Chemosensitivity Testing Research Articles

Chemotherapy for Malignant Gliomas Based on Histoculture Drug Response Assay : A Pilot Study

The Histoculture Drug Response Assay (HDRA), which measures chemosensitivity using minced tumor tissue on drug-soaked gelfoam, has been expected to overcome the limitations of in vitro chemosensitivity test in part. We analyzed interim results of HDRA in malignant gliomas to see if the test can deserve further clinical trials. Thirty-three patients with malignant gliomas were operated and their tumor samples were examined for the chemosensitivity to 10 chosen drugs by HDRA. The most sensitive chemotherapy regimen among those pre-established was chosen based on the number of sensitive drugs or total inhibition rate (IR) of the regimen. The response was evaluated by 3 month magnetic resonance image. Among 13 patients who underwent total resection of the tumor, 12 showed no evidence of disease and one patient revealed progression. The response rate in 20 patients with residual tumors was 55% (3 complete and 8 partial responses). HDRA sensitivity at the cut-off value of more than one sensitive drug in the applied regimen showed a sensitivity of 100%, specificity of 60% and predictability of 70%. Another cut-off value of >80% of total IR revealed a sensitivity of 100%, specificity of 69%, and predictability of 80%. For 12 newly diagnosed glioblastoma patients, median progression-free survival of the HDRA sensitive group was 21 months, while that of the non-sensitive group was 6 months (p=0.07). HDRA for malignant glioma was inferred as a feasible method to predict the chemotherapy response. We are encouraged to launch phase 2 clinical trial with chemosensitivity on HDRA.

Involvement of ARLTS1 in chemotherapy and apoptosis in ovarian cancer cell line

Recent evidence suggests that ADP-ribosylation factor-like tumor suppressor gene 1(ARLTS1) may act as a tumor suppressor gene. However, its role in tumor chemotherapy remains unclear. The aim of this study is to investigate the effects of ARLTS1 gene in regulation of chemosensitivity in ovarian cystadenocarcinoma cell line SKOV3. We stably expressed wild-type (wt) ARLTS1 and empty vector (neo) in SKOV3 cells. Chemosensitivity test was carried out with four chemotherapeutic agents. Cell proliferation, cycle kinetics and apoptosis were evaluated by MTT assay and flow cytometry. Apoptosis-related proteins caspase-3 and bcl-2 were determined by Western blot analysis. The proliferation of wtARLTS1 clones was more dramatically inhibited by all the cytotoxic agents than parental cells (P < 0.05). Increased sensitivity to chemotherapy by two to threefolds was detected in wtARLTS1 cells. The rate of apoptosis in wtARLTS1 was 60.2% treated with DDP (10× peak plasma concentration, PPC), which was dramatically higher than that of neo and parental cells (P is 0.017 and 0.020, respectively). Expression of caspase-3 and bcl-2 in parental cells declined modestly when treated with DDP, while in wtARLTS1 clones the expression of caspase-3 and bcl-2 levels declined more dramatically and become undetectable at lower DDP doses (P = 0.023 and <0.001, respectively). Our findings suggested that ARLTS1 may facilitate chemosensitivity in ovarian cancer cells by acting synergistic with chemotherapeutic agents to induce the apoptosis signaling pathway and regulate apoptosis-related proteins.

High-Dose Cyclophosphamide (Cy), Rituximab, and a Cancer Vaccine for Relapsed Classical Hodgkin's Lymphoma (cHL)

AbstractAbstract 3954Background: Curative but less toxic regimens are needed for relapsed cHL, as autologous stem cell transplantation (auto SCT) carries ∼ 5% mortality risk and nearly 100% risk of infertility. Work at our institution showed that 1) high-dose Cy, equivalent to transplantation doses, can be given safely without SCT and the potential of reinfusing tumor cells, does not preclude any form of salvage SCT, and can spare fertility; and 2) unlike Hodgkin and Reed Sternberg (HRS) cells, cHL cancer stem cells may have a CD20+, memory B cell phenotype and circulate in surprisingly high frequencies (Jones RJ et al, Blood 2009;113:5920), potentially explaining rituximab's activity in this disease. Derived from the K562 cell line, the HL vaccine is a novel, allogeneic (allo), GM-CSF producing vaccine that expresses antigens commonly present in cHL including survivin. In addition, the vaccine expresses the EBV antigens LMP2 and EBNA1, commonly present in EBV+ cHL. Patients and Methods: A phase I/II study of high-dose therapy and immunotherapy for relapsed cHL was developed with curative intent. Eligibility included chemosensitive relapse (chemosensitivity testing was not required for low-volume relapse), no prior SCT, no primary refractory disease, and adequate end-organ function. Chemotherapy consisted of rituximab 375 mg/m2 IV on d 1 and 4, high-dose Cy (50 mg/kg/d IV on d 8–11) with mesna, filgrastim, and rituximab 375 mg/m2 IV weekly for 4 weeks upon platelet recovery. HL vaccine (1 × 10exp8 cells/dose) was administered 1 d after Cy completion and 4, 8, 12, 16, and 24 weeks later. Consolidative radiation was permitted. All treatment was intended to be outpatient. Results: Clinical outcomes of the first 25 pts accrued are described. Median age was 40 (range 18–67) y; 10 were female. Stage was IIB-IV in 14 pts (56%) upon diagnosis, and 9 pts (36%) relapsed with extranodal disease. HRS cells were CD20+ in 4/25 cases (16%), EBV+ in 6/24 evaluable cases (25%). Median time between first-line therapy completion and relapse was 12 (range 2–94) mo; 22 pts had CR or PR and 2 had stable disease to salvage therapy consisting chiefly of ICE or ESHAP (median 2 cycles, range 1–4), and 1 had untested relapse. With median 31 (range 7–53) mo follow-up for all pts, actuarial 1 y and 2 y overall survival is 100%, while actuarial event-free survival is 79% at 1 y and 66% at 2 y following high-dose Cy (Figure). The regimen was well-tolerated, and there was no treatment-related mortality. Eleven admissions occurred, 10 for infections (8 neutropenic, 2 non-neutropenic) which all resolved without sequelae, with 2 other neutropenic fevers managed without admission. One pt developed idiopathic protracted pancytopenia (gradually improving), cardiomyopathy, and chronic kidney disease. Vaccine toxicities were largely limited to mild local skin reactions. Median time to neutrophil recovery after the last dose of Cy was 17 (range 10–26) d; median time to sustained platelets >= 20,000/μL, without transfusion in the preceeding week, was 21 (range 0–46) d, with 5 pts not requiring platelet transfusion. Grade 3 or 4, late-onset neutropenia, presumably rituximab-related, was documented in 11 pts (44%; grade 4 in 5 pts) at a median of 3 (range 1–18) mo following study initiation. In the 7 relapsed pts, median time to relapse was 7 (range 5–16) mo after high-dose Cy; 1 died of disease after auto and allo SCT, 1 died in the course of allo SCT, 1 is alive with disease after allo SCT, 3 are event-free 7 mo - 3 y after allo SCT, and 1 has allo SCT planned. Early stopping rules for safety and efficacy have not been met. One pt had a successful pregnancy 33 mo after high-dose Cy. Conclusion: A novel outpatient regimen incorporating high-dose Cy and rituximab produces encouraging outcomes on preliminary analysis in relapsed cHL pts. Early efficacy appears comparable to that of auto SCT, with potentially less toxicity. Analyses of immunologic endpoints and HL biomarkers are in progress. [Display omitted] Disclosures:Kasamon:Genentech: Research Funding. Off Label Use: Rituximab, cyclophosphamide: off-label use. Cancer vaccine: investigational. Levitsky:HL vaccine: Patents & Royalties. Borrello:HL vaccine: Patents & Royalties. Swinnen:Genentech: Consultancy, Research Funding.

Chemosensitivity test for 5-fluorouracil and 5-chloro-2, 4-dihydroxypyridine predicts outcome of gastric cancer patients receiving S-1 postoperatively

Chemosensitivity tests have long been discussed but remain a topic of research. In this study, we investigated the correlation between the results of a chemosensitivity test for 5-fluorouracil and 5-chloro-2, 4-dihydroxypyridine and the clinical outcomes of gastric cancer patients treated with S-1, an oral fluoropyrimidine, as adjuvant chemotherapy. For gastric cancer patients, we performed surgical treatment and a lymph node dissection of D2 or more. Afterwards, a chemosensitivity test for 5-fluorouracil and 5-chloro-2, 4-dihydroxypyridine was performed, using the collagen gel droplet embedded culture drug-sensitivity test (CD-DST), in surgical specimens. All the patients received postoperative adjuvant chemotherapy with S-1 for 1 year, and the overall survival (OS), relapse-free survival (RFS), and adverse events were investigated. The chemosensitivity test was performed for 27 patients. The growth inhibition rate (IR) was 50% or more (high-sensitivity group) in 59.3% (16 cases) and it was under 50% (low-sensitivity group) in 40.7% (11 cases). The 3-year OS rate was 100% in the high-sensitivity group and 62.34% in the low-sensitivity group. The 3-year RFS rate was 83.33% in the high-sensitivity group and 24.24% in the low-sensitivity group. Thus, the 3-year OS rate and the 3-year RFS rate were higher in the high-sensitivity group than in the low-sensitivity group. No adverse events of grade 3 or greater severity were observed. The results of the chemosensitivity test were correlated with the patient outcome. Therefore, such results might be useful for individualizing cancer chemotherapy and for determining future indications for postoperative adjuvant chemotherapy.

Advanced dynamic monitoring of cellular status using label-free and non-invasive cell-based sensing technology for the prediction of anticancer drug efficacy

Quantitative evaluation of anticancer drug efficacy using in vitro cell-based assays is useful for cancer patients, particularly those who show unconventional cancer development. Nevertheless, conventional chemosensitivity testing often requires widely used labeling agents and time-consuming laboratory procedures that provide low reliability. Label-free non-invasive cell-based assays are desired for dynamic monitoring of cellular status. This critical review first describes conventional chemosensitivity testing and then advanced label-free cell-based technology used to screen anticancer drugs through dynamic monitoring of cellular status, focusing on dosage and the use of drug-resistant cancer cells. Results from label-free cell-based approaches are compared with those of conventional chemosensitivity testing. The cellular statuses, addressed in terms of respective mechanisms and disadvantages, are extracellular fluxes of proton (H(+)), O(2), and anticancer drugs, cell morphology changes, cell-environment interaction, and mitochondrial membrane potential. Finally, a cell-based systems outlook is presented. This paper represents a step toward efficient and accurate initial screening of anticancer drugs and development of compounds and their combined use to achieve pharmacodynamic and pharmacokinetic interactions, and chemotherapy evaluation of particular anticancer drugs for individual patients.

Influence of methoxy groups on the antiproliferative effects of [Fe III(salophene-OMe)Cl] complexes

We synthesized methoxy-substituted iron(III)-salophene complexes ([Fe III(OMe-salophene)Cl] with salophene = N, N′-bis(salicylidene)-1,2-phenylenediamine) and analyzed their biological activity in MCF-7 and MDA-MB-231 breast cancer as well as in HT-29 colon carcinoma cells. The results obtained in a time-dependent chemosensitivity test clearly demonstrated the correlation between the cytotoxicity of the complexes and the position of methoxy substituents in the salicylidene moieties: 3-OCH 3 ( 4) < 5-OCH 3 ( 8) < H ( 2) < 4-OCH 3 ( 6) = 6-OCH 3 ( 10). Compounds 6 and 10 caused cytocidal effects already at a concentration of 0.5 μM. Both lead compound 2 and complex 8 showed similar time response curves, however, with a 5-fold lower activity compared to 6 and 10, respectively. Referring to [Fe III(salophene)Cl] ( 2), methoxy substitution was accompanied with the loss of tumor cell selectivity. Moreover, the free ligands ( 1, 3, 5, 7, and 9) were inactive.

Chemoresistance testing of human ovarian cancer cells and its in vitro model

Ovarian carcinoma represents the most common cause of death from gynecological malignancies in Europe and North America, being the third most frequent and the first as to the mortality. The standard chemotherapeutical regimen for ovarian cancer involves the administration of platinum derivate (carboplatin, cisplatin), in advanced stage is platinum derivate combined with paclitaxel. Introducing chemoresistance testing of ovarian tumour cells may help to choose optimal chemotherapeutic drug and customize the individual chemotherapeutical regimens in patients. One of approaches of individualization of chemotherapy is in vitro chemosensitivity testing. In our study, we evaluated the cytotoxic effects of selected chemotherapeutics in cells isolated from ovarian tumours and ascites of individual patients. Panel of chemotherapeutics used in the study included cisplatin, paclitaxel, carboplatin, topotecan, gemcitabine and etoposide and their effects on cell viability were determined by the MTT assay. In the total number of 32 clinical samples of tumour and ascites cells, the highest sensitivity showed cells to topotecan, sensitivity to cisplatin was higher than to carboplatin and paclitaxel used in clinical practice showed most often only the marginal reactivity. Resistance to carboplatin and most of the time to gemcitabine and etoposide was commonly present. When the same test on cells that have been frozen for several weeks was repeated it was found that in 20 cases chemosensitivity increased while in 18 cases decreased. In remaining cases there was no change in reactivity to cytostatics. Moreover, chemosensitivity of cells isolated from solid tumour and ascites from the same patient did not show any significant difference with exaption of paclitaxel.

Metastatic Lymph Node Targeted Chemosensitivity Test for Gastric Cancer

The objective of this study was to compare the chemosensitivity of primary tumor and metastasized lymph node from patient with gastric adenocarcinoma. We studied 26 gastric cancer patients with lymph node metastasis who underwent gastric resection at the Korea University Guro Hospital from Feb 2007 to July 2008. The chemosensitivity of primary tumor and metastatic lymph node were studied using an adenosine triphosphate-based chemotherapy response assay (ATP-CRA). The concordance rate of the ATP-CRA test was 30.8% (8/26). The concordance rate between primary tumor and metastatic N2 group lymph node was only 9.1% (1/11). The metastatic tumor inhibition rates with 5-fluorouracil, cisplatin, doxorubicin, and oxaliplatin were higher than the inhibition rates for primary tumor. Tumor inhibition rates was significantly different between primary tumor and metastatic tumor after doxorubicin treatment (27.734±20.95 versus 38.403±26.87, P=0.021). We detected simple correlations of tumor inhibition rates between primary and metastatic tumors with cisplatin (r=0.661, P<0.001) and doxorubicin (r=0.475, P=0.031). We observed differences between first choice chemotherapeutic agents based on ATPCRA tests of primary tumor and metastatic tumor in lymph node. Therefore, chemotherapeutic agents should be carefully selected for adjuvant chemotherapy using a chemosensitivity test.

ER-60 (PDIA3) is highly expressed in a newly established serous ovarian cancer cell line, YDOV-139

Characterization of a newly established serous ovarian cancer cell line, YDOV-139 was performed and ER-60 (PDIA3), which was highly expressed in YDOV-139, was evaluated as novel biomarker for ovarian cancer. The YDOV-139 cell line was established using ascites samples from a 67-year-old Korean woman with recurrent ovarian cancer, and was characterized with respect to various biological and genetic features. Gene expression profiles were analyzed using cDNA microarrays, and proteomic evaluation was performed by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time of flight peptide mass fingerprinting (MALDI-TOF/PMF). Four candidate markers that were strongly up-regulated in YDOV-139 were validated by real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC). The epithelial-like characteristics of YDOV-139 were evident from morphologic studies, and the average population doubling time was 120 h. When transplanted into nude mice, YDOV-139 cells successfully induced tumor masses in all three animals. Chemosensitivity tests showed that gemcitabine had the highest chemosensitivity index against YDOV-139 cells. HLA typing revealed A*24/A*31, B*07/B*35, Cw03*(09)/w*07, and DRB1*01/DRB1*15 alleles. Compared with human ovarian surface epithelial (HOSE) cells, 2,520 genes and 23 protein spots were differentially expressed in YDOV-139. Validation by real-time PCR showed that mRNA expression of LCN2, MDK, SLCO4A1, and ER-60 (PDIA3) were strongly elevated in ovarian cancers. In IHC analysis, ER-60 (PDIA3) was significantly overexpressed in both borderline tumors and invasive ovarian cancers (P<0.001). The molecular characteristics of YDOV-139 may have implications for future ovarian cancer research and ER-60 (PDIA3) should be investigated further as a potential biomarker of ovarian cancer.

Oxaliplatin‐based chemotherapy (dexamethasone, high‐dose cytarabine, and oxaliplatin) ± rituximab is an effective salvage regimen in patients with relapsed or refractory lymphoma

Patients affected by relapsed or primary refractory lymphomas currently have a poor prognosis and no standard salvage treatment options. This study was carried out to assess the efficacy and safety of a dexamethasone, high-dose cytarabine, and oxaliplatin as salvage therapy in those patients, replacing cisplatin with oxaliplatin in the standard dexamethasone, cytarabine, and cisplatin scheme. Seventy patients with relapsed or refractory aggressive non-Hodgkin or Hodgkin lymphoma were treated from September 2001 to September 2007. The median age of patients was 51 years (range, 19-75 years). Histological subtypes were: diffuse large B-cell lymphoma (n=47) and Hodgkin lymphoma (n=23). The overall response rate was 73% (51 of 70), with 30 (43%) complete remissions and 21 (30%) partial remissions. Fifty-two patients were treated with dexamethasone, high-dose cytarabine, and oxaliplatin as second-line chemotherapy. Forty-eight patients were enrolled in an autologous stem cell transplantation program; forty (83%) finally proceeded to high-dose consolidation and autografting. No grade 3 or 4 nonhematological toxicity was demonstrated; in particular, no renal or neurotoxicity was reported. After a median follow-up period of 21 months (range, 2-87 months), 22 (31%) patients had died. Probabilities of 2-year progression-free survival (PFS) and overall survival (OS) were 44% and 71%, respectively. In the chemosensitive patients, the PFS and OS were 52% and 83%, respectively. The only factor that significantly correlated with better OS was the response to therapy. This study confirms that dexamethasone, high-dose cytarabine, and oxaliplatin ± rituximab is an effective and feasible outpatient regimen for salvage therapy in patients affected by relapsed or refractory lymphoma. Moreover, the feasibility and efficacy of this scheme as an in vivo chemosensitive test in patients in autotransplantation programs was confirmed.

The MTS vs. the ATP assay for in vitro chemosensitivity testing of primary glioma tumour culture

The MTS vs. the ATP assay for in vitro chemosensitivity testing of primary glioma tumour culture

Chemosensitivity testing to predict chemosensitivity for gemcitabine, using the biopsy specimens obtained by EUS-FNA from unresectable pancreatic cancer.

e14640 Background: The purpose of this prospective study is to evaluate the feasibility of chemosensitivity testing using EUS-FNA samples and determine the optimal cut-off value of gemcitabine. Methods: Thirty-two consecutive patients, who were undergone EUS-FNA in our institute between October 2006 and July 2009, with unresectable pancreatic cancer were enrolled. All patients were treated with gemcitabine. Chemosensitivity (treated/control ratio: T/C) was calculated as the percentage of quantity of ATP of a tumor treated with gemcitabine and of control. Concentration of gemcitabine was 20 μg/mL according to the peak plasma concentration. To identify the cut-off value that could predict 180 days of PFS, receiver operating characteristic (ROC) curve and corresponding area under the curve (AUC) was calculated. Results: Thirty-three times assays were performed on 32 patients. Twenty-eight patients (87.5%) were successful. Four patients of 28 patients were excluded because of impossible to keep the protocol, therefore, 24 patients could be prospectively followed. After 2 cycles chemotherapy, tumor response were PR in 2, SD in 13, and PD in 9. Response (PR) rate was 8.3%, progression arrest (PR+SD) rate was 62.5%. Median PFS was 108 days, and Median OS was 264 days. The cut-off value was determined as 74% using the ROC analysis (area under the curve = 0.727; 95% CI = 0.457-0.997). From above results, we could predict 180 days PFS with a sensitivity and specificity of 71.4% and 88.2%, respectively. When patients were divided into two groups at T/C ratio 74%, there was a significant difference between 2 groups about PFS (median: 77 vs. 205 days, p = 0.0059), on the other hand, there was no significant difference about OS. With respect to tumor response, there was no significant difference in response rate, however, there was a significant difference in progression arrest (p = 0.015). In addition, T/C ratio < 74% was only significant prognostic factor that influence PFS. Conclusions: Chemosensitivity testing usig EUS-FNA sample is feasible. According to this definition, it might be possible to select the patients who are expected favorable result by gemcitabine treatment. No significant financial relationships to disclose.

Combining molecular analysis, chemo sensitivity testing in vitro, and therapy monitoring in vivo on disseminated tumor cells in breast cancer patients.

e21116 Background: Most breast cancer patients do not die of their primary tumor but from metastases developing sometimes years after the primary tumor has been removed. Cells from the tumor seem to be disseminated continuously during tumor growth and the first spread of tumor cells detectable with conventional methods is in the lymph nodes. Patients with lymph node positive disease have a poorer disease free survival than patients without affected lymph nodes. An increase in circulating epithelial tumor cell (CETC) numbers during adjuvant therapy is correlated with an 11 to 16-fold increase in hazard of relapse indicating increasing resistance to the applied drugs. Therefore, it would be desirable to better characterize these cells, test them for alternate drugs and monitor the actual response to the therapy decided on. Methods: For this purpose we have developed a test system comprising isolation of individual CETC from lymph nodes and blood from breast cancer patients for molecular characterization, chemo sensitivity testing of cells from the same sample defining the effectivity of the drugs as percentage of cells killed and measurement of the response of the CETC to the drugs in these patients treated according to guide lines. Results: The molecular characteristics and the sensitivity to the applied drugs of the tested cells and the response of the CETC were subsequently compared to clinical outcome. Conclusions: The response of CETC to therapy correlated highly with the previously tested chemo sensitivity and the molecular characteristics of these cells. Correlation to relapse or progression free survival is under investigation. No significant financial relationships to disclose.

Abstract 4146: Quantitative cell signaling analysis for cetuximab sensitivity prediction in colorectal cancer

Abstract [Background] Anti-human EGFR monoclonal antibody (cetuximab) has been widely used in the treatment of colorectal cancer (CRC). However it is thought that a reliable predictor for therapeutic efficacy should be established because the benefit of cetuximab treatment is often still uncertain. Our preliminary study showed that the prediction of therapeutic efficacy using a conventional chemosensitivity test using growth suppression assay is generally not informative because cetuximab does not seem to have as strong a cytotoxicity as other anticancer drugs. KRAS mutation status has been used as a biomarker to predict therapeutic efficiency but it is not sensitive enough to determine the absolute indication. [Purpose] We hypothesized that we could predict therapeutic efficacy by evaluating expected molecular reaction when cetuximab is administrated in vitro. In the present study, we build up an in vitro EGFR signaling model for a new chemosensitivity test based on protein monitoring in response to cetuximab administration. [Materials and Methods] Three growth factors (EGF, TGF-α, and IGF) and the inhibitor (cetuximab) were added to HT29 (KRAS wild type, EGFR positive) in six combinations. Cell pellets were harvested at 5 time points over a 15min time course, each of which was processed for cell lysate. The lysates were analyzed by Western blotting to see if the proteins involved in the EGFR signaling pathway responded over time. [Results] We confirmed the activation of signaling stimulated by EGF, TGF-α, and IGF; and the effect on signaling by cetuximab in HT29 with Western blotting. Intriguingly, the phosphorylation level of EGFR was rapidly elevated and declined by EGF stimulating in the absence of cetuximab whereas dephosphorylation was prolonged in the presence of cetuximab. In the administration of either TGF-α or IGF, the phosphorylation pattern of EGFR, c-RAF, MEK, MAPK, ERK, PI3K, AKT, PTEN, and STAT was different from that of EGF alone. [Conclusions] These results shows that:(i) EGF is not EGFR's only ligand; (ii) cetuximab can't inhibit every signaling pathway starting from EGFR; and (iii) TGF-α and IGF activate signaling pathway starting from EGFR partially but not thoroughly. An additional reliable predictor of cetuximab efficacy should be identified based on functional protein characteristics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4146.

Abstract 5502: Therapeutic efficacy prediction by quantitative protein analysis in response to anti-cancer agents

Abstract Background: The conventional chemosensitivity test employs a growth suppression assay. However, surgically removed specimens often have low viability which makes the assay unreliable, since the poor cell growth may be associated with the low viability instead of the chemosensitivity of the cell. On the other hand, we do know that the level of proteins involved in signal transduction is changed prior to cellular responses to the anti-cancer agents. In the present study, we investigated if protein signaling within a 24-hour exposure of the anti-cancer agents can predict the cellular fate at 72-hour time points. Materials and Methods: The human colon cancer cell line, HCT116, was exposed to three different DNA-damaging anti-cancer agents including gamma irradiation, ultraviolet, and doxorubicin. The anti-cancer agent-exposed cell pellets from different conditions (e.g., doses, and time-points) were collected to produce a lysate array, which finally accommodated more than 500 samples on a single glass slide. Array image and quantitative protein analyses were performed with the P-SCAN and ProteinScan programs. Results: A total of 19 protein species were tested on a lysate array, providing kinetic information of the protein. In response to all three agents, p53 protein was stabilized and the level increased over time in a dose-dependent manner. Upon detailed time-course analysis, however, an oscillation was seen in response to gamma irradiation, indicating the presence of a feedback loop. Comparing the rate of increasing levels of p21 and CyclinD3 within 24 hours in response to doxorubicin administration, the rates are well associated with the status of cell fate. In other words, when the rate of p21 is higher than CyclinD3, it is associated with a G2 arrest at 72 hours, whereas a higher CyclinD3 rate is associated with apoptosis at 72 hours after drug administration. Conclusion: Some events occurring at 72 hours can be predicted by seeing the quantitative level of protein kinetics within 24 hours. Most of the primary culture cells can survive for only a short period of time. The protein kinetics may be a useful clinical chemosensitivity predictor even when using low viability specimens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5502.

Abstract A2: Circulating epithelial tumor cells (CETC) for molecular analysis, chemo sensitivity testing in vitro and therapy monitoring in vivo

Abstract Most breast cancer patients do not die of their primary tumor but from metastases developing sometimes years after the primary tumor has been removed. The first spread of tumor cells detectable with conventional methods is in the lymph nodes and patients with lymph node positive disease have a poorer disease free survival than patients without affected lymph nodes. Cells from the tumor seem also to be disseminated continuously into blood during tumor growth and an increase in circulating epithelial tumor cell (CETC) numbers during adjuvant therapy is correlated with an 11- 16 fold increase in hazard of relapse. A reincrease in numbers indicates increasing resistance to the applied drugs. Therefore, it would be desirable to better characterize these cells, test them for alternative drugs and monitor the actual response to the therapy decided on. For this purpose we have developed a method to isolate individual CETC from blood from breast cancer patients and to successfully perform molecular characterization. Cells from the same sample were subject to chemo sensitivity testing and the drugs with the highest effectivity defined. Patients were subsequently treated according to guide lines. The molecular characteristics, sensitivity to the applied drugs and response of the CETC to these drugs were subsequently compared to clinical outcome. The response of CETC to therapy correlated highly with the previously tested chemo sensitivity and with the reduction of an index metastasis in metastatic breast cancer patients and progression free survival. The molecular characteristics of these cells are under investigation. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A2

Chemosensitivity testing with ChemoFx and overall survival in primary ovarian cancer

We sought to determine the association between tumor responses in vitro to platinum therapy by using the ChemoFx drug response marker and overall survival (OS) after first-line platinum-based chemotherapy in patients with advanced-stage primary ovarian cancer (POC). Chemosensitivity testing was performed in vitro on tumors from 192 POC patients. Tumors were classified as responsive, intermediately responsive, and nonresponsive to chemotherapy. Physicians made all management decisions. Survival status was retrieved from the Social Security Death Index. OS was modeled using Cox proportional hazard regression analysis. Median OS was 72.5 months for patients with tumors categorized as responsive, 48.6 months for intermediately responsive, and 28.2 months for nonresponsive (P = .03; hazard ratio, 0.70; 95% confidence interval, 0.50-0.97). The ChemoFx prediction of responses to platinum agents was an independent predictor of OS. Results of chemosensitivity testing with a drug response marker for therapy was predictive of OS in POC patients.

Complete response of anaplastic pancreatic carcinoma to paclitaxel treatment selected by chemosensitivity testing

A 58-year-old woman was admitted to our institute in June 2007 because of obstructive jaundice. A mass in the pancreatic head and multiple swollen lymph nodes surrounding the common hepatic artery were identified using computed tomography (CT). Subsequently, endoscopic ultrasonography-guided fine-needle aspiration biopsy (EUS-FNA) was done. Histopathologic results revealed anaplastic carcinoma of the pancreas. Using specimens obtained with EUS-FNA, chemosensitivity testing with adenosine triphosphate assay was performed. Testing indicated high chemosensitivity to paclitaxel (PTX). According to this result, chemotherapy using PTX was performed, and the primary lesion and lymph node metastases disappeared completely on CT. The patient has been doing well with no recurrence for 1 year and 11 months since initial chemotherapy.

Chemosensitivity of lung cancer: Differences between the primary lesion and lymph node metastasis

In this study, chemosensitivity tests were performed on both primary lesions (PLs) and lymph node metastases (LMs) from surgically resected non-small cell lung cancer (NSCLC). Differences between the results obtained were evaluated. Operative specimens were obtained from 13 patients with NSCLC [6 with squamous cell carcinoma (SQ) and 7 with adenocarcinoma (AD)] whose lymph nodes were confirmed to be positive for metastasis. Both the PL and LM from the same patient were examined immediately after resection. The collagen gel droplet-embedded culture drug sensitivity test (CD-DST) was used as the chemosensitivity test against six anticancer drugs [5-fluorouracil (5-FU), cisplatin, gemcitabine, docetaxel, vinorelbine and SN-38 (an active metabolite of irinotecan)]. When the growth rate, determined by the T/C ratio (T, signal for viable cells in the treated group and C, signal in the control) was less than 50%, the tumor cells were considered to be sensitive to the drug. Only in 4 cases (2 SQ and 2 AD) was the chemosensitivity of the primary lesion identical to that of LM. In the SQ cases, chemosensitivity of the primary lesions to 5-FU tended to be consistent with that of LMs. In contrast, the primary lesions in 4 of the 7 AD cases were negative for chemosensitivity to 5-FU; however, LMs were sensitive. In many cases, the chemosensitivity of the PLs to each anticancer drug differed from that of the LMs. In conclusion, both primary and metastatic tumors should be examined to ensure maximum clinical efficacy of in vitro drug-sensitivity testing for adjuvant chemotherapy after complete resection of n1 and n2 NSCLC.

Response evaluation in head and neck oncology

In this issue you will find the review article ‘‘assay-basedresponse evaluation in head and neck oncology: require-ments for better decision making’’ by Dietz and co-workers[1]. This interesting review addresses a major issue in headand neck cancer therapy. A major problem in the treatmentof head and neck cancers, particularly head and necksquamous cell carcinomas (HNSCCs), accounting for over90% of all malignancies in this area, is unpredictableresistance to chemotherapy and failure after multimodalitytreatment in general. Being able to identify responders andnon-responders prior to therapy induction is therefore ofutmost importance.The authors indicate the difficulties in predicting thetreatment outcome of tumors since these are frequentlyhighly heterogeneous in their biological behavior [2] andemphasize the need for a reliable predictive assay. Theyintroduce and discuss different types of assays that havebeen used for prediction of chemosensitivity in solid can-cers such as breast, lung, ovarian, brain and HNSCCtumors [3–5]. The authors particularly point out that inthese so-called ex vivo assays, which use the patients owncancer tissue, all cell types that build up a tumor mass arepresent such as fibroblasts, endothelial cells and the epi-thelial carcinoma cells themselves. This should have aclear advantage since these assays will likely mimic thereal in vivo situation of the tumor. The authors also indi-cate that for the same reason cell lines are not suitable forthe development of reliable assays since cell lines arehighly selected tumor stem cells and important cellulartumor tissue constituents such as fibroblasts and endothelialcells are lacking.However, as much as ex vivo tissues appear suitable tomimic the real in vivo situation there is one importantdrawback. By having at least three different cell types,chemosensitivity testing can be distorted if one does notdistinguish between the single cell types. Stromal cells ofthe tumor usually are derived from normal body cells,which in general are significantly more resistant to che-motherapy than the respective tumor cells. Therefore, it isessential that a suitable HNSCC chemosensitivity test iscapable of selectively testing the epithelial tumor cellpopulation.Next to these features Dietz et al. define further testcriteria such as high feasibility, high predictive accuracy,robust SOPs, quantitative results, getting the results in areasonable time and being able to perform the tests onsmall amounts of tumor tissues such as those obtained fromtumor biopsies. The authors also discuss the importantquestion if a single biopsy is representative of the wholetumor. Their own data supports the view that single biop-sies indeed are representative of the whole tumor.Lastly, the authors explain why the chemosensitivitytest, known as the FLAVINO assay is advantageous oreven superior to other test systems. Particularly, they stressthe tests ability to account for the flavin-dependent inhib-iting effect of cell culture media on chemotherapeuticagents, its ability to correct for the presence of stromal cellsand that it carries control (KB) cells for quality purposes.Dietz et al. conclude that chemosensitivity tests are nowsuitable and technically mature to be investigated as anadditional tool for therapeutic decision making in clinicaltrials. The authors blamed the low acceptance of chemo-sensitivity assays as stated by the ASCO in 2004 on thesevere lack of controlled clinical studies [6]. Therefore, to