ml of streptomycin at 37°C, 5% CO₂ in 75 cm² tissue culture flasks or Petri dishes (Corning). After seeding on plasticware for 48–72 hours, nonadherent cells and contaminant inflammatory cells were gently removed from the culture by multiple washings with PBS.

Table 1.

Clinicopathological features of the tumors from whom cell lines were established

PATIENT	Age (years)	Race	FIGO stage*	HISTOLOGY
UMMT-ARK-1	70	AA†	IC	Homologous
UMMT-ARK-2	46	C‡	IIB	Homologous
OMMT-ARK-1	55	AA	IV	Heterologous
OMMT-ARK-2	78	C	IV	Homologous

^*FIGO: International Federation of Gynecology and Obstetrics

^†AA: African-American

^‡C: Caucasian

Immunostaining of Cell Blocks

Cell blocks were obtained from all four carcinosarcoma cell lines and reviewed by a surgical pathologist. The level of Her2/neu expression and that of multiple additional markers including p53, estrogen receptor (ER), cytokeratins, CD10 and the smooth muscle antigens (SMA) were evaluated using standard IHC techniques as previously described²². Briefly, HER2/neu immunohistochemical staining was performed on paraffin-embedded 5 µm sections of cell blocks after deparaffinisation and rehydration, using the c-erb-2 antibody (Thermo Fisher Scientific, Fremont, CA) at 1:800 dilution. HER2/neu intensity of staining was graded as 0 (negative = no staining observed), 1+ (light staining = weak, incomplete membrane staining in any proportion of tumor cells or weak complete membrane staining in <10% of tumor cells), 2+ (moderate staining = weak complete membrane staining in at least 10% of tumor cells or intense complete membrane staining in 30% or less of tumor cells), or 3+ (strong staining = uniform, intense membrane staining in >30% of tumor cells). Appropriate positive and negative controls were used with each case.

Fluorescent in situ Hybridization

Fluorescent in situ hybridization (FISH) analysis was performed using the PathVysion Her-2 DNA FISH Kit (Abbott Molecular Inc., Abbott Park, IL, USA) according to the manufacturer’s instructions. Cell block sections of 5 µm were deparaffinized and rehydrated, followed by acid pretreatment and proteinase K digestion. A probe mix containing an orange probe directed against the HER2 gene (Vysis, Inc., Downers Grove, IL, USA, LSI Her2/neu) and a green probe directed against the pericentromeric region of chromosome 17 (Vysis CEP 17) were added and specimens were denatured for 5 minutes at 73°C. Slides were then incubated overnight in a humidity chamber at 37°C and washed the day after when a fluorescence mounting medium, containing 4,6-diamidino-2-phenylindole (DAPI), was applied. Fluorescent signals in at least 30 non-overlapping interphase nuclei with intact morphology were scored using a Zeiss Axioplan 2 microscope (Carl Zeiss Meditec, Inc., Dublin, CA, USA) with a 100× planar objective, using a triple band-pass filter that permits simultaneous blue, green, and red colors. Tumor cells were scored for the number of orange (HER2/neu) and green (chromosome 17) signals. A case was scored as amplified when the ratio of the number of fluorescent signals of HER2/neu to chromosome 17 was ≥2.

Quantitative Real-Time Polymerase Chain Reaction

RNA isolation from all four primary carcinosarcoma cell lines, normal endometrium and ovarian epithelial cell control tissues were performed using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions as previously described²³. The endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Assay on Demand Hs99999905_m1 (Applied Biosystems, Foster City, CA) was used to normalize variations in cDNA quantities from different samples. The comparative threshold cycle (C_T) method was used for the calculation of amplification fold as specified by the manufacturer. Quantitative real-time PCR (qRT-PCR) was done with a 7500 Real-time PCR System using the protocols recommended by the manufacturer (Applied Biosystems) to evaluate expression of HER2/neu in all samples. Briefly, 5 µg of total RNA from each sample was reverse transcribed using SuperScript III first-strand cDNA synthesis (Invitrogen). Five µl of reverse transcribed RNA samples (from 500 µl of total volume) were amplified by using the TaqMan Universal PCR Master Mix (Applied Biosystems) to produce PCR products specific for HER2. The C_T method (Applied Biosystems) was used to determine gene expression in each sample relative to the value observed in a control cell line known to express HER2, using GAPDH (Assay ID Hs99999905_m1) RNA as internal controls.

Flow Cytometry

The clinically marketed anti-HER2/neu trastuzumab (Herceptin, Genentech, San Francisco, CA), a humanized monoclonal antibody (mAb) of the G1 type that binds with high affinity to the extracellular domain of the HER2/neu receptor and commercially available antibodies against membrane-bound complement regulatory protein CD46 (cat#555948), CD55 (cat#555691) and CD59 (cat#555761) (BD Pharmingen, San Diego, CA), were used for our study. All four primary carcinosarcoma cell lines obtained from the above described patients were incubated with 2.5 µg/ml of trastuzumab or 1µg/200 µl of anti-CD46, anti-CD55 or anti-CD59 for 30 min on ice. A total of 5 µg/ml of the chimeric anti-CD20 mAb rituximab (Rituxan; Genentech, San Francisco, CA) was used as a negative control. A fluorescein isothiocyanate-conjugated goat antihuman F(ab1)2 immunoglobulin (BioSource International, Camarillo, CA, USA) or goat-anti-mouse FITC (BD Pharmingen, San Diego, CA) were used as secondary reagents. Analysis was conducted with a FACScalibur, using Cell Quest software (BD Biosciences, San Diego, CA, USA).

Tests for ADCC

A standard 5-hour chromium (⁵¹Cr) release assay was performed to measure the cytotoxic reactivity of Ficoll-PaqueTM PLUS (GE Healthcare, Uppsala, Sweden) separated peripheral blood lymphocytes (PBL) obtained from several healthy donors against all four cell lines. The release of ⁵¹Cr from the target cells was measured as evidence of tumor cell lysis after exposure of tumor cells to 5 µg/ml of trastuzumab. Controls included the incubation of target cells alone or with PBL or mAb separately. The chimeric anti-CD20 mAb rituximab was used as a negative control for trastuzumab in all bioassays. ADCC was calculated as percentage killing of target cells obtained after trastuzumab incubation with effector cells compared with ⁵¹Cr release from target cells incubated alone.

Complement-Mediated Target Cell Lysis and γ-Globulin Inhibition

To evaluate primary carcinosarcoma cell lines for their sensitivity to complement-mediated cytotoxicity, carcinosarcoma cell lines were challenged by adding diluted human plasma (1:2) in the presence or absence of the effector cells and trastuzumab to standard 5 hours ⁵¹Cr-release assays. Diluted human plasma was used as a source of complement. In additional experiments, human plasma was heat-inactivated by incubation at 56°C for 60 minutes and added in the presence of effector PBL in order to clarify the role of physiological concentration of γ-globulin. Controls included the incubation of target cells alone or with either lymphocytes or mAb separately. Rituximab was used as a control mAb.

Statistical Analysis

For qRT-PCR data, the right skewing was removed by taking copy number ratios relative to the lowest-expressing normal endometrium (NEC) or normal ovarian sample (NOVA) (relative copy number), analyzing them with log2 that lead to transformation into ΔCTs, and comparing the results by means of unequal-variance t-test for uterine or ovarian carcinosarcomas versus controls. Group means with 95% confidence intervals (CIs) were calculated by computing them on the ΔCTs and then reverse-transforming the results to obtain means (with 95% CIs) of relative copy numbers. Differences in HER2/neu expression by flow cytometry were analyzed by the unpaired t-test, and a p-value of <0.05 among samples was considered to be significant. The Wilcoxon rank-sum (WRS) test was used to compare carcinosarcoma with controls for differences in IHC HER2/neu staining intensities. Sample-type differences were expressed as odds ratios accompanied by 95% confidence limits. Kruskal-Wallis test and chi-square analyses were used to evaluate differences in trastuzumab-induced ADCC levels in primary tumor cell lines. Statistical analysis was performed using PASW Version 18 (SPSS, Chicago, IL).

RESULTS

Morphologic and Immunocytochemical Findings

The immunophenotypes and morphologic characteristics of OMMT-ARK-1, OMMT-ARK-2, UMMT-ARK-1 and UMMT-ARK-2 primary cell lines are shown in Table 2 and Figure 1, respectively. OMMT-ARK-2 cell line showed diffuse, strong positivity for both CK AE1/AE3 and CK7, while it was negative for vimentin, indicating that the cells growing in culture represent the epithelial component of the tumor. Cell line UMMT-ARK-2 on the other hand was negative for CK immunostains and positive for vimentin, indicating that this cell line was composed of the sarcomatous component of the original tumor. P53 was found strongly positive in OMMT-ARK-1 and UMMT-ARK-2 cell lines while UMMT-ARK-1 was focally positive for p53 expression. In contrast, OMMT-ARK-2 showed absence of p53 expression, raising the possibility of a “null-mutation”.

Table 2.

Phenotypes of primary carcinomasarcoma cell lines

PATIENT	Her2	CKAE1/AE3	CK7	Vimentin	P53	ER	SMA	CD10
UMMT-ARK-1	0	90%	0%	100%	5%	0%	0%	0%
UMMT-ARK-2	0	0%	0%	95%	95%	0%	0%	0%
OMMT-ARK-1	1+	30%	30%	100%	80%	0%	10%	20%
OMMT-ARK-2	3+	100%	100%	0%	0%	0%	0%	40%

Figure 1.

Morphologic appearance of primary uterine and ovarian carcinosarcoma cell lines. A: UMMT-ARK-1, B: UMMT-ARK-2, C: OMMT-ARK-1 and D: OMMT-ARK-2. Note that cells growing as monolayer of the UMMT-ARK-1, OMMT-ARK-1 and OMMT-ARK-2 primary cultures are polygonal and epithelioid in shape while UMMT-ARK-2 cells are spindle-shape. Morphologic appearance is consistent with the phenotype reported in Table 2 showing that UMMT-ARK-2 cell line is composed exclusively of sarcomatous cells.

HER2/neu Expression by Immunohistochemistry

Immunohistochemical analysis was performed on cell blocks of all primary cell lines derived from patients harboring uterine (two patients) and ovarian (two patients) carcinosarcomas. Membranous HER2/neu expression was 0, negative in both UMMT samples (Table 2 and 3). In contrast, OMMT-ARK-1 was found to have a low positivity (1+) for HER2/neu protein (Table 2 and 3), while, as representatively shown in Figure 2, OMMT-ARK-2 showed strong (3+) HER2/neu positivity by IHC. HER2/neu expression results on the cell blocks were in agreement with those found by IHC on the tumor tissue blocks from which the cell lines were derived (data are not shown).

Table 3.

mRNA and protein HER2 expression in primary carcinosarcoma cell lines

SAMPLE	IHC* (Cell block)	RT-PCR† (mRNA copy number)	Flow Cytometry
			(%‡)	MFI**
UMMT-ARK-1	0	0.6	54.2	10.5
UMMT-ARK-2	0	5.0	59.5	16.2
OMMT-ARK-1	1+	33.0	100	24.4
OMMT-ARK-2	3+	6020.1	100	191.6

^*IHC: immunohistochemistry

^†RT– PCR: real-time PCR

^‡percentage of positive cells

^**MFI=mean fluorescence intensity

Figure 2.

Representative HER2/neu protein expression and gene amplification by IHC and FISH, respectively, in OMMT-ARK-2 primary cell line. Left panel (2a): OMMT-ARK-2 shows strong (3+) staining for HER2/neu (magnification 20×). Right panel (2b): high amplification (4.0) of the ErbB2 gene in OMMT-ARK-2 (magnification 20×).

ErbB2 gene amplification by FISH

Fluorescent in situ hybridization was performed on the cell blocks obtained from all four primary cell lines used in cytotoxicity experiments. Consistent with the IHC results, ErbB2 gene amplification was detected in OMMT-ARK-2 cells with a high level of amplification (Her2/CEP17 ratio = 4.0) (Figure 2), suggesting that the strong receptor expression and high HER2/neu mRNA level (see RT-PCR results below) are likely caused by gene amplification. In contrast, the remaining cell lines (i.e., OMMT-ARK-1, UMMT-ARK-1 and UMMT-ARK-2) were found to be negative for erbB2 gene amplification.

HER2/neu Transcript Levels in Carcinosarcomas

All ovarian and uterine carcinosarcoma cell lines were tested for HER2/neu expression by qRT-PCR. HER2/neu expression was significantly higher in two carcinosarcoma cell lines (i.e., OMMT-ARK-2 and OMMT-ARK-1) when compared with normal control tissues (p = 0.002). Among the four carcinosarcomas tested, OMMT-ARK-2 demonstrated high mRNA copy number (i.e., 6020.1) while OMMT-ARK-1 demonstrated low mRNA copy number (i.e., 33.0). In contrast, negligible HER2/neu expression by qRT-PCR (i.e., 0.6 to 5.0 mRNA copy numbers), was detected in the two remaining cell lines (i.e., UMMT-ARK-1, UMMT-ARK-2), respectively (Table 3).

HER2/neu and Membrane-Bound Complement-Regulatory-Protein Expression by Flow Cytometry

To determine whether the expression of HER2/neu mRNA detected by qRT-PCR in carcinosarcoma cell lines also resulted in expression of the protein on the surface of tumor cells, flow cytometry was performed on all primary cell lines. In addition, primary MMT cell lines were also evaluated for CD46, CD55, and CD59 mCRP expression by FACS analysis. HER2/neu surface expression results from flow cytometry analysis were found to be in good agreement with HER2/neu expression results found by qRT-PCR in all four primary cell lines (Table 3). The difference in HER2/neu surface protein expression tested by flow cytometry between the cell lines with low/negligible and positive HER2/neu expression was statistically significant (p<0.001). Figure 3 representatively shows the low and high reactivity against HER2/neu found in OMMT-ARK-1 versus OMMT-ARK-2, respectively, using trastuzumab. When mCRP surface expression was evaluated by flow cytometry, with no exception, we found all MMT primary cell lines tested to express high levels of CD46, CD55 and CD59 (Table 4).

Figure 3.

Flow cytometry histogram of OMMT-ARK-1 (upper panel, 3a) and OMMT-ARK-2 (lower panel, 3b) showing low and high expression of HER2/neu protein on the surface of tumor cells, respectively. Rituximab-anti-CD20 control antibody (black solid); trastuzumab (solid line).

Table 4.

mCRP expression in primary carcinosarcoma cell lines

SAMPLE	Flow Cytometry
	CD46		CD55		CD59
	(%‡)	MFI*	(%‡)	MFI*	(%‡)	MFI*
UMMT-ARK-1	100	21.2	100	18.1	100	80.9
UMMT-ARK-2	100	24.2	100	19.5	100	27.1
OMMT-ARK-1	100	88.4	100	27.8	100	40.4
OMMT-ARK-2	100	58.7	100	45.5	100	25.4

^‡percentage of positive cells

^*MFI=mean fluorescence intensity

Trastuzumab-mediated ADCC

All four carcinosarcoma cell lines were tested for their sensitivity to natural killer (NK) cell activity by exposure to PBL collected from several healthy donors. Carcinosarcoma cell lines were found highly resistant to NK-mediated lysis when exposed to PBL in the presence or absence of rituximab control antibody: mean killing 4.0±1.5% (Figure 4). In contrast, after incubation with trastuzumab, the FISH positive HER2/neu overexpressing cell line (i.e., OMMT-ARK-2) demonstrated a high level of killing: mean 45.6±13.5% (range: 32.3–72.6%; p<0.001) (Figure 4). A lower level of killing was detected against the low HER2/neu expressing cell line OMMT-ARK-1: mean 26.5±3.9% (range: 21.0–31.8%; p = 0.001) (Figure 4). Consistent with their negligible HER2/neu expression seen by qRT-PCR and flow cytometry, UMMT-ARK-1, UMMT-ARK-2 demonstrated no significant killing after incubation with PBL with trastuzumab (data not shown).

Figure 4.

Representative cytotoxicity experiments against the low HER2/neu expressing cell line OMMT-ARK-1 (upper panel, 4a) and the high HER2/neu expressing cell line OMMT-ARK-2 (lower panel, 4 b) at different effector to target cell ratios in the presence or absence of trastuzumab in 5-hr ⁵¹Cr-release cytotoxicity assays. Consistent with their HER2/neu expression levels, low degrees of ADCC was detected against OMMT-ARK-1 while a high degree of ADCC was detected against OMMT-ARK-2 cell line. Negligible cytotoxicity was detectable in the absence of trastuzumab or in the presence of rituximab control antibody against both cell lines.

Complement and IgG effects on trastuzumab-mediated ADCC

To evaluate the potential cytotoxic effect of complement in the presence or absence of trastuzumab and the potential inhibition of ADCC by physiological IgG serum concentrations, MMT cell lines were incubated with human plasma diluted 1:2 (with and without heat inactivation) in the presence or absence of trastuzumab during standard 5h-⁵¹Cr-release cytotoxicity assays. As shown in the upper panel of Figure 5 for all the MMT cell lines, the addition of untreated plasma in the presence or absence of trastuzumab (and in the absence of PBL) did not induce any significant cytotoxicity against the carcinosarcoma cell lines (mean 2.1±1.3 % (range 1.2–3.1%). Importantly, addition of physiological concentrations of IgG by adding heat-inactivated plasma to PBL in the presence of trastuzumab did not significantly decrease trastuzumab-mediated killing against OMMT-ARK-2 when compared to incubation without plasma: mean 43.8±0.54% (range 43.4–54.3% p = 0.76) (Figure 5, lower panel). These data suggest that these primary carcinosarcoma cell lines are highly resistant to CDC in vitro while physiological amounts of IgG do not significantly alter the ability of trastuzumab to mediate ADCC against carcinosarcoma cell lines overexpressing HER2/neu.

Figure 5.

Upper panel (5a, 5b, 5c, 5d): Representative cytotoxicity experiments against all 4 primary carcinosarcoma cell lines by adding human plasma diluted 1:2 as source of complement (with or without heat inactivation) in the presence or absence of trastuzumab. Negligible levels of CDC were detected against the four primary cell lines. Lower panel (5e): representative cytotoxicity experiments against OMMT-ARK-2 cell line by adding human plasma diluted 1:2 (with or without heat inactivation) in the presence of the effector cells and either trastuzumab or rituximab control antibody in standard 5-hr ⁵¹Cr-release assays. Addition of untreated plasma (diluted 1:2) to PBL in the presence of trastuzumab and of heat-inactivated plasma diluted 1:2 to PBL did not significantly alter the degree of ADCC achieved against OMMT-ARK-2 in the presence of trastuzumab and PBL (p = 0.76).

DISCUSSION

As a key gene for cell survival, growth and differentiation, HER2/neu gene amplification and protein overexpression lead to malignant transformation^{15, 16}. It has been previously demonstrated that HER2/neu is overexpressed in different human cancers including breast cancer as well as uterine carcinosarcomas^24–28. The reported rate of overexpression and amplification in uterine carcinosarcoma ranged from 17 to 43%²⁵. Indeed, in uterine carcinosarcomas, Raspollini et al.²⁶ found HER2/neu expression in 7 of 24 cases (29%), Sawada et al.²⁷ in 7 of 24 cases (29.2%), Amant et al.²⁸ in 3 of 7 (43%) considering both 2+ and 3+, while Livasy et al.²⁵ reported a 25% rate (14/55) considering only 3+ positive cases. HER2/neu expression in ovarian carcinosarcomas has not been well investigated thus far. However, a study from Zorzou et al.¹⁰ has reported no HER2/neu receptor expression by immunohistochemistry in any of the nine ovarian carcinosarcoma tissue blocks tested.

In this study, using multiple techniques, we investigated HER2/neu expression in a total of four primary uterine and ovarian carcinosarcoma cell lines recently established in our laboratory. In addition we have evaluated the in vitro potential of trastuzumab as a novel immunotherapeutic treatment against chemotherapy-resistant MMT. Our findings demonstrate that HER2/neu is highly expressed in 25% of the carcinosarcoma cell lines. Indeed, we found overexpression of HER2/neu by IHC (i.e., 3+) and c-erbB-2 amplification by FISH in one out of four available cell lines (i.e., OMMT-ARK-2) while low HER2/neu expression (i.e., 1+ by IHC in the absence of gene amplification) was found in OMMT-ARK-1. No HER2/neu expression was found in the remaining carcinosarcoma cell lines. This percentage of HER2/neu positivity in primary MMT cell lines is in agreement with the reported rate of overexpression and amplification in gynecologic carcinosarcomas^24–28. Importantly, such results were confirmed by evaluating HER2/neu gene expression levels (RT-PCR) as well as surface protein expression (flow cytometry) on all MMT cell lines. These data further support the notion that anti-HER2/neu antibody-based therapy may have potential anti-tumoral effect in carcinosarcoma patients in vivo.

Trastuzumab, a humanized monoclonal anti-HER2/neu antibody, has been shown to induce apoptosis in breast cancer cells through multiple mechanisms including antibody-dependent cellular cytoxicity^{32, 33} and our research group has recently demonstrated that high levels of ADCC are achievable in vitro against biologically aggressive Type II (i.e., uterine serous) carcinoma cell lines after incubation with trastuzumab²². In the current study, we report for the first time the possibility to achieve high levels of ADCC in vitro against a FISH positive carcinosarcoma cell line expressing high copy numbers of HER2/neu mRNA by PCR as well as high surface levels of HER2/neu expression by IHC and flow cytometry. Of interest, while OMMT-ARK-2 was found to be highly sensitive to ADCC in the presence of trastuzumab in vitro, negligible killing was detected in the presence of allogeneic PBL in the absence of trastuzumab or in the presence of rituximab control antibody. Importantly, in this regard, OMMT-ARK-2 cell line was established from a biopsy collected from a patient experiencing disease progression after treatment with multiple chemotherapy regimens and the tumor was confirmed to be highly resistant to multiple cytotoxic drugs including platinum, paclitaxel and adryamicin in vitro by MTT assays. Thus, our experimental results clearly suggest that carcinosarcoma cell lines confirmed to be highly resistant to multiple clinically available chemotherapeutic agents in vitro and in vivo and shown to be highly resistant to NK cell-induced cytotoxicity in vitro, may become highly sensitive to lysis by NK cells when these cells are engaged by the HER2/neu specific antibody.

For effective in vivo ADCC, effector cells must be able to interact with the therapeutic antibody at the target site, even in the presence of high concentrations of human IgG. Moreover, because complement activation may be an additional effector mechanism triggered by trastuzumab, inhibition of complement activation by tumor cells due to overexpression of membrane-bound complement regulatory protein such as CD46, CD55 and CD59 may potentially reduce in vivo trastuzumab therapeutic efficacy by decreasing complement-dependent cellular cytotoxicity^29–31. Consistent with this view, in this study we have evaluated the expression of CD46, CD55 and CD59 mCRP on the surface of multiple carcinosarcoma cell lines as well as the effect of complement and high concentration of irrelevant IgG during trastuzumab-mediated killing in vitro. Of interest, we found that regardless to their HER2/neu expression levels, primary carcinosarcoma cell lines express high levels of CD46, CD55 and CD59 mCRPs, and in agreement with these findings, are extremely resistant to CDC in the presence or absence of trastuzumab Mab. Thus, these results suggest that CDC is unlikely to play a significant role against carcinosarcomas during trastuzumab therapy in vivo. Nevertheless, these data suggest, as previously postulated for ovarian cancer gene therapy³³, that silencing of mCRPs may potentially synergize with trastuzumab-ADCC in vitro as well as in vivo against chemotherapy resistant carcinosarcomas.

Finally, in order to mimic the in vivo situation, heat inactivated human plasma diluted 1:2 was added to carcinosarcoma cell lines in the presence of effector cells during cytotoxicity assays. Consistent with the results of El-Sahwi et al. using trastuzumab against uterine serous tumors²², in this study we showed that ADCC against OMMT-ARK-2 line was not inhibited by high concentrations (up to 50%) of human serum. These data, therefore, suggest that trastuzumab efficacy in vivo against MMT overexpressing HER2/neu should not be significantly affected by physiological amounts of γ-globulins. On the basis of these findings, we can therefore speculate that the binding of trastuzumab to the Fc receptor on mononuclear effector cells is likely to occur in vivo.

In conclusion, this is the first report on HER2/neu gene and protein expression in primary MMT cell lines and the first demonstration of trastuzumab-mediated antibody-dependent cellular cytotoxicity against uterine and ovarian carcinosarcomas in vitro. Although in vivo data will ultimately be necessary to validate the therapeutic potential of trastuzumab against HER2/neu expressing carcinosarcomas, our in vitro results strongly suggest that targeting cancer cells with high surface expression of HER2/neu can be an attractive and novel strategy to treat residual and/or resistant gynecologic carcinosarcomas refractory to salvage chemotherapy.