Lymphocyte Apoptosis Assay Research Articles

Predictors of radiation-induced complications in radiation oncology based on cell survival tests after ex vivo exposure: literature review

Background. Among cancer patients receiving radiotherapy about 5–15 % may have adverse reactions in normal tissues and organs that limit their treatment in a full, originally scheduled regimen. The development of biomarkers and assays for radiation oncology allowing the prediction of patients’ normal tissue toxicity requires a lot of resourses, threfore its current status amd potential directions for future research have to be periodically analyzed and re-evaluated.
 Purpose – this review summarizes the methodological approaches and developments in the area of functional laboratory assays based on ex vivo cell survival for the prediction of the individual clinical radiosensitivity.
 Materials and methods. Data for the analysis and systematization were obtained from the full-text articles published in peer review international scientific journals (in English) in 1990–2020, which were selected by the extensive search in PubMed information database and cross references on the topic “Functional cellular tests for intrinsic radiosensitivity to predict adverse radiation effects and radiotherapy complications”.
 Results. In theory, it might be expected that clonogenic cell survival after ex vivo irradiation can surve as the best individual predictor of radiation toxicity, as it is an integral indicator of cell damage and decline of their regenerative potential. Tendentially, fibroblasts, as a test system for such studies, did not show significant advantages over lymphocytes either in detecting inter-individual variations in the intrinsic cellular radiosensitivity or in predicting clinical radiation toxicity, even for that in skin. It was found that clonogenic cell survival assay, being very time consuming and technically demanding, also suffers from the lack of sensitivity and specificity, essential uncertainty and low reproducibility of the results, and thus is not suitable for the sceening for the abnormal intrinsic radiosensitivity. However, this type of assays is applicable for the radiobiological expertise post factum in individual cases with unexpected, extreme radiation lesions. Radiation-induced lymphocyte apoptosis assay seems to be more promising however still requires further fundamental research for better understanding of its background and more validation studies in order to assess the optimum patient groups, radiotherapy regimens and adverse effects for its confident use in clinical practice. Changes in the regulation of cell cycle check-points (radiationinduced delay) ex vivo can have either positive or inverted association, or no correlation with clinical radiation responses in tissues, thus so far cannot be included in the toolbox of applied radiobiological tests.
 Conclusions. To date, in the practice of clinical radiobiology, there are no fully validated and standardized functional tests based on the cell survival after ex vivo irradiation, which would allow a sufficiently accurate prediction of adverse radiation effects in normal tissues of radiotherapy patients. In general, ex vivo tests based on the evaluation of only one form of cell death in one cell type are not fully reliable as a “stand alone” assay, because different pathways of cell death probably play different roles and show different dose response within the overal reaction of the irradiated tissue or critical organ. Such tests should become a part of the multiparametric predictive platforms.

Multi-centre technical evaluation of the radiation-induced lymphocyte apoptosis assay as a predictive test for radiotherapy toxicity

Predicting which patients will develop adverse reactions to radiotherapy is important for personalised treatment. Prediction will require an algorithm or nomogram combining clinical and biological data. The radiation-induced lymphocyte apoptosis (RILA) assay is the leading candidate as a biological predictor of radiotherapy toxicity. In this study we tested the potential of the assay for standardisation and use in multiple testing laboratories.The assay was standardised and reproducibility determined using samples from healthy volunteers assayed concurrently in three laboratories in Leicester (UK), Mannheim (Germany) and Montpellier (France). RILA assays were performed on samples taken prior to radiotherapy from 1319 cancer patients enrolled in the REQUITE project at multiple centres. The patients were being treated for breast (n = 753), prostate (n = 506) or lung (n = 60) cancer.Inter-laboratory comparisons identified several factors affecting results: storage time, incubation periods and type of foetal calf serum. Following standardisation, there was no significant difference in results between the centres. Significant differences were seen in RILA scores between cancer types (prostate > breast > lung), by smoking status (non-smokers > smokers) and co-morbidity with rheumatoid arthritis (arthritics > non-arthritics).An analysis of acute radiotherapy toxicity showed as expected that RILA assay does not predict most end-points, but unexpectedly did predict acute breast pain. This result may elucidate the mechanism by which the RILA assay predicts late radiotherapy toxicity.The work shows clinical trials involving multiple laboratory measurement of the RILA assay are feasible and the need to account for tumour type and other variables when applying to predictive models.

Prediction of Radiation-induced Breast Fibrosis by the Radiation-induced Lymphocyte Apoptosis Assay: Relation to the Surgical Area

Prediction of Radiation-induced Breast Fibrosis by the Radiation-induced Lymphocyte Apoptosis Assay: Relation to the Surgical Area

Radiation Induced Lymphocyte Apoptosis Assay and Predictive Breast Fibrosis Nomogram: First Implementation in Daily Practice in France

The radiation-induced CD8-lymphocyte apoptosis assay (RILA) adjusted by the NovaGray Breast® nomogram has been shown to significantly predict breast fibrosis risk. Recent prospective trials confirmed the predictive role of RILA in urinary, sexual or anorectal toxicity risk after prostate radiotherapy. This study aims at evaluating the daily use of this assay and its impact on current practice. Between 2007 and 2016, 304 patients benefited from the RILA assay outside of clinical trial setting. We analyzed the reasons for assay prescription, late toxicity (assessed by CTCAEv3.0 grading scale) and clinical impact of the result. For non-breast demands, RILA cutoffs for high and low values were taken from the results of the REQUITE trial and Ozsahin et al. (Clin Cancer Res, 2005) For breast tumors, all RILA results were adjusted by the nomogram developed by NovaGray based on the recent results of the multicenter prospective trial (Azria et al., Ebiomedecine 2015). 149 patients with sufficient available data were included in the analysis. 51 patients were assessed because of late toxicities after radiotherapy (RT). Of these, 36 (70.6%) had low and 15 (29.4%) intermediate/high RILA values. For breast cancer, 27 of 29 patients with toxicities (93.1%) fitted with the predictive nomogram. 13 patients were assessed before re-irradiation, and 2 saw their RT treatment cancelled because of low RILA values (one had surgery and one refused treatment). 85 patients were assessed before initial RT treatment. Of these, 36 (42.4%) were assessed because of medical history (i.e.: systemic sclerosis, lupus or polymorphous light eruption), 33 (38.9%) because the patient asked for the test, 7 (8.2%) to aid clinical decision (i.e.: proximity of organs at risk or unclear RT indication), 4 (4.7%) because of history of surgery or trauma in the treated area, 4 (4.7%) before inclusion in clinical trials which they ended excluded from, and one (1.2%) because of severe toxicity after chemotherapy. 23 of the 85 patients (27%) had treatment changes because of the test results. Seven patients with low RILA (30.4%) saw their RT treatment replaced by other treatment modalities (mastectomy, radical prostatectomy, focal treatment, hormone therapy or brachytherapy). Treatment was canceled altogether because of low RILA in 5 cases (21.8%). Eight patients (34.8%) with low RILA had changes in the RT regimen (2 total dose reduction, 3 fractionation change, 3 RT modality change towards IMRT). Three patients (13%) had the RT indication maintained despite the test results. This is the first daily clinical implementation of the RILA assay and breast nomogram risk. Interestingly, half of the patients were assessed before treatment, with a high rate of treatment change based on the results, showing the importance of predictive markers before treatment decision.

PO-0959: REQUITE: Radiation Induced Lymphocyte Apoptosis assay as a predictor for radiotherapy side effects

PO-0959: REQUITE: Radiation Induced Lymphocyte Apoptosis assay as a predictor for radiotherapy side effects

A multicenter study of biological effects assessment of pharmacy workers occupationally exposed to antineoplastic drugs in Pharmacy Intravenous Admixture Services

This multi-centered study was designed to evaluate the biological effects of exposure to antineoplastic drugs (ADs) at PIVAS (Pharmacy Intravenous Admixture Service) across ten Chinese hospitals. 8-hydroxy-2-deoxyguanosine (8-OHdG) was used as a biomarker of DNA oxidative damage and lymphocyte apoptosis assays using peripheral lymphocyte cells were used to detect primary DNA damage. The mutagenicity activity was estimated with the Ames fluctuation test. 158 exposed and 143 unexposed workers participated in this study. The urinary 8-OHdG/Cr concentrations of the exposed group was 22.05±17.89ng/mg Cr, which was significantly higher than controls of 17.36±13.50ng/mg Cr (P<0.05). The rate of early lymphocyte apoptosis was slightly increased in exposed group than that of the control group (P=0.087). The mutagenic activity was significantly higher in the exposed group relative to the control group (P<0.05). Moreover, while no statistically significant difference was observed, higher concentrations of 8-OHdG/Cr in urine and an early lymphocyte apoptosis rate were found in exposed group II as compared to exposed group I. In addition, a significant correlation between early lymphocyte apoptosis and exposure time to ADs was also observed (P<0.05). In conclusion, our study identified elevated biomarkers in PIVAS workers exposed to ADs. However whether these findings could lead to increased incidence of genotoxic responses remains to be further investigated.

Incorporating Genetic Biomarkers into Predictive Models of Normal Tissue Toxicity

There is considerable variation in the level of toxicity patients experience for a given dose of radiotherapy, which is associated with differences in underlying individual normal tissue radiosensitivity. A number of syndromes have a large effect on clinical radiosensitivity, but these are rare. Among non-syndromic patients, variation is less extreme, but equivalent to a ±20% variation in dose. Thus, if individual normal tissue radiosensitivity could be measured, it should be possible to optimise schedules for individual patients. Early investigations of in vitro cellular radiosensitivity supported a link with tissue response, but individual studies were equivocal. A lymphocyte apoptosis assay has potential, and is currently under prospective validation. The investigation of underlying genetic variation also has potential. Although early candidate gene studies were inconclusive, more recent genome-wide association studies are revealing definite associations between genotype and toxicity and highlighting the potential for future genetic testing. Genetic testing and individualised dose prescriptions could reduce toxicity in radiosensitive patients, and permit isotoxic dose escalation to increase local control in radioresistant individuals. The approach could improve outcomes for half the patients requiring radical radiotherapy. As a number of patient- and treatment-related factors also affect the risk of toxicity for a given dose, genetic testing data will need to be incorporated into models that combine patient, treatment and genetic data.

A new functional assay for the diagnosis of X-linked inhibitor of apoptosis (XIAP) deficiency

X-linked inhibitor of apoptosis (XIAP) deficiency, caused by mutations in BIRC4, is an immunodeficiency associated with immune dysregulation and a highly variable clinical presentation. Current diagnostic screening tests such as flow cytometry for XIAP expression or lymphocyte apoptosis assays have significant limitations. Based on recent evidence that XIAP is essential for nucleotide-binding and oligomerization domains (NOD)1/2 signalling, we evaluated the use of a simple flow cytometric assay assessing tumour necrosis factor (TNF) production of monocytes in response to NOD2 stimulation by muramyl dipeptides (L18-MDP) for the functional diagnosis of XIAP deficiency. We investigated 12 patients with XIAP deficiency, six female carriers and relevant disease controls. Irrespective of the diverse clinical phenotype, the extent of residual protein expression or the nature of the mutation, the TNF response was severely reduced in all patients. On average, L18-MDP induced TNF production in 25% of monocytes from healthy donors or female carriers, while fewer than 6% of monocytes responded in affected patients. Notably, the assay clearly discriminated affected patients from disease controls with other immunodeficiencies accompanied by lymphoproliferation, hypogammaglobulinaemia or inflammatory bowel disease. Functional testing of the NOD2 signalling pathway is an easy, fast and reliable assay in the diagnostic evaluation of patients with suspected XIAP deficiency.

Radiation-Induced Lymphocyte Apoptosis to Predict Radiation Therapy Late Toxicity in Prostate Cancer Patients

To examine a potential correlation between the in vitro apoptotic response of lymphocytes to radiation and the risk of developing late gastrointestinal (GI)/genitourinary (GU) toxicity from radiotherapy for prostate cancer. Prostate cancer patients formerly enrolled in a randomized study were tested for radiosensitivity by using a radiation-induced lymphocyte apoptosis assay. Apoptosis was measured using flow cytometry-based Annexin-FITC/7AAD and DiOC(6)/7AAD assays in subpopulations of lymphocytes (total lymphocytes, CD4+, CD8+ and CD4-/CD8-) after exposure to an in vitro dose of 0, 2, 4, or 8 Gy. Patients with late toxicity after radiotherapy showed lower lymphocyte apoptotic responses to 8 Gy than patients who had not developed late toxicity (p = 0.01). All patients with late toxicity had apoptosis levels that were at or below the group mean. The negative predictive value in both apoptosis assays ranged from 95% to 100%, with sensitivity values of 83% to 100%. Apoptosis at lower dose points and in lymphocyte subpopulations had a weaker correlation with the occurrence of late toxicity. Lymphocyte apoptosis after 8 Gy of radiation has the potential to predict which patients will be spared late toxicity after radiation therapy. Further research should be performed to identify the specific subset of lymphocytes that correlates with late toxicity, followed by a corresponding prospective study.

Pyrimethamine treatment does not ameliorate lymphoproliferation or autoimmune disease in MRL/lpr−/− mice or in patients with autoimmune lymphoproliferative syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is an inherited disorder of lymphocyte apoptosis leading to childhood onset of marked lymphadenopathy, hepatosplenomegaly, autoimmune cytopenias, and increased risk of lymphoma. Most cases are associated with heterozygous mutations in the gene encoding Fas protein. Prolonged use of immunosuppressive drugs that do ameliorate its autoimmune complications fail to consistently lessen lymphoproliferation in ALPS. A case series had described children with ALPS, whose spleens (SPL) and lymph nodes decreased in size when treated weekly with pyrimethamine and sulfadoxine; parallel in vitro studies showed only pyrimethamine to promote apoptosis. On the basis of that experience, we undertook additional in vitro lymphocyte apoptosis assays, and measured SPL weights, lymphocyte numbers, and immunophenotypes in Fas-deficient MRL/lpr-/- mice to gain further insights into the utility of combined pyrimethamine/sulfadoxine or pyrimethamine alone. Moreover, seven children with ALPS enrolled in a study of escalating dose of pyrimethamine alone given twice weekly for 12 weeks to determine if their lymphadenopathy and/or splenomegaly would diminish, as assessed by standardized computerized tomography. Neither pyrimethamine alone or with sulfadoxine in the MRL/lpr-/- mice, nor pyrimethamine alone in ALPS patients proved efficacious. We conclude that these drugs do not warrant further use empirically or as part of clinical trials in ALPS Type Ia as a lympholytic agent.

Po‐Poster ‐ 29: Radiation‐induced apoptosis of lymphocytes to predict late toxicity from radiotherapy

An important challenge arises in radiotherapy when certain individuals present with abnormal radiosensitivities. Radiosensitive individuals may not tolerate normal therapy and encounter complications whereas radioresistant patients may not respond adequately to standard treatments regimes because of intrinsic cellular resistance. It has been shown (Crompton et al. IJROBP 2003; 45:707–714 and IJROBP 2001; 49:547–554) that late normal tissue toxicity was correlated to low level apoptosis in CD4 and CD8 lymphocytes. The assay developed by this group used a simple flow cytometry based assay. Our group has researched the sensitivity of alternative apoptosis assays (DiOC6, Caspase‐3, Annexin‐V, PI, 7AAD and Comet), and have shown that both the Annexin V‐FITC and the DiOC6 assay have the greatest suitability in clinical use with respect to speed, simplicity and sensitivity. In our present study, 80 prostate cancer patients will be tested for radiosensitivity using lymphocyte apoptosis assays. These patients were formerly enrolled in a randomized dose‐escalation study (Sathya et al. JCO 2005; 45:1192–1199). Toxicity was recorded prospectively. The aim of this study is to determine a correlation between the proportion of T‐lymphocytes undergoing apoptosis when irradiated (2, 4 and 8Gy) and the occurrence of late toxicity (Grade II and above). We will present our current results showing a large inter‐individual variation within our patient cohort. Data obtained using the Annexin Assay measuring CD4 lymphocyte apoptosis receiving 8Gy show a wide range of responses (mean = 34% apoptosis, σ = 8.2) with z‐scores ranging from −1.5 to 2.4.

Finding our sensitive patients

The practice of radiation oncology requires balancing tumor control probability against the risk of normal tissue injury. Using radiation to eradicate disease is acceptable only if life-threatening or severe normal tissue injury is avoided. Despite a number of advances in the field of radiation oncology, the understanding of the genetic and epigenetic phenomenon that determine an individual’s risk for normal tissue radiation injury remains rudimentary. Patients display a diverse spectrum of toxicity for any given treatment schedule. While toxicity risks for populations of patients are known, the determination of an individual’s normal tissue radiosensitivity is rarely possible prior to treatment. Therefore, current practice standards commonly prescribe radiation dose schedules according to clinical scenarios, without regard to the genotype or phenotype of the individual being irradiated. Moving away from considering normal tissue risk of radiation injury as a population phenomenon towards determining the risk for the individual being irradiated is a worthy task. For patients with a radiosensitive genotype or phenotype, the risk-benefit ratio of radiation could be more clearly defined, and lower dosages or alternatives to radiation could be explored. Equally important, the ability to identify the most radiosensitive patients has the potential to improve the tumor control probability in the remaining population. In general, practice standards limit radiation dosages to those predicted to cause serious late complications in 10% or less of the treated population. Accordingly, this dose is below the threshold for radiation injury in the remaining 90%. A normal tissue predictive assay that successfully identified the 10% most radiosensitive patients would allow radiation dosages to be escalated in the remaining 90%. Tucker et al. have mathematically modeled this concept using a set of human fibroblast radiosensitivity data (1). Their results estimated that dose escalation of 10% would be possible if the identified radiosensitive patients were excluded from treatment. For clinical situations in which dose escalation would occur over the steep portion of the tumor control probability curve, this dose increase could achieve clinically meaningful results. Despite the theoretical appeal, normal tissue predictive assays have not proven clinically useful. The major limitation of laboratory-based normal tissue predictive assays is that most radiation complications result from a number of genetic and epigenetic phenomenon. Target cells have different intrinsic radiosensitivity and cellular environments dramatically affect radiation response. In addition, host factors that impair microvasculature (diabetes, atherosclerosis) or alter immune response (scleroderma, lupus) are known to predispose to radiation injury. The contributions of epigenetic factors and the cellular environment to radiation response are difficult to reproduce in laboratory settings. Correspondingly, most in vitro predictive assays have focused on studying the genetic predisposition of an individual to radiation injury. The excitement for this strategy arose after individuals with ataxia telangiectasia (AT) were found to have a radiosensitive phenotype. In vitro studies of fibroblasts and lymphocytes from these individuals revealed an exquisite normal tissue radiosensitivity that was directly attributable to a specific genetic defect. Based on this paradigm, a number of functional normal tissue predictive assays have been developed. These assays commonly use such cells as fibroblasts and lymphocytes to predict clinical injuries that develop independently of the cell type used in the assay. The theory underlying these assays is that germline factors affecting radiosensitivity will be present in any cell, and therefore fibroblasts and lymphocytes are appropriate surrogates for the target cell of the normal tissue injury. In this issue of the IJROBP, Crompton et al. report a pilot study using a new predictive assay based, in part, on this paradigm. Their study examines the value of a lymphocyte apoptosis assay in predicting radiation injury risk. The methodology of the study compares the degree of in vitro radiation-induced apoptosis of peripheral lymphocytes in a 12-patient radiosensitive cohort with age-matched controls and with a cohort of 9 patients with AT. Their results indicated a decreased rate of radiation-induced apoptosis in the AT and hypersensitive patients compared with controls. This result seems paradoxical, in that unlike fibroblast clo-