Long-term effects of aromatase inhibitors on body mass index among postmenopausal breast cancer survivors in Africa: observational cohort study.

PURPOSE: this study was conducted to assess the impact of AIs on body mass index and high sensitivity as prognostic predictors to be incorporated into point of care technology (POCT) testing in postmenopausal breast cancer women after a 24 month follow up in Africa. An observational cohort study was conducted; including 126 female BC patients with stages ranging from 0-III initially subjected to AIs and subsequently followed up for 24 months. Multiple imputation model was conducted to predict missing data. RESULTS: Random effects model was used to monitor the changes over the time. The study revealed stronger statistically association between BMI and homocysteine (p = 0.021, 95%CI: 0.0083 to 0.1029). Weight and total body fat were strongly associated after 24 months follow up. Hs-CRP was associated with BMI (p = 0.0001), and hs-CRP was associated with other biomedical markers such as calcium (p = 0.021, 95% CI: 0.01 to 0.10), phosphate (p = 0.039, 95%CI: 0.01 to 0.10), and ferritin (p = 0.002, 95%CI: 0.02 to 0.08) and calcium. The patients subjected to AIs are likely to develop cardiovascular adverse events. POCT of care strategy which include clinical, biomedical and genetic predictor's measurement is required to improve BC survivorship.

BAG1, MGMT, FOXO1, and DNAJA1 as potential drug targets for radiosensitizing cancer cell lines.

BACKGROUND AND PURPOSE: Activation of some signaling pathways can promote cell survival and have a negative impact on tumor response to radiotherapy. Here, the role of differences in expression levels of genes related to the poly(ADP-ribose) polymerase-1 (PARP-1), heat shock protein 90 (Hsp90), B-cell lymphoma 2 (Bcl-2), and phosphoinositide 3-kinase (PI3K) pathways in the survival or death of cells following X-ray exposure was investigated. METHODS: Eight human cell cultures (MCF-7 and MDA-MB-231: breast cancers; MCF-12A: apparently normal breast; A549: lung cancer; L132: normal lung; G28, G44 and G112: glial cancers) were irradiated with X-rays. The colony-forming and real-time PCR based on a custom human pathway RT2 Profiler PCR Array assays were used to evaluate cell survival and gene expression, respectively. RESULTS: The surviving fractions at 2 Gy for the cell lines, in order of increasing radioresistance, were found to be as follows: MCF-7 (0.200 ± 0.011), G44 (0.277 ± 0.065), L132 (0.367 ± 0.023), MDA-MB-231 (0.391 ± 0.057), G112 (0.397 ± 0.113), A549 (0.490 ± 0.048), MCF-12A (0.526 ± 0.004), and G28 (0.633 ± 0.094). The rank order of radioresistance at 6 Gy was: MCF-7 < L132 < G44 < MDA-MB-231 < A549 < G28 < G112 < MCF-12A. PCR array data analysis revealed that several genes were differentially expressed between irradiated and unirradiated cell cultures. The following genes, with fold changes: BCL2A1 (21.91), TP53 (8743.75), RAD51 (11.66), FOX1 (65.86), TCP1 (141.32), DNAJB1 (3283.64), RAD51 (51.52), and HSPE1 (12887.29) were highly overexpressed, and BAX (-127.21), FOX1 (-81.79), PDPK1 (-1241.78), BRCA1 (-8.70), MLH1 (-12143.95), BCL2 (-18.69), CCND1 (-46475.98), and GJA1 (-2832.70) were highly underexpressed in the MDA-MB-231, MCF-7, MCF-12A, A549, L132, G28, G44, and G112 cell lines, respectively. The radioresistance in the malignant A549 and G28 cells was linked to upregulation in the apoptotic, DNA repair, PI3K, and Hsp90 pathway genes BAG1, MGMT, FOXO1, and DNAJA1, respectively, and inhibition of these genes resulted in significant radiosensitization. CONCLUSIONS: Targeting BAG1, MGMT, FOXO1, and DNAJA1 with specific inhibitors might effectively sensitize radioresistant tumors to radiotherapy.

Feasibility of point of care testing for prevention and management of breast cancer therapy associated comorbidities in 6 African countries: short communication.

OBJECTIVE: Obesity and mediators of inflammation have been identified as the most important risk and predictive factors in postmenopausal breast cancer (BC) survivors using aromatase inhibitors (AIs). This study was conducted to assess the impact of point of care technology (PCOT) as part of pathology supported genetic testing (PSGT) to prevent BC therapy-associated comorbidities in African settings. RESULTS: The study revealed that high sensitivity C-reactive protein (hs-CRP) and body mass index (BMI) are predictors of cardiovascular (CVD) related adverse events in obese postmenopausal patients subjected to AIs. There were statistically significant variations in total body fat (TBF), weight, hs-CRP, body mass index (BMI), homocysteine, ferritin, and calcium between baseline and after 24 months of follow-up. The above inflammatory markers can be incorporated in pathology supported genetic testing (PSGT) using HyBeacon® probe technology at POC for prediction and management of AI-associated adverse events among postmenopausal breast cancer survivors and associated comorbidities. The barriers for implementation of POCT application among six African countries for diagnosis of breast cancer were documented as insufficient of BC diagnosis and management capacity at different levels of health system.

The Role of Positron Emission Tomography and Computed Tomographic (PET/CT) Imaging for Radiation Therapy Planning: A Literature Review.

PET/CT is revolutionising radiotherapy treatment planning in many cancer sites. While its utility has been confirmed in some cancer sites, and is used in routine clinical practice, it is still at an experimental stage in many other cancer sites. This review discusses the utility of PET/CT in cancer sites where the role of PET/CT has been established in cases such as head and neck, cervix, brain, and lung cancers, as well as cancer sites where the role of PET/CT is still under investigation such as uterine, ovarian, and prostate cancers. Finally, the review touches on PET/CT utilisation in Africa.

Selective therapeutic benefit of X-rays and inhibitors of EGFR, PI3K/mTOR, and Bcl-2 in breast, lung, and cervical cancer cells.

Cancer continues to be a growing burden, especially in the resource limited regions of the world, and more effective and affordable therapies are highly desirable. In this study, the effect of X-ray irradiation and four inhibitors, viz. those against epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), mammalian target of rapamycin (mTOR) and B-cell lymphoma 2 (Bcl-2) was evaluated in lung, breast, and cervical cancer cell lines, including normal cell lines to determine and compare the potential therapeutic benefit of these treatment modalities. A clonogenic survival assay was used to determine the radiosensitivity and cytotoxicity of inhibitors of EGFR, PI3K/mTOR, and Bcl-2 in the cell lines. From the data, the equivalent dose at which 50% of the cell populations were killed, for cancer and normal cells, was used to determine the relative cellular sensitivity to X-ray irradiation and inhibitor treatment. It was found that breast cancer cell lines were more sensitive to X-ray irradiation, whilst cervical and lung cancer cell lines were more sensitive to EGFR and PI3K/mTOR inhibitor therapy. These data suggest that patients with breast cancer possessing similar characteristics to MDA-MB-231 and MCF-7 cells may derive therapeutic benefit from X-ray irradiation, whilst EGFR and PI3K/mTOR inhibitor therapy may potentially benefit cancer patients possessing cancers similar to HeLa and A549 cells.

The malignancy index is a robust predictor of prostate cancer.

The objective of this study was to validate the results from our published work and to test the robustness of our unique malignancy index as a (non-invasive) predictor of prostate cancer in fresh blood samples obtained from patients diagnosed with prostate cancer (PCa), benign prostatic hyperplasia (BPH), and healthy volunteers (Controls). The malignancy index was obtained by dividing the product of three biomarker values, [urokinase plasminogen activator (uPA), plasminogen activator inhibitor type-1 (PAI-1), and prostate-specific antigen (PSA)], by the age of the patient/healthy volunteer, using enzyme-linked immunosorbent (ELISA) assay methodology. The results confirmed earlier findings that the malignancy index discriminates prostate cancer from non-prostate cancer. The index significantly separated the PCa group from the Control group with values of 0.0701 (n=54) and 0.0007 (n=47), respectively, by a factor of 100. The malignancy index of the small BPH cohort was found to be 0.0016 (n=20), differing by a factor of 44 from the Control group. When data from the earlier study and the current study data were collectively analyzed, the index again significantly separated the PCa group from the Control group by a factor of 15, with values of 0.0624 (n=125) and 0.0042 (n=110), respectively. However, the same could not be said of the BPH data since the sample size (n=20) was well below par, for comparison. In the initial blood study, the PCa group was significantly separated from the Control group by a factor of 8.5. The data presented here concur with findings in needle biopsies and transurethral resection tissue, reported elsewhere (Bohm et al., 2013; Akudugu et al., 2015). At this preliminary stage, the malignancy index has potential and merit as a prostate cancer biomarker.

Chemotherapy for elderly colorectal cancer patients at a tertiary hospital in South Africa.

INTRODUCTION: surgical treatment of colorectal cancer (CRC) in elderly patients has improved, but data on the tolerability and benefits of adjuvant and palliative chemotherapy in this growing population remains scarce. METHODS: we conducted a retrospective study to compare chemotherapy-associated toxicities in CRC patients aged < 70 years and ≥ 70 years at Tygerberg Hospital (South Africa). We also assessed tumor-related mortality, progression free survival (PFS), and overall survival (OS) including predictive factors of OS. RESULTS: a total of 50 patients received either adjuvant or palliative chemotherapy. There was no difference in overall toxicity between the two groups. Out of the 50 patients, 8 (16%) had Grade 3-4 toxicity. 4 of these patients made up 15% of the < 70 years age group, whereas the other 4 made up 17% of the ≥ 70 years age group. The mean follow-up time was 47.5 months (95% CI 41.5 - 53.5 months). The 5-year over-all survival rate for stage II and III patients < 70 years and ≥ 70 years were 80.9% and 69.5%, respectively, and not significantly different (P = 0.52). Furthermore, the 5-year progression-free survival rates of the < 70 and ≥ 70 age groups were 70.7% and 58.8%, respectively, and also not statistically significantly different (P = 0.49). For stage IV patients, there were no significant differences in survival between the two age groups. CONCLUSION: the benefits from adjuvant and palliative chemotherapy for elderly CRC patients are similar to that of younger patients. Therefore, standardized adjuvant and palliative chemotherapy is recommended for elderly patients.

A preliminary study on treatment of human breast cancer xenografts with a cocktail of paclitaxel, doxorubicin, and 131I-anti-epithelial cell adhesion molecule (9C4).

Triple-negative breast cancer often has devastating outcomes and treatment options remain limited. Therefore, different treatment combinations are worthy of testing. The efficacy of a cocktail of paclitaxel, doxorubicin, and 131I-anti-epithelial cell adhesion molecule (EpCAM) (9C4) to treat breast cancer was tested. Efficacy was tested with an MDA-MB-231 human breast cancer xenograft model. Anti-EpCAM (9C4) was demonstrated to bind to MDA-MB-231 human adenocarcinoma cells in vitro. Subsequently, mice-bearing MDA-MB-231× enografts were treated with either 131I-anti-EpCAM (9C4), unlabeled anti-EpCAM (9C4), paclitaxel, doxorubicin, or a cocktail of all of the agents. Tumor volume was measured for up to 70-day postinjection. Exponential regression was performed on tumor growth curves for each of the therapy groups. Statistical comparison of the growth constants λ of the regression models for each of the treatment groups with that of the cold antibody and control groups was done using extra sum-of-square F-tests. Biexponential clearance of 131I-anti-EpCAM (9C4) was observed with biological clearance half-times of 1.14 and 17.6 days for the first and second components, respectively. The mean growth rate of the tumors in animals treated with a cocktail of all of the agents was slower than in those treated with unlabeled anti-EpCAM (9C4) (P = 0.022). These preliminary data suggest that a cocktail of 131I-anti-EpCAM (9C4), paclitaxel, and doxorubicin may be suitable for treating breast cancers with high expression of EpCAM.

The malignancy index in plasma samples as a prostate cancer biomarker.

No unambiguous role of the involvement of uroplasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) in prostate cancer has emerged, with current evidence suggesting that neither biomarker is likely of significant clinical value, save as an overall contributor. In this study, we attempt to discriminate prostate cancer from non-cancer in a cohort of plasma samples, using the Imubind ELISA assay. In this cohort, PAI-1 levels are higher in prostate cancer patients than healthy donors; uPA levels are higher in healthy donors than prostate cancer patients; and the uPA/PAI-1 ratio is higher in healthy donors than in prostate cancer patients. To date, and across three prostate sample types, i.e. transurethral resection tissue, needle biopsies, and blood plasma, data have been disparate. Given the inconsistency, a malignancy index was created by dividing the product of three biomarkers [uPA, PAI-1, and prostate specific antigen (PSA)] by the age of the patient/donor. The malignancy index clearly distinguishes prostate disease from non-disease in peripheral blood plasma samples (P=0.0127), concurring with findings in core needle biopsies and transurethral resection tissue, reported elsewhere. This is an important finding given the gravity of prostate cancer and the legions of over-diagnosed and over-treated men worldwide.

Strong synergism between small molecule inhibitors of HER2, PI3K, mTOR and Bcl-2 in human breast cancer cells.

Targeting pro-survival cell signaling components has been promising in cancer therapy, but the benefit of targeting with single agents is limited. For malignancies such as triple-negative breast cancer, there is a paucity of targets that are amenable to existing interventions as they are devoid of the human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR), and estrogen receptor (ER). Concurrent targeting of cell signaling entities other than HER2, PR and ER with multiple agents may be more effective. Evaluating modes of interaction between agents can inform efficient selection of agents when used in cocktails. Using clonogenic cell survival, interaction between inhibitors of HER2 (TAK-165), phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) (NVP-BEZ235), and the pro-survival gene (Bcl-2) (ABT-263) in three human breast cell lines (MDA-MB-231, MCF-7 and MCF-12A) ranged from strong to very strong synergism. The strongest synergy was demonstrated in PR and ER negative cells. Inhibition of PI3K, mTOR and Bcl-2 could potentially be effective in the treatment of triple-negative cancers. The very strong synergy observed even at lowest concentrations of inhibitors indicates that these cocktails might be able to be used at a minimised risk of systemic toxicity. Concurrent use of multiple inhibitors can potentiate conventional interventions like radiotherapy and chemotherapy.

Flow cytometry-assisted quantification of γH2AX expression has potential as a rapid high-throughput biodosimetry tool.

Large-scale radiological events require immediate and accurate estimates of doses received by victims, and possibly the first responders, to assist in treatment decisions. Although there are numerous efforts worldwide to develop biodosimetric tools to adequately handle triage needs during radiological incidents, such endeavours do not seem to actively involve sub-Saharan Africa which currently has a significant level of nuclear-related activity. To initiate a similar interest in Africa, ex vivo radiation-induced γH2AX expression in peripheral blood lymphocytes from fourteen healthy donors was assessed using flow cytometry. While the technique shows potential for use as a rapid high-throughput biodosimetric tool for radiation absorbed doses up to 5 Gy, significant inter-individual differences in γH2AX expression emerged. Also, female donors exhibited higher levels of γH2AX expression than their male counterparts. To address these shortcomings, gender-based in-house dose-response curves for γH2AX induction in lymphocytes 2, 4, and 6 h after X-ray irradiation are proposed for the South African population. The obtained results show that γH2AX is a good candidate biomarker for biodosimetry, but might need some refinement and validation through further studies involving a larger cohort of donors.

Vitamins A, C, and E May Reduce Intestinal 210Po Levels after Ingestion.

Damage to the gut mucosa is a probable contributory cause of death from ingested Po. Therefore, medical products are needed that can prevent, mitigate, and/or repair gastrointestinal (GI) damage caused by high-LET radiation emitted by Po. The present studies investigated the capacity of a diet highly enriched with vitamins A, C, and E (vitamin ACE) to protect against intestinal mucosal damage indicated by functional reductions in nutrient transport caused by orally ingested Po. Mice were gavaged with 0 or 18.5 kBq Po-citrate and fed a control or vitamin ACE-enriched diet (the latter beginning either 96 h before or immediately after gavage). Mouse intestines significantly retained Po on day 8 post-gavage. The concentration of Po in intestinal tissues was significantly (p<0.05) lower in all vitamin ACE groups compared to control. There were borderline significant Po-induced reductions in intestinal absorption of D-fructose. The combination of vitamins A, C, and E may reduce Po incorporation in the intestines when given before, or enhance decorporation when provided after, Po gavage.

Towards Establishing Capacity for Biological Dosimetry at Ghana Atomic Energy Commission.

The aim of this study was not only to obtain basic technical prerequisites for the establishment of capacity of biological dosimetry at the Ghana Atomic Energy Commission (GAEC) but also to stimulate interest in biological dosimetry research in Ghana and Sub-Saharan Africa. Peripheral blood from four healthy donors was exposed to different doses (0-6 Gy) of gamma rays from a radiotherapy machine and lymphocytes were subsequently stimulated, cultured, and processed according to standard protocols for 48-50 h. Processed cells were analyzed for the frequencies of dicentric and centric ring chromosomes. Radiation dose delivered to the experimental model was verified using GafChromic® EBT films in parallel experiments. Basic technical prerequisites for the establishment of capacity of biological dosimetry in the GAEC have been realized and expertise in the dicentric chromosome assay consolidated. We successfully obtained preliminary cytogenetic data for a dose-response relationship of the irradiated blood lymphocytes. The data strongly indicate the existence of significant linear (α) and quadratic (ß) components and are consistent with those published for the production of chromosome aberrations in comparable absorbed dose ranges.

Further evaluation of uPA and PAI-1 as biomarkers for prostatic diseases.

PURPOSE: To assay for uPA and PAI-1 in prostate tissue from 40 patients with prostatic disease and to examine the robustness of the correlation of the uPA/PAI-1 ratio with benign prostatic hyperplasia (BPH) and prostate cancer (PCa), previously identified in a different cohort of 62 patients. METHODS: uPA and PAI-1 were extracted from liquid N2 frozen homogenised prostate tissue with TRIS/Triton pH 8.5 buffer and measured by ELISA (FEMTELLE). RESULTS: The concentration of uPA (mean ± SD) was found to be 0.1177 ± 0.0266 (range 0.0070-0.7200; n = 30) and 0.1092 ± 0.0130 (range 0.0040-0.7800; n = 70) for PCa and BPH patients, respectively. The concentration of PAI-1 was found to be 5.236 ± 0.688 ng/mg protein (range 1.10-15.19; n = 30) and 4.975 ± 0.501 ng/mg protein (range 0.20-25.00; n = 70) for PCa and BPH patients, respectively. The mean uPA/PAI-1 ratio was found to be 0.0479 ± 0.0060 (range 0.0043-0.1200; n = 30) in PCa samples and was significantly higher than BPH samples where the ratio was 0.0332 ± 0.0023 (range 0.0040-0.0860; n = 70) (P = 0.0064). In PCa patients older than 68 years, the uPA/PAI-1 ratio was above 0.050 reaching 0.100 in 73-year-old patients. CONCLUSIONS: Evaluation of 100 patients with prostatic pathologies (70 PCa; 30 BPH) shows the uPA/PAI-1 ratios in PCa patients to be significantly higher than in BPH patients. This is fully consistent with a previous study on 62 patients (16 were PCa; 46 BPH) where the ratios were 0.055 and 0.031 for PCa and BPH patients, respectively (P = 0.0028). In older PCa patients, uPA/PAI-1 ratios tend to be higher.

The advantage of antibody cocktails for targeted alpha therapy depends on specific activity.

UNLABELLED: Nonuniform dose distributions among disseminated tumor cells can be a significant limiting factor in targeted α therapy. This study examines how cocktails of radiolabeled antibodies can be formulated to overcome this limitation. METHODS: Cultured MDA-MB-231 human breast cancer cells were treated with different concentrations of a cocktail of 4 fluorochrome-conjugated monoclonal antibodies. The amount of each antibody bound to each cell was quantified using flow cytometry. A spreadsheet was developed to "arm" the antibodies with any desired radionuclide and specific activity, calculate the absorbed dose to each cell, and perform a Monte Carlo simulation of the surviving fraction of cells after exposure to cocktails of different antibody combinations. Simulations were performed for the α-particle emitters (211)At, (213)Bi, and (225)Ac. RESULTS: Activity delivered to the least labeled cell can be increased by 200%-400% with antibody cocktails, relative to the best-performing single antibody. Specific activity determined whether a cocktail or a single antibody achieved greater cell killing. With certain specific activities, cocktails outperformed single antibodies by a factor of up to 244. There was a profound difference (≤16 logs) in the surviving fraction when a uniform antibody distribution was assumed and compared with the experimentally observed nonuniform distribution. CONCLUSION: These findings suggest that targeted α therapy can be improved with customized radiolabeled antibody cocktails. Depending on the antibody combination and specific activity of the radiolabeled antibodies, cocktails can provide a substantial advantage in tumor cell killing. The methodology used in this analysis provides a foundation for pretreatment prediction of tumor cell survival in the context of personalized cancer therapy.

uPA/PAI-1 ratios distinguish benign prostatic hyperplasia and prostate cancer.

PURPOSE: Urokinase plasminogen activator (uPA) and its inhibitor type 1 (PAI-1) are associated with tumour metabolism and are widely considered to be informative for the identification of cancer. We have analysed prostate tissue resections from patients with prostate cancer (PCa) and with benign prostatic hyperplasia (BPH) for protein levels of uPA and PAI-1, and searched for distinctions between these two clinical manifestations. METHODS: Prostate tissue was deep frozen in liquid N2 and homogenized in a stainless steel punch homogenizer. The tissue powder was extracted with a pH 8.5 TRIS/Triton X-100 buffer, and the extract analysed by FEMTELLE assay to generate uPA and PAI-1 readings in ng/mg protein. The uPA/PAI-1 ratio was calculated for each sample, and the mean ratios for the two diagnostic groups were compared. RESULTS: The concentration of uPA (mean ± SD) was found to be 0.19 ± 0.04 ng/mg protein (range 0.05-0.72 ng/mg) and 0.15 ± 0.02 ng/mg protein (range 0.03-0.78 ng/mg) in PCa and BPH samples, respectively. The concentration of PAI-1 was found to be 4.93 ± 0.90 ng/mg (range 1.10-11.80 ng/mg) and 5.87 ± 0.70 ng/mg (range 0.2-25.0 ng/mg) in PCa and BPH samples, respectively. A consistent finding being that PAI-1 concentrations exceed uPA concentrations by far giving rise to characteristic uPA/PAI-1 ratios. In BPH samples, there was a trend of PAI-1 to increase with uPA content, while in PCa samples, PAI-1 remained fairly constant. The mean uPA/PAI-1 ratio in PCa samples was found to be 0.06 ± 0.01 and was significantly higher than in BPH samples where the mean uPA/PAI-1 ratio was 0.03 ± 0.003 (p = 0.0028). R(2) = 0.1389. CONCLUSION: Using a contingent of 62 patients of which 46 were BPH and 16 were PCa, we report definitive concentrations of uPA and PAI-1 in tumour tissue extracts and show that the uPA/PAI-1 ratio emerges as a candidate marker to distinguish between BPH and PCa.

In vitro radiosensitization by pentoxifylline does not depend on p53 status.

PURPOSE: The mode by which the xanthine derivative, pentoxifylline, induces a radiosensitizing effect in cell cultures is a key and controversial radiobiological issue and requires further elucidation. MATERIALS AND METHODS: Six human glioblastoma cell lines were tested for the effect of pentoxifylline treatment at maximum G2/M block on the basis of cell survival, mitotic activity, and micronucleus formation after exposure to gamma radiation. Cell survival was measured by the colony-forming assay. Micronucleus formation (an indicator of DNA damage) and the proportion of binucleated cells (a representation of mitotic activity) were determined using the cytokinesis-block assay. RESULTS: Remarkably, exposure to a single dose of 4 Gy produced strong G2/M blocks in both p53 mutant and wild-type cells. Addition of pentoxifylline at the peak of radiation-induced G2/M blocks resulted in a p53-independent reduction in cell survival in all cell lines. This radiosensitization was strongly correlated with the magnitude of the radiation-induced G2/M block. The changes observed in mitotic activity and micronucleus yield were also p53-independent. CONCLUSIONS: These results are at variance with the view that pentoxifylline preferentially sensitizes p53 mutant cells, and that sensitization occurs only when cells are irradiated in the presence of the drug. The data suggest that the effectiveness of pentoxifylline as radiosensitizer depends on the proportion of cells that are arrested in the G2/M phase transition following exposure to ionizing radiation. These findings can assist in the identification of cancers that may benefit from therapies using G2/M checkpoint abrogators.

A method to predict response of cell populations to cocktails of chemotherapeutics and radiopharmaceuticals: validation with daunomycin, doxorubicin, and the alpha particle emitter (210)Po.

UNLABELLED: There is considerable interest in the use of α-emitting radionuclides in radioimmunotherapy. However, the high toxicity of α-emitting radionuclides often does not permit administration of high activities for fear of normal tissue toxicity. Accordingly, targeting procedures need to be optimized for improved tumor control and minimized normal tissue toxicity. To guide design of effective cocktails of α-emitting radiopharmaceuticals and chemotherapy drugs, approaches that can predict biological response of a cell population on a cell-by-cell basis are needed. METHODS: Cells were concomitantly treated with the α-particle emitting radiochemical (210)Po-citrate and daunomycin, or with (210)Po-citrate and doxorubicin. The responses of the treated cell populations were measured with a colony forming assay. The nonuniform cellular incorporation of the radiochemical and drugs was determined simultaneously on a cell-by-cell basis using flow cytometry. Monte Carlo methods were used to simulate cell survival on the basis of individual cell incorporation of each cytotoxic agent and validated by direct comparison with the experimental clonogenic cell survival. RESULTS: Both daunomycin and doxorubicin enhanced the toxicity of the α-particles with a magnitude greater than expected based on single-agent toxicities. Cell survival obtained by Monte Carlo simulation was in good agreement with clonogenic cell survival for the combination treatments. CONCLUSION: Flow cytometry assisted Monte Carlo simulations can be used to predict toxicity of cocktails of α-emitting radiopharmaceuticals and chemotherapy drugs in a manner that takes into account the effects of nonuniform distributions of agents within cell populations.

Induction of lethal bystander effects in human breast cancer cell cultures by DNA-incorporated Iodine-125 depends on phenotype.

PURPOSE: This study uses a three-dimensional cell culture model to investigate lethal bystander effects in human breast cancer cell cultures (MCF-7, MDA-MB-231) treated with (125)I-labeled 5-iodo-2 -deoxyuridine ((125)IdU). These breast cancer cell lines respectively form metastatic xenografts in nude mice in an estrogen-dependent and independent manner. MATERIALS AND METHODS: In the present study, these cells were cultured in loosely-packed three-dimensional architecture in a Cytomatrix™ carbon scaffold. Cultures were pulse-labeled for 3 h with (125)IdU to selectively irradiate a minor fraction of cells, and simultaneously co-pulse-labeled with 0.04 mM 5-ethynyl-2'-deoxyuridine (EdU) to identify the radiolabeled cells using Click-iT(®) EdU and flow cytometry. The cultures were then washed and incubated for 48 h. The cells were then harvested, serially diluted, and seeded for colony formation. Aliquots of cells were subjected to flow cytometry to determine the percentage of cells labeled with (125)IdU/EdU. Additional aliquots were used to determine the mean (125)I activity per labeled cell. The percentage of labeled cells was about 15% and 10% for MCF-7 and MDA cells, respectively. This created irradiation conditions wherein the cross-dose to unlabeled cells was small relative to the self-dose to labeled cells. The surviving fraction relative to EdU-treated controls was measured. RESULTS: Survival curves indicated significant lethal bystander effect in MCF-7 cells, however, no significant lethal bystander effect was observed in MDA-MB-231 cells. CONCLUSIONS: These studies demonstrate the capacity of (125)IdU to induce lethal bystander effects in human breast cancer cells and suggest that the response depends on phenotype.

Flow cytometry-assisted Monte Carlo simulation predicts clonogenic survival of cell populations with lognormal distributions of radiopharmaceuticals and anticancer drugs.

PURPOSE: Although the distribution of therapeutic agents within cell populations may appear uniform at the macroscopic level, the distribution at the multicellular level is nonuniform. As such, the mean agent concentration in tissue may not be a suitable quantity for use in predicting biological effects. Failure in chemotherapy and targeted radionuclide therapy has been attributed, in part, to the ubiquity of lognormal distributions of therapeutic agents. To improve capacity to predict biological response, this work develops approaches that determine the fate of a cell population on a cell-by-cell basis. METHODS: Incorporation of the α-particle emitting radiochemical ((210)Po-citrate) and two anticancer drugs (daunomycin and doxorubicin) by Chinese hamster V79 cells was determined using flow cytometry. Monte Carlo simulation was used to estimate cell survival on the bases of mean and individual cell incorporation of each cytotoxic agent. The interrelationships between the Monte Carlo simulated cell survival and clonogenic cell survival were evaluated. RESULTS: Cell survival obtained by Monte Carlo simulation based on individual cell incorporation was in good agreement with clonogenic cell survival for all agents. However, the agreement was poor when the simulation was carried out using the mean cell incorporation of the agents. CONCLUSION: These data indicate that, with the aid of flow cytometry, Monte Carlo simulations can be used to predict the toxicity of therapeutic agents in a manner that takes into account the effects of lognormal and other nonuniform distributions of agents within cell populations.