Patent Application
20090286276
The present invention relates to individualizing cancer treatment, and particularly to individualizing cancer treatment by evaluating a patient for responsiveness to an EGFR inhibitor prior to therapy with such agent.
Epidermal growth factor receptor (EGFR) inhibitors have been approved or tested for treatment of a variety of cancers, including non-small cell lung cancer (NSCLC), head and neck cancer, colorectal carcinoma, and Her2-positive breast cancer, and are increasingly being added to standard therapy. EGFR inhibitors, which may target either the intracellular tyrosine kinase domain or the extracellular domain of the EGFR target, are generally plagued by low population response rates, leading to ineffective or non-optimal chemotherapy in many instances, as well as unnecessary drug toxicity and expense. For example, a reported clinical response rate for treatment of breast carcinoma with lapatinib (a small molecule EGFR tyrosine kinase inhibitor) is about 10% [New England J. Med. 2006; 355:2733-43], a reported clinical response rate for treatment of colorectal carcinoma with cetuximab (a chimeric monoclonal antibody targeting the extracellular domain of EGFR) is about 11% [New England J. Med. 2004; 351:337-45], and a reported clinical response rate for treatment of NSCLC with erlotinib is about 8.9% [13].
Thus, there is a need for predicting patient responsiveness to EGFR inhibitors prior to treatment with such agents, so as to better individualize patent therapy.
For example, small molecules including gefitinib and erlotinib have been developed that inhibit the intracellular tyrosine kinase domain of EGFR, thus blocking EGFR signaling. The addition of gefitinib or erlotinib to first-line platinum-based chemotherapy in patients with NSCLC did not show a clear survival benefit [6,7,8-11]. However, patients that reported never smoking did benefit with the addition of erlotinib [10]. Erlotinib showed a survival advantage when administered as monotherapy in the second- or third-line setting [12,13] and was approved for this indication by the FDA in 2004. For gefitinib, subgroup analyses showed a statistically significant survival benefit in Asians and those reporting having never smoked. Of course, not all patients that fit these demographic criteria respond to the inhibitor, and thus more predictive and/or definitive tests are necessary to guide the use of these agents selectively in responsive subpopulations.
The identification of surrogate biomarkers is one potential strategy for identifying responsive patients prior to treatment. Investigations are ongoing to identify biomarkers of response to EGFR inhibitors, including for NSCLC (reviewed in [14]). Several somatic mutations in the tyrosine kinase domain of EGFR may be associated with likelihood of response to EGFR tyrosine kinase inhibition [15-17]. However, validating and performing multiple molecular analyses to guide treatment can be complicated and costly. Furthermore, such biomarkers may not correlate to clinical response for all patients, and thus may be better suited to provide prognostic, not predictive, information.
A chemotherapy sensitivity and resistance assay (CSRA) that is able to predict tumor response to EGFR inhibitors is needed to assist clinical decision making.
The present invention provides methods for individualizing chemotherapy for cancer treatment, and particularly for evaluating a patient's responsiveness to one or more epidermal growth factor receptor (EGFR) inhibitors prior to treatment with such agents. Particularly, the invention provides an in vitro chemoresponse assay for predicting a patient's response to an EGFR inhibitor, such as an EGFR tyrosine kinase inhibitor or a molecule targeting the extracellular domain of EGFR. The method generally comprises culturing malignant cells from a patient's specimen (e.g., biopsy specimen), contacting the cultured cells with an EGFR inhibitor that is a candidate treatment for the patient, and evaluating the cultured cells for a response to the drug. In certain embodiments, monolayer(s) of malignant cells are cultured from explants prepared by mincing tumor tissue, and the cells of the monolayer are suspended and plated for chemosenstivity testing. The in vitro response to the drug as determined by the method of the invention is correlative with the patient's in vivo response upon receiving the EGFR inhibitor during chemotherapeutic treatment (e.g., in the course of standardized or individualized chemotherapeutic regimen).
In certain embodiments, the EGFR inhibitor is a tyrosine kinase inhibitor such as erlotinib, gefitinib, or lapatinib, or a molecule that targets the EGFR extracellular domain (e.g., cetuximab). While such agents may have relatively low population response rates, the invention provides a convenient in vitro assay to predict whether a particular patient will be responsive to an EGFR inhibitor, thus avoiding ineffective treatment as well as unnecessary toxicity and expense. As described herein, the method of the invention predicts responsiveness to EGFR inhibitors at a rate that matches reported clinical response rates for the EGFR inhibitors.
The present invention provides methods for individualizing chemotherapy for cancer treatment, and particularly, provides an in vitro chemoresponse assay for evaluating a patient's responsiveness to one or more EGFR inhibitors prior to treatment with such agents. The method generally comprises culturing malignant cells from a patient's specimen (e.g., biopsy), contacting the cultured cells with an EGFR inhibitor, and evaluating the cultured cells for a response to the drug. The in vitro response to the drug as determined by the method of the invention is correlative with an in vivo response upon receiving the EGFR inhibitor during chemotherapeutic treatment.
The present invention supports individualized chemotherapy decisions for cancer patients, and particularly with candidate EGFR inhibitors. The patient generally has a cancer for which an EGFR is a candidate treatment, for example, alone or in combination with other therapy. For example, the cancer may be selected from breast, ovarian, colorectal, endometrial, thyroid, nasopharynx, prostate, head and neck, liver, kidney, pancreas, bladder, brain, and lung. In certain embodiments, the tumor is a solid tissue tumor and/or is epithelial in nature. For example, the patient may be a Her2-positive breast cancer patient, a colorectal carcinoma patient, NSCLC patient, head and neck cancer patient, or endometrial cancer patient.
The present invention involves conducting chemoresponse testing with one or a panel of chemotherapeutic agents on cultured cells from a cancer patient, including one or more EGFR inhibitors. In certain embodiments, the chemoresponse method is as described in U.S. Pat. Nos. 5,728,541, 6,900,027, 6,887,680, 6,933,129, 6,416,967, 7,112,415, and 7,314,731 (all of which are hereby incorporated by reference in their entireties). The chemoresponse method may further employ the variations described in US Published Patent Application Nos. 2007/0059821 and 2008/0085519, both of which are hereby incorporated by reference in their entireties. Such chemoresponse methods are commercially available as the ChemoFx® Assay (Precision Therapeutics, Inc, Pittsburgh, Pa.).
Briefly, in certain embodiments, cohesive multicellular particulates (explants) are prepared from a patient's tissue sample (e.g., a biopsy sample) using mechanical fragmentation. This mechanical fragmentation of the explant may take place in a medium substantially free of enzymes that are capable of digesting the explant. However, in some embodiments, some enzymatic treatment may be conducted. Generally, the tissue sample is systematically minced using two sterile scalpels in a scissor-like motion, or mechanically equivalent manual or automated opposing incisor blades. This cross-cutting motion creates smooth cut edges on the resulting tissue multicellular particulates. The tumor particulates each measure from about 0.25 to about 1.5 mm3, for example, about 1 mm3.
After the tissue sample has been minced, the particles are plated in culture flasks (e.g., about 5 to 25 explants per flask). For example, about 9 explants may be plated per T-25 flask, or about 20 particulates may be plated per T-75 flask. For purposes of illustration, the explants may be evenly distributed across the bottom surface of the flask, followed by initial inversion for about 10-15 minutes. The flask may then be placed in a non-inverted position in a 37° C. CO2 incubator for about 5-10 minutes. Flasks are checked regularly for growth and contamination. Over a period of a few weeks a cell monolayer will form. Further, it is believed (without any intention of being bound by the theory) that tumor cells grow out from the multicellular explant prior to stromal cells. Thus, by initially maintaining the tissue cells within the explant and removing the explant at a predetermined time (e.g., at about 10 to about 50 percent confluency, or at about 15 to about 25 percent confluency), growth of the tumor cells (as opposed to stromal cells) into a monolayer is facilitated. Further, in certain embodiments, the tumor explant may be agitated to substantially release tumor cells from the tumor explant, and the released cells cultured to produce a cell culture monolayer. The use of this procedure to form a cell culture monolayer helps maximize the growth of representative tumor cells from the tissue sample.
Prior to the chemotherapy assay, the growth of the cells may be monitored, and data from periodic counting may be used to determine growth rates which may or may not be considered parallel to growth rates of the same cells in vivo in the patient. If growth rate cycles can be documented, for example, then dosing of certain active agents may be customized for the patient. Monolayer growth rate and/or cellular morphology and/or epithelial character may be monitored using, for example, a phase-contrast inverted microscope. Generally, the monolayers are monitored to ensure that the cells are actively growing at the time the cells are suspended for drug exposure. Thus, the monolayers will be non-confluent when the cells are suspended for chemoresponse testing.
A panel of active agents may then be screened using the cultured cells, including one or more EGFR inhibitors. Generally, the agents are tested against the cultured cells using plates such as microtiter plates. For the chemosensitivity assay, a reproducible number of cells is delivered to a plurality of wells on one or more plates, preferably with an even distribution of cells throughout the wells. For example, cell suspensions are generally formed from the monolayer cells before substantial phenotypic drift of the tumor cell population occurs. The cell suspensions may be, without limitation, about 4,000 to 12,000 cells/ml, or may be about 4,000 to 9,000 cells/ml, or about 7,000 to 9,000 cells/ml. The individual wells for chemoresponse testing are inoculated with the cell suspension, with each well or “segregated site” containing about 102 to 104 cells. The cells are generally cultured in the segregated sites for about 4 to about 30 hours prior to contact with an agent.
Each test well is then contacted with at least one pharmaceutical agent, or a sequence of agents. In addition to at least one EGFR inhibitor (as discussed in more detail below), the panel of chemotherapeutic agents may comprise at least one agent selected from a platinum-based drug, a taxane, a nitrogen mustard, a kinase inhibitor, a pyrimidine analog, a podophyllotoxin, an anthracycline, a monoclonal antibody, and a topoisomerase I inhibitor. For example, the panel may comprise 1, 2, 3, 4, or 5 agents selected from bevacizumab, capecitabine, carboplatin, cecetuximab, cisplatin, cyclophosphamide, docetaxel, doxorubicin, epirubicin, etoposide, 5-fluorouracil, gemcitabine, irinotecan, oxaliplatin, paclitaxel, panitumumab, tamoxifen, topotecan, and trastuzumab, in addition to other potential agents for treatment. In certain embodiments, the chemoresponse testing includes one or more combination treatments, such combination treatments including one or more agents described above. Generally, each agent in the panel is tested in the chemoresponse assay at a plurality of concentrations representing a range of expected extracellular fluid concentrations upon therapy.
The efficacy of each agent in the panel is determined against the patient's cultured cells, by determining the viability of the cells (e.g., number of viable cells). For example, at predetermined intervals before, simultaneously with, or beginning immediately after, contact with each agent or combination, an automated cell imaging system may take images of the cells using one or more of visible light, UV light and fluorescent light. Alternatively, the cells may be imaged after about 25 to about 200 hours of contact with each treatment. The cells may be imaged once or multiple times, prior to or during contact with each treatment. Of course, any method for determining the viability of the cells may be used to assess the efficacy of each treatment in vitro.
While any grading system may be employed, in certain embodiments the grading system may employ from 2 to 10 response levels, e.g., about 3, 4, or 5 response levels. For example, when using three response grades, the three grades may correspond to a responsive grade, an intermediate responsive grade, and a non-responsive grade. In certain embodiments, the patient's cells show a heterogeneous response across the panel of agents, making the selection of an agent particularly crucial for the patient's treatment.
The output of the assay is a series of dose-response curves for tumor cell survivals under the pressure of a single or combination of drugs, with multiple dose settings each (e.g., ten dose settings). To better quantify the assay results, the invention employs in some embodiments a scoring algorithm accommodating a dose-response curve. Specifically, the chemoresponse data are applied to an algorithm to quantify the chemoresponse assay results by determining an adjusted area under curve (aAUC) (see U.S. application Ser. No. 12/252,073, which is hereby incorporated by reference in its entirety).
In some embodiments, the agents are designated as, for example, sensitive, or resistant, or intermediate, by comparing the aAUC test value to one or more cut-off values for the particular drug (e.g., representing sensitive, resistant, and/or intermediate aAUC scores for that drug). The cut-off values for any particular drug may be set or determined in a variety of ways, for example, by determining the distribution of a clinical outcome within a range of corresponding aAUC reference scores. That is, a number of patient tumor specimens are tested for chemosenstivity/resistance to a particular drug prior to treatment, and aAUC quantified for each specimen. Then after clinical treatment with that drug, aAUC values that correspond to a clinical response (e.g., sensitive) and the absence of significant clinical response (e.g., resistant) are determined. Cut-off values may alternatively be determined from population response rates. For example, where a patient population is known to have a response rate of 30% for the tested drug, the cut-off values may be determined by assigning the top 30% of aAUC scores for that drug as sensitive. Further still, cut-off values may be determined by statistical measures.
In accordance with the present invention, cultured cells may be tested for their responsiveness to any candidate EGFR inhibitor (e.g., an EGFR inhibitor that is a candidate treatment for the patient). The EGRF inhibitor may be an EGFR tyrosine kinase inhibitor, or may alternatively target the extracellular domain of the EGFR target.
In certain embodiments, the EGFR inhibitor is a tyrosine kinase inhibitor such as Erlotinib, Gefitinib, or Lapatinib, or a molecule that targets the EGFR extracellular domain (e.g., Cetuximab or Panitumumab).
Erlotinib hydrochloride (e.g., as marketed as Tarceva™) is used to treat non-small cell lung cancer, pancreatic cancer and several other types of cancer. Erlotinib specifically targets the epidermal growth factor receptor (EGFR) tyrosine kinase, which is highly expressed and occasionally mutated in various forms of cancer. It binds in a reversible fashion to the adenosine triphosphate (ATP) binding site of the receptor to inhibit receptor signaling. Erlotinib has shown a survival benefit in the treatment of lung cancer in phase III trials. It has been approved for the treatment of locally advanced or metastatic non-small cell lung cancer that has failed at least one prior chemotherapy regimen. The FDA has further approved the use of erlotinib in combination with gemcitabine for treatment of locally advanced, unresectable, or metastatic pancreatic cancer. It has been reported that responses among patients with lung cancer are seen most often in females who were never smokers, particularly Asian women and those with adenocarcinoma cell type.
Gefitinib acts in a similar manner to erlotinib (marketed as Tarceva), and is marketed under the trade name Iressa™. Research on gefitinib-sensitive non-small cell lung cancers has shown that a mutation in the EGFR tyrosine kinase domain may be responsible for activating anti-apoptotic pathways. These mutations may confer increased sensitivity to tyrosine kinase inhibitors such as gefitinib and erlotinib. Of the types of non-small cell lung cancer histologies, adenocarcinoma most often harbors these mutations. These mutations are more commonly seen in Asians, women, and non-smokers (who also tend to more often have adenocarcinoma).
Gefitinib is indicated for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC) in patients who have previously received chemotherapy. There is also potential for use of gefitinib in the treatment of other cancers where EGFR overexpression is involved.
Lapatinib inhibits the tyrosine kinase activity associated with two oncogenes, EGFR (epidermal growth factor receptor) and HER2/neu (Human EGFR type 2). Over expression of HER2/neu can be responsible for certain types of high-risk breast cancers in women. Lapatinib is a protein kinase inhibitor shown to decrease tumor-causing breast cancer stem cells. Lapatanib inhibits receptor signal processes by binding to the ATP-binding pocket of the EGFR/HER2 protein kinase domain, preventing self-phosphorylation and subsequent activation of the signal mechanism.
Lapatinib is used as a treatment for women's breast cancer in patients who have HER2-positive advanced breast cancer that has progressed after previous treatment with other chemotherapeutic agents, such as anthracycline, taxane-derived drugs, or trastuzumab (Herceptin,Genentech).
Cetuximab is a chimeric monoclonal antibody targeting EGFR, and is given by intravenous injection for treatment of metastatic colorectal cancer and head and neck cancer. Cetuximab may act by binding to the extracellular domain of the EGFR, preventing ligand binding and activation of the receptor. This blocks the downstream signaling of EGFR resulting in impaired cell growth and proliferation. Cetuximab has also been shown to mediate antibody dependent cellular cytotoxicity (ADCC).
Cetuximab is used in metastatic colon cancer and is given concurrently with the chemotherapy drug irinotecan (Camptosar), a form of chemotherapy that blocks the effect of DNA topoisomerase I, resulting in fatal damage to the DNA of affected cells. Cetuximab was approved by the FDA for use in combination with radiation therapy for treating squamous cell carcinoma of the head and neck (SCCHN) or as a single agent in patients who have had prior platinum-based therapy.
Panitumumab is a recombinant, human IgG2 kappa monoclonal antibody that binds specifically to the human epidermal growth factor receptor (EGFR). Panitumumab is indicated as a single agent for the treatment of EGFR-expressing, metastatic colorectal carcinoma with disease progression on or following fluoropyrimidine-, oxaliplatin-, and irinotecan-containing chemotherapy regimens.
EGFR inhibitors are generally plagued by low population response rates, leading to ineffective or non-optimal chemotherapy in many instances, as well as unnecessary drug toxicity and expense. For example, a reported clinical response rate for treatment of breast carcinoma with lapatinib is 10% [New England J. Med. 2006; 355:2733-43], a reported clinical response rate for treatment of colorectal carcinoma with cetuximab is 11% [New England J. Med. 2004; 351:337-45], and a reported clinical response rate for treatment of NSCLC with erlotinib is 8.9% [13].
The method of the invention predicts patient responsiveness to EGFR inhibitors at rates that match reported clinical response rates for the EGFR inhibitors.
Three human lung tumor-derived immortalized cell lines were tested in this study: H292, H358, and Calu3 (American Type Culture Collection, Manassas, Va.). These cell lines were seeded at 40,000 cells in T25 flasks (PGC Scientifics, Frederick, Md.) and allowed to grow for one week to approximately 90% confluence.
Patient tumor specimens: Primary cell cultures were established using tumor specimens procured for research purposes from the following sources: National Disease Research Interchange (Philadelphia, Pa.), Cooperative Human Tissue Network (Philadelphia, Pa.), Forbes Regional Hospital (Monroeville, Pa.), Jameson Hospital (New Castle, Pa.), Saint Barnabas Medical Center (Livingston, N.J.), Hamot Medical Center (Erie, Pa.), and Windber Research Institute (Windber, Pa.). The tumors were removed from the patient at the time of surgery, placed in the supplied 125-mL bottle containing sterile McCoy's shipping medium (Mediatech, Herndon, Va.), and shipped overnight to Precision Therapeutics, Inc. laboratories (Pittsburgh, Pa.).
Erlotinib hydrochloride was kindly provided by OSI Pharmaceuticals (Melville, N.Y.) as a lyophilized powder. The drug was reconstituted to 5 mM in 100% DMSO and frozen at −80° C.
Cell lines and tissue specimens were processed and tested with the ChemoFx® assay as described elsewhere [21]. Also see, U.S. Pat. Nos. 5,728,541, 6,900,027, 6,887,680, 6,933,129, 6,416,967, 7,112,415, and 7,314,731; and U.S. application Ser. Nos. 10/399,563, 11/504,098, 11/595,967, 11/713,662, and 11/785,984, all of which are hereby incorporated by reference in their entireties, and especially with regard to tissue processing and cell culturing techniques, and assays. Ten doses of erlotinib were prepared by serial dilution. The same 10-dose concentration range was used for the cell lines and the tissue specimens. For each dose, a cytotoxic index (CI) was calculated according to the following formula: CI=Mean cell countdose x/mean cell countcontrol, which represents the ratio of cells killed as a result of the treatment. Cell counts were the average of 3 replicates at each dose for primary cultures and 9 replicates at each dose for immortalized cell lines. Dose response curves were generated using the CI at each dose. Adjusted areas under the curve (aAUC) were calculated for each dose-response curve as previously described [20]. Assay results were classified as responsive (R; assay score ≧7.48), intermediate responsive (IR; assay score 6.89-7.47), or non-responsive (NR; assay score ≦6.88).
The chemoresponse assay was performed on 3 NSCLC lung cancer cell lines (H292, H358, and Calu-3) to determine whether the assay was able to detect sensitivity of the cells to erlotinib. The 3 cell lines exhibited a heterogeneous response to erlotinib (
Each dose-response curve includes 9 replicates at each dose for each cell line tested; each assay included 3 replicates, and 3 assays were run per cell line. The coefficient of variance (CoV) was calculated for each cell line using the Log EC50 values (by dose number) for each assay. H292 had a CoV of 7%, H358 was 9%, and Calu-3 was 3%.
The in vitro responsiveness to erlotinib of the 3 NSCLC cell lines was compared with published reports of the responsiveness of human tumor xenografts [22]. The sensitivity of tumor growth inhibition in xenografts derived from these cell lines is consistent with the in vitro prediction of response in the same cell lines (Table 1).
Of the 34 lung cancer patient specimens evaluated in this study, 22 (64.7%) were confirmed to be NSCLC, 11(32.4%) were of unconfirmed lung cancer subtype, and 1 (2.9%) was confirmed as not NSCLC (mesothelioma). The 34 tumor specimens exhibited heterogeneity of in vitro response to erlotinib (
These results indicate that the assay described herein is able to distinguish tumor response to erlotinib in patients with lung carcinoma. The invention is thus useful as a decision support tool to assist oncologists in making treatment decisions involving erlotinib in lung cancer patients. The approach described above was to first conduct the assay on NSCLC cell lines to determine its ability to distinguish in vitro response to erlotinib. The responses of the three NSCLC cell lines tested (H292, H358, Calu-3) to erlotinib in the current study were similar to the responses observed in previously published studies using other types of chemoresponse assays [23,24]. In addition, the assay is shown to be highly reproducible (i.e. low process variability) in assessing chemoresponse to erlotinib in 3 separate NSCLC cell lines.
To confirm the range of responses observed, we next compared the in vitro sensitivity of these cell lines to the observed outcomes of ex vivo human tumor xenografts derived from those same cell lines as an estimation of correlation with clinical response. Corresponding sensitivities support the hypothesis that in vitro response may predict clinical response.
Having evidence that the assay prediction of response to erlotinib of the NSCLC cell lines corresponded to responsiveness of human tumor xenografts produced from the same cell lines, we next examined human lung tumor specimens. The assay was able to distinguish sensitivity to erlotinib among 34 human tumor specimens. Our finding that 8.8% of the tumors were responsive to erlotinib is similar to the 8.9% reported response rate in a phase 3, randomized, double blind, placebo-controlled study of previously treated NSCLC patients [13].
Currently, erlotinib is FDA approved only as second- or third-line treatment for advanced NSCLC. Reports from clinical trials to date have not shown a benefit of erlotinib in first line treatment [8-11]. However, subgroup analyses have shown that groups of patients differed in their sensitivity and clinical response to erlotinib [10,13,25]. Investigators have speculated about the potential findings had the large, first-line studies been conducted on selected populations showing increased sensitivity [6]. Thus, an accurate and reliable test to identify erlotinib-sensitive subpopulations of NSCLC patients would be of crucial benefit. Much interest has been focused on identifying patients sensitive to EGFR inhibition using molecular profiles, such as EGFR mutations and amplifications as well as increased gene number [26,27]. A chemoresponse assay that can reproducibly and reliably identify sensitive patients by in vitro tumor response would be a superior alternative to support clinical decision making, in some embodiments, may be performed alongside molecular profiling.
Lapatinib (Tykerb®) is a small molecule tyrosine kinase inhibitor which targets the intracellular domain of both the epidermal growth factor receptor and Her2, thereby inhibiting cell growth and proliferation. Lapatinib is currently FDA-approved to treat Her2 positive breast cancer which has previously been treated with anthracycline and taxane therapies and trastuzumab. Due to the low population response rate of lapatinib, a biomarker which can identify patients with an increased likelihood for response would be of great clinical utility. This example demonstrates an in vitro chemoresponse assay developed to predict sensitivity and resistance of primary cultures of human breast tumor specimens to lapatinib.
The chemoresponse assay for lapatinib was developed using four different immortalized cell lines (SK-OV3, BT474, MDA-MB-231, MCF7). In addition to cell lines, the chemoresponse assay was also performed on 55 primary cultures of breast carcinomas. All cultures were confirmed to contain keratin-positive epithelial cells using fluorescence immunocytochemistry. Cell lines and specimens were treated with a 10 dose concentration range of lapatinib for 72 hours and stained with DAPI; remaining live cells were counted on an inverted fluorescent microscope. Resulting dose-response curves were analyzed and categorized as responsive, intermediate responsive, or non-responsive.
All four cell lines (BT474, MDA-MB-231, MCF7, SK-OV3) were responsive to lapatinib treatment, with EC50 values of approximately 10 uM (
In conclusion, these results demonstrate that in vitro chemoresponse testing is useful in predicting patient response to lapatinib. This test may increase the efficacy of the current chemotherapy decision-making process for patients.
Cetuximab (Erbitux®) is a chimeric monoclonal antibody that binds to the extracellular domain of the epidermal growth factor receptor. This interaction interferes with binding of the ligand and causes internalization of the receptor which blocks the downstream signaling of EGFR, resulting in impaired cell growth and proliferation. Cetuximab has also been shown to mediate antibody dependent cellular cytotoxicity. Cetuximab is FDA-approved to treat head and neck cancer and colorectal carcinomas; it is also being evaluated in clinical trials for use in other cancers, including non-small cell lung and endometrial cancer. Due to the low population response rate of cetuximab, a test that can identify patients with an increased likelihood for response would be of great clinical utility. The current example demonstrates an in vitro chemoresponse assay to predict response of primary cultures of human colorectal tumor specimens to cetuximab.
The chemoresponse assay was developed using four different immortalized cell lines (NCI-H292, NCI-H522, NCI-H1666, Calu3). The chemoresponse assay was also performed on 54 primary cultures of human colorectal tumor specimens. Cell lines and specimens were treated with a 10 dose concentration range of cetuximab for 72 hours, stained with a nuclear dye, and remaining post-treatment live cells were counted. Resulting dose-response curves were analyzed.
Two of the examined cell lines showed response to cetuximab treatment; EC50 values for NCI-H292 and NCI-H1666 were 825 nM and 13 nM, respectively (
These results demonstrate that in vitro chemoresponse testing is useful for predicting patient responsiveness to cetuximab. Use of these embodiments in practice could increase the efficacy of the current chemotherapy decision-making process for patients.
The following references are hereby incorporated by reference in their entireties for all purposes:
This application claims priority to U.S. Provisional Application 61/142,809 filed Jan. 6, 2009 and U.S. Provisional Application No. 61/053,094 filed May 14, 2008, each of which is hereby incorporated by reference in its entirety and for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 61142809 | Jan 2009 | US | |
| 61053094 | May 2008 | US |
