Objective. Treatment of primary and metastatic colorectal carcinoma (CRC) based on 5-fluorouracil and folinic acid (5FU + FA), combined with irinotecan (FOLFIRI) or oxaliplatin (FOLFOX), provides response rates approaching 50% and a 20-month overall survival. Approximately 50% of CRC patients fail to respond to one or more drugs in either regimen, in many cases due to inherent or acquired drug resistance. We therefore characterized in vitro drug response and cross-resistance in primary and metastatic CRC lesions. Materials and methods. The in vitro Extreme Drug Resistance Assay (EDRA) identifies extreme drug resistance (EDR) in solid tumors with over 99% accuracy and appears to mimic the clinical experience. We analyzed EDRA results from 4854 freshly resected CRC biopsies, including 1740 primary and 847 liver metastases. Results. Primary and metastatic CRCs responded similarly to single agents 5FU + FA, irinotecan, and oxaliplatin. Primary and metastatic tumors expressing EDR to 5FU + FA demonstrated up to 58% cross-resistance to a variety of chemotherapy agents, with the lowest percentages for oxaliplatin (11% and 8%, respectively) and irinotecan (16% and 14%). Importantly, approximately 20% of tumors showed EDR to either FULFOX or FOLFIRI. Conclusion. Overall data analyses indicated that EDRA results obtained at initial diagnosis may be useful in guiding therapy selection for metastatic disease. Pre-testing of tumors before treatment may provide essential drug cross-resistance information for better chemotherapy selection. Prospective clinical trials employing the EDRA are needed to substantiate these data.

Over the last 30 years, significant progress was made in the development of monoclonal antibodies (mAbs) initiated by the pioneer studies of Caesar Mil-stein on the structure and genetics of immunoglobulins in the 1960s and 1970s. Table 1 summarizes the most significant advancements in the hybridoma field since the first publication by Kohler and Milstein in 1976 (1). The variety of research and diagnostic mAbs was expanded from “classic” mouse-mouse monohybridomas through the use of rat-rat, hamster-hamster and increasingly popular rabbit-rabbit monohybridomas (2–4), as well as relatively simple to make and efficient mouse-rat and rat-mouse heterohybridomas (2). After many years of persistent development complicated by difficulties in identifying reliable fusion partner cell lines, chicken (5) and, especially, human (6) monohybridomas are also coming closer to fruition. In the therapeutic arena, human mAb versions originated in transgenic mice (7) and recombinant mAbs expressed in vitro using phage displays (8), ribosome displays (9) and baculovirus expression vectors (10) are now playing a major role in the discovery and clinical applications of new immunotherapeutic agents. A new and rapidly evolving approach with a very promising future in multiple biomedical ventures is mAb manufacturing in non-animal system, including yeast (11) and plant (12) cells. Finally, fully synthetic mAbs prepared using amino acid (13) and nonamino acid (14) diversity platforms have been added to the arsenal of research, diagnostic and therapeutic mAbs.

14509 Background: Lymph Node (LN) status is the most important prognostic factor in colon cancer (Cca). Angiogenesis Index (AI) has been studied as a prognostic marker in various solid tumors with conflicting results. Hence, a retrospective analysis was done to evaluate the role of AI as a prognostic marker in Cca. Methods: Pts with Cca who underwent SLNM to determine LN status were included. A portion of tumor was sent for tumor marker analysis including p53, Thrombospondin-1 and CD31 by IHC. AI was derived for each specimen by summing the biomarker specific score for the three tumor markers. AI of -ve 6 was taken as cut off for significance based on previous studies on solid tumors. Metastatic foci in SLNs and non-SLNs were measured in greatest diameter by ocular micrometer; and were added for each pt to estimate overall tumor burden in SLNs and non-SLNs. Results: A total of 111 consecutive pts with Cca were included in the study. SLNM was successful in 100% pts. Pts with distant metastasis (mets) (n=18), Tis (n=1) and skip mets (n=9) were excluded from final analysis. Out of the remaining 83 pts, an AI of -6 or less was found in 22 pts (26.5%) while 61 pts (73.5%) had AI of more than (>) -6. Of the 61 pts with AI > -6, 37.7% pts were SLN +ve while 62.3% pts were SLN -ve (p=0.05)( Table 1A ). Of the 22 pts with AI less than or -6, 45.5% pts were SLN +ve and 54.5% pts were SLN -ve (p=0.65). Size of the metastatic tumor burden in lymph nodes was available in 69.7% of SLN +ve pts. Total average tumor burden for pts with AI > -6 (n=15) was 2.04cm as compared to 1.48cm in pts with AI of -6 or less (n=8)(p=0.66). Average SLN met size was 0.73cm in pts with AI > -6 and 0.63cm in pts with AI of -6 or less (p=0.66)( Table 1B ). Conclusions: AI did not correlate with nodal positivity or tumor burden in LNs in pts with Cca. LN status remains the most important prognostic marker in Cca. Further larger trials are required to determine the role of AI as a prognostic marker in Cca. [Table: see text] [Table: see text] No significant financial relationships to disclose.

16055 Background: The reliability of molecular biomarkers as predictors of treatment outcome remains unclear and, in relation to drug resistance even less is known. Immunohistochemical (IHC) expression of biomarkers and in vitro drug resistance (IVDR) relative to clinical outcome were evaluated in patients with stage III-IV epithelial ovarian cancer (OC) who received combination carboplatin (CAR) plus paclitaxel (TAX) chemotherapy. Methods: We correlated clinical outcome with histopathology and biomarker expression of MIB-1, p53, BCL2, EGFR, ER and PR in 98 OC patients with tumors tested for IVDR by extreme drug resistance (EDR) assay. IVDR was determined following exposures to single agents CAR, cisplatin (CP), TAX, taxotere (TXT), gemcitabine (GCB), topotecan (TP), liposomil doxorubicin, and cytoxan (CT). Percentage cell growth inhibition (PCI) for each drug was compared with PCI from untreated control cultures. Results: Tumors with prior chemotherapy (n=29) were more resistant to CAR (unpaired t-test, p=0.046), and CT (p=0.038), and more sensitive to liposomil doxorubicin (p=0.009), compared to treatment naive tumors (n=53). For 65 tumors, there was a positive relationship between MIB-1 vs p53 (r=0.419; p=0.001), and between ER vs PR (r=0.339; p=0.011). Correlation between biomarkers and drug response showed a significantly inverse relationship between MIB-1 vs liposomil doxorubicin, p53 vs liposomil doxorubicin, EGFR vs CAR, CP, GCB and TAX; and between ER vs TXT and CT, and PR vs CT and TP. With a 53% (33/61) response rate to CAR+TAX, time to progression (TTP) for responders (R) with EGFR+ tumors was longer (median TTP 15.6 mo); and shorter for R with EGFR- tumors (p=0.008). For R and non-R, BCL2+ tumors had longer overall survival (OS) (median OS 23.3 mo; p=0.006). No other significant correlations were seen. Conclusion: Prior chemotherapy increased IVDR to CAR and CT, and increased sensitivity to liposomil doxorubicin. Increased expression of proliferation biomarkers, particularly EGFR, correlated with resistance to several chemotherapeutic agents. EGFR expression in responders was associated with longer TTP. The significance of in vitro correlates of prognostic biomarker expression and IVDR for predicting chemoresistance warrants prospective studies to assess its value in clinical settings. [Table: see text]

2532 Background: To determine the biomarker expression and drug resistance patterns of undifferentiated ovarian cancers. Methods: Biomarker expression was determined by immunohistochemistry. In vitro drug resistance profiles were analyzed by assays exposing tumors to suprapharmacologic doses of chemotherapy. Results: Of 177 ovarian cancer specimens, 63 were classified as undifferentiated tumors and 114 well-differentiated cancers were used for comparison. Of the undifferentiated tumors, 46.7% were primary cancers and the remainder were recurrent cancers. Most of the well-differentiated tumors were obtained from primary (82%) vs. recurrent disease specimens. The average DNA index and S-phase fraction were significantly higher in the undifferentiated vs. well-differentiated tumors (1.42 vs. 1.23, p=0.025 for DNA index; 1.69 vs. 3.06, p<0.0001 for S phase fraction). In addition, the percent of tumors with p53 mutations was also higher in the undifferentiated compared to the well-differentiated tumors (47.8% vs. 17.8%; p=0.005). The mean percent cell inhibition was significantly lower in the undifferentiated tumors compared to well-differentiated cancers after exposure to etoposide (54%±21 vs. 80%±18; p=0.0002), doxorubicin (59%±28 vs. 80.0%±21; p=0.07), and topotecan (61.7%±20 vs. 71%±21; p=0.015). Compared to well-differentiated tumors, undifferentiated tumors tended to be more resistant to etoposide (40% vs. 11%), doxorubicin (22% vs. 9%), and topotecan (19% vs. 16%). However, the drug resistance patterns after exposure to taxanes (paclitaxel and taxotere) and platinums (carboplatin and cisplatin) were similar. Conclusions: Undifferentiated tumors of the ovary have a significantly different biomarker expression and drug resistance profiles compared to well-differentiated tumors. Given that undifferentiated cancers provide a treatment challenge for the clinician, this data can provide additional prognostic and therapeutic information, and warrants further investigation. No significant financial relationships to disclose.

OBJECTIVES:Empiric chemotherapy for patients with non-small cell lung cancer who have undergone resection is recommended without knowledge of the tumor's specific biologic characteristics, and many patients may not benefit. In vitro chemotherapy resistance is associated with clinical unresponsiveness in some tumors, and in lung cancer, chemotherapy resistance is prevalent. Multiple-agent chemotherapy resistance and association of chemotherapy resistance with molecular markers are described.METHODS:Chemotherapy resistance to doublets--carboplatin and paclitaxel, cisplatin and navelbline, cisplatin and docetaxel, and cisplatin and gemcitabine--was analyzed in 4571 non-small cell lung cancer tumors with the extreme drug resistance assay. Chemotherapy resistance is defined as follows: extreme drug resistance, 1 SD above the median chemotherapy resistance; intermediate drug resistance, between the median and extreme drug resistances; and low drug resistance, 1 SD below the median. Chemotherapy resistance was compared with DNA ploidy measured by flow cytometry, and markers p53 and epithelial growth factor receptor were assayed by immunohistochemistry.RESULTS:Tumors with extreme or intermediate drug resistance were noted in 30% to carboplatin-paclitaxel, in 24% to cisplatin-navelbline, in 42% to cisplatin-gemcitabine, and in 27% to cisplatin-docetaxel. Extreme or intermediate drug resistance to at least one drug occurred in 74% to carboplatin-paclitaxel, in 68% to cisplatin-navelbline, in 88% to cisplatin-gemcitabine, and in 68% to cisplatin-docetaxel. More intermediate plus extreme chemotherapy resistances occurred in aneuploid tumors to etoposide (53% vs 36%, P = .0002) and topotecan (48% vs 36%, P = .0094), with less intermediate or extreme chemotherapy resistance to gemcitabine (88% vs 81%, P = .0345). p53-Positive tumors had more intermediate or extreme resistance to etoposide (57% vs 44%, P = .0009) and doxorubicin (73% vs. 58%, P = .0324) and less intermediate or extreme resistance to cisplatin (44% vs 54%, P = .0125), to carboplatin (47% vs 57%, P = .0129), to taxol (47% vs 57%, P = .0056), and to gemcitabine (78% vs 87%, P = .0108). Fewer epithelial growth factor receptor-positive tumors were extremely drug resistant to cisplatin (13% vs 26%, P = .0074) and carboplatin (13% v. 30%, P = .0008).CONCLUSIONS:Multi-drug chemotherapy resistance in non-small cell lung cancer tumor cultures is common, and associations between molecular markers and in vitro chemotherapy resistance are noted. Clinical validation through integration of such testing into clinical trials seems warranted.

Cell fusion protocols that were developed by Kohler and Milstein in the mid-1970s and aimed at producing and characterization of mouse monoclonal antibodies (MAbs) remain the gold standard of hybridoma development. Despite tremendous progress in using MAbs in multiple research, diagnostic, and therapeutic areas, major experimental flaws in designing and carrying out hybridoma experimentation often result in the production of hybridomas exhibiting poor growth parameters and secreting low-specificity and low-affinity antibodies. This methodology chapter is built around the conventional hybridoma protocol, with a special emphasis on tissue culture and biochemical techniques aimed at producing truly monospecific and highly active mouse MAbs.

Acquired and intrinsic multidrug resistance is the major reason for the failure of anticancer chemotherapy. The most important component of clinical multidrug resistance is mediated by P-glycoprotein (Pgp), an ABC transporter encoded by the MDR1 gene and expressed on the membrane of tumor and normal cells. Sensitive and reproducible detection of Pgp expression and function are critical for the development of new MDR1 drugs and clinical protocols aimed at modulating Pgp-mediated multidrug resistance. The most commonly used methods for detecting Pgp distribution and functional activity have major flaws when used in routine clinical diagnostics. In this chapter, we describe and compare these techniques and introduce a new method for simultaneous detection of Pgp expression and function--the UIC2 Shift assay.

Depending on the tumour type, a larger or smaller number of cancer patients receive chemotherapy with systemic toxicity as the only effect. In that situation, an alternative, not necessarily medical, treatment would have been a better choice – and toxicity (and financial resources) could have been spared by withholding ineffective drugs. One of the reasons for this apparent paradigm is that the tumour cells of each cancer pa- tient may show different sensitivity/resistance towards different chemotherapeutic drugs, i.e. breast cancer or colorectal cancer is not only breast or colorectal cancer. With our increasing biological insight and understanding, it has become apparent that each patient's tumour tissue is unique and as a consequence, each patient's tumour cell sensitivity/resistance to- wards chemotherapeutic drugs may be different. As of today there is no method in routine clinical use to predict the sensitivity/resistance to chemotherapy in its broad sense in the individual patient. This chapter will describe several different DNA, RNA, protein and cell based assay methodologies and marker molecules that have been brought forward as potential predictive assays/markers to be used to select the most effective drugs for the individual cancer patient.

A201 Ovarian cancer is one of the most aggressive cancers in women. The majority of ovarian cancer patients are diagnosed at late stages and they are usually treated with cytoreductive surgery followed by chemotherapy with carboplatin and taxol. Although ovarian cancer is initially responsive to chemotherapeutic agents, most patients eventually develop chemoresistant tumors and succumb to their recurrent diseases. We have previously identified NAC-1 as a tumor recurrence-associated gene in ovarian serous carcinomas. NAC-1 is a transcription repressor belonging to the BTB/POZ family which participates in a variety of biological activities including regulation of transcription and maintenance of stem cell phenotype. Our previous studies demonstrated that NAC-1 was highly expressed in recurrent ovarian carcinomas and its expression in primary tumors predicts early tumor recurrence (PNAS 2006, 103:18739; Hum Pathol 2007, 38:1030). In this study, we determine whether NAC-1 expression contributes to chemoresistance in ovarian cancer. Firstly, we showed that NIH3T3 cells over-expressing NAC-1 either by stable transfection or by retroviral delivery of an NAC-1 expression cassette are more resistance to taxol but not to carboplatin as compared to their control cells without NAC-1 expression (IC50 = 5000 nM and 100 nM, respectively). Secondly, we demonstrate that NAC-1 is upregulated in SKOV3taxol cells that have been induced to become taxol resistant in culture and in post taxol-treated SKOV3 xenografts in nude mice as compared to control naive cells based on real-time PCR and immunohistochemistry, respectively. Thirdly, using immunohistochemistry on 160 ovarian cancer specimens in which their in vitro drug resistance status is available, we found that NAC-1 immunointensity correlates with the most taxol resistant status with a p-value of 0.05 (Fisher’s Exact test). Lastly, down-regulation of NAC-1 by NAC-1 specific shRNA sensitizes SKOV3taxol cells to taxol killing. In summary, we provide new evidence that NAC-1 overexpression contributes to tumor recurrence by equipping cancer cells with taxol-resistant phenotypes in ovarian carcinomas. Furthermore, NAC-1 might be considered as a molecular target for future development of novel therapeutic reagents to sensitize the anti-tumor effect of taxol in recurrent ovarian carcinomas overexpressing NAC-1.

5037 Background: Although chemotherapy is widely used to treat advanced and recurrent endometrial cancer, no patient-tailored protocols were developed, resulting in intermittent and incomplete clinical response. The utility of the Extreme Drug Resistance (EDR) assay to predict clinical resistance to chemotherapy was proven in several cancer types, including ovarian cancer. This study is the first attempt to evaluate the efficacy of the EDR assay in endometrioid (E) and papillary serous (PS) tumors and correlate it with the expression of two major biomarkers, DNA ploidy and p53. Methods: 2520 E and 586_PS adenocarcinomas were tested in the EDR assay using in vitro incubation in suprapharmacologic exposures of 4-HC (the active species of cyclophosphamide), cisplatin, carboplatin, and doxorubicin. The results were defined as EDR (one standard deviation more resistant than the database median), intermediate drug resistance (IDR - between the median and EDR), and low drug resistance (LDR - lower than the median). DNA ploidy was assessed in flow cytometry; p53 expression was quantitated on paraffin sections by immunohistochemistry. Results: Compared to E, the more clinically aggressive PS tumors were significantly associated with high (IDR + EDR) in vitro resistance to all EDR-tested drugs: 4-HC (p = 0.0010; χ2 statistics), cisplatin (p = 0.0064), carboplatin (p = 0480), doxorubicin (p = 0.0001). PS tumors showed significantly higher non-diploid status (p < 0.0001) and p53-positivity (p < 0.0001). For all EDR-tested drugs, increased in vitro resistance closely correlated with the non-diploid and high p53 status. Conclusions: These data document close correlation between the EDR assay results and biological behavior of endometrial tumors in terms of their clinical aggressiveness and biomarker expression and justify further studies on combined EDR and biomarker testing in assay-guided chemotherapy. [Table: see text] No significant financial relationships to disclose.

Purpose: To investigate the association between parameters obtained from dynamic contrast enhanced MRI (DCE-MRI) of breast cancer using different analysis approaches, as well as their correlation with angiogenesis biomarkers (vascular endothelial growth factor and vessel density).Materials and Methods: DCE-MRI results were obtained from 105 patients with breast cancer (108 lesions). Three analysis methods were applied: 1) whole tumor analysis, 2) regional hot-soot analysis, and 3) intratumor pixel-by-pixel analysis. Early enhancement intensities and fitted pharmacokinetic parameters were studied. Paraffin blocks of 71 surgically resected specimens were analyzed by immunohistochemical staining to measure microvessel counts (with CD31) and vascular endothelial growth factor (VEGF) expression levels.Results: MRI parameters obtained from the three analysis methods showed significant correlations (P < 0.0001), but a substantial dispersion from, the linear regression line was noted (r = 0.72-0.97). The entire region of interest (ROI) vs. pixel population analyses had a significantly higher association compared to the entire ROI vs. hot-spot analyses. Cancer specimens with high VEGF expression had significantly higher CD31 microvessel densities than did specimens with low VEGF levels (P < 0.005). No significant association was found between MRI parameters obtained from the three analysis strategies and IHC based measurements of angiogenesis.Conclusion: A consistent analysis strategy was important in-the DCE-MRI study. In this series, none of these strategies yielded results for MRI based quantitation of tumor vascularity that were associated with IHC based measurements., Therefore, different analyses could not account for the lack of association.

During transport‐associated adenosine triphosphate hydrolysis, P‐glycoprotein (Pgp) undergoes conformation transitions detected by UIC2, a functional anti‐Pgp monoclonal antibody. A newly developed UIC2 shift assay is based on increased UIC2 reactivity in the presence of Pgp substrates. All peripheral blood leukocytes express low Pgp levels. The existing antibody‐based detection methods are limited in their sensitivity and require additional techniques to simultaneously analyze Pgp expression and efflux, making it difficult to ascertain the physiologic role of Pgp‐mediated transport.

P-glycoprotein (Pgp), a membrane pump often responsible for the multidrug resistance of cancer cells, undergoes conformational changes in the presence of substrates/modulators, or upon ATP depletion, reflected by its enhanced reactivity with the UIC2 monoclonal antibody. When the UIC2-shift was elicited by certain modulators (e.g. cyclosporin A or vinblastine, but not with verapamil or Tween 80), the subsequent binding of other monoclonal anti-Pgp Ig sharing epitopes with UIC2 (e.g. MM12.10) was abolished [Nagy, H., Goda, K., Arceci, R., Cianfriglia, M., Mechetner, E. & Szabó Jr, G. (2001) Eur. J. Biochem. 268, 2416-2420]. To further study the relationship between UIC2-shift and the suppression of MM12.10 binding, we compared, on live cells, how ATP depletion and treatment of cells with phosphate analogues (sodium orthovanadate, beryllium fluoride and fluoro-aluminate) that trap nucleotides at the catalytic site, affect the two phenomena. Similarly to modulators or ATP depleting agents, all the phosphate analogues increased daunorubicin accumulation in Pgp-expressing cells. Prelabeling of ATP depleted cells with UIC2 completely abolished the subsequent binding of MM12.10, in accordance with the enhanced binding of the first mAb. Vanadate and beryllium fluoride, but not fluoro-aluminate, reversed the effect of cyclosporin A, preventing UIC2 binding and allowing for labeling of cells with MM12.10. Thus, changes in UIC2 reactivity are accompanied by complementary changes in MM12.10 binding also in response to direct modulation of the ATP-binding site, confirming that conformational changes intrinsic to the catalytic cycle are reflected by both UIC2-related phenomena. These data also fit a model where the UIC2 epitope is available for antibody binding throughout the catalytic cycle including the step of ATP binding, to become unavailable only in the catalytic transition state.

The mechanisms responsible for regulating epithelial ATP permeability and purinergic signaling are not well defined. Based on the observations that members of the ATP-binding cassette (ABC)1 family of proteins may contribute to ATP release, the purpose of these studies was to assess whether multidrug resistance-1 (MDR1) proteins are involved in ATP release from HTC hepatoma cells. Using a bioluminescence assay to detect extracellular ATP, increases in cell volume increased ATP release ∼3-fold. The MDR1 inhibitors cyclosporine A (10 μm) and verapramil (10 μm) inhibited ATP release by 69% and 62%, respectively (p < 0.001). Similarly, in whole-cell patch-clamp recordings, intracellular dialysis with C219 antibodies to inhibit MDR1 decreased ATP-dependent volume-sensitive Cl− current density from −33.1 ± 12.5 pA/pF to −2.0 ± 0.3 pA/pF (−80 mV, p≤ 0.02). In contrast, overexpression of MDR1 in NIH 3T3 cells increased ATP release rates. Inhibition of ATP release by Gd3+ had no effect on transport of the MDR1 substrate rhodamine-123; and alteration of MDR1-substrate selectivity by mutation of G185 to V185 had no effect on ATP release. Since the effects of P-glycoproteins on ATP release can be dissociated from P-glycoprotein substrate transport, MDR1 is not likely to function as an ATP channel, but instead serves as a potent regulator of other cellular ATP transport pathways.

Conformational changes accompanying P-glycoprotein (Pgp) mediated drug transport are reflected by changes in the avidity of certain monoclonal antibodies (mAbs). More of the UIC2 mAb binds to Pgp-expressing cells in the presence of substrates or modulators [Mechetner, E.B., Schott, B., Morse, S.B., Stein, W., Druley, T., Dvis, K.A., Tsuruo, T. & Roninson, I.B. (1997) Proc. Natl Acad. Sci. USA 94, 12908-12913], while the binding of other mAbs (e.g. MM12.10, MRK16, 4E3) is not conformation sensitive. Pre-staining of Pgp+ cells with UIC2 decreased the subsequent binding of MM12.10 mAb by about 30-40%, suggesting that there are Pgp molecules available for both UIC2 and MM12.10, and others accessible only for MM12.10. In the presence of certain substrates/modulators such as vinblastin, cyclosporin A or valinomycin, the MM12.10 reactivity was completely abolished by preincubation with UIC2. However, verapamil, Tween-80 and nifedipine did not influence the ratio of bound mAbs significantly. This is the first assay to our knowledge, sharply distinguishing two classes of modulators. The conformational changes accompanying the mAb competition phenomenon appear to be closely related, though not identical to those accompanying the UIC2-shift, as suggested by the simultaneous assessment of the two phenomena.