Age Estimation Based on Computed Tomography Analysis of the Scapula.

Background and Objectives: Age estimation from skeletal remains and in living individuals is an important issue for human identification, and also plays a critical role in judicial proceedings for migrants. Forensic analysis of ossification centers is the main evaluation method for age estimation, and ossification degree can be determined using computed tomography analysis. The purpose of this study is to investigate the applicability of CT (computed tomography) in the analysis of left scapula ossification centers, for forensic age estimation in Turkish society. Materials and Methods: We analyzed six ossification centers of the left scapula and these ossification centers are the coracoid, subcoracoid, coracoid apex, acromial, glenoid, and inferior angle ossification centers. A pediatric radiologist analyzed these six ossification centers of the scapula by using a staging method defined by Schmeling et al. in 2004. Two months after the first assessment, 20 randomly selected cases was reanalyzed by the first observer and by another pediatric radiologist. Correlation between the age and ossification stage was assessed using Spearman's nonparametric correlation test. Linear regression analysis was performed using a backwards model. Cohen's kappa coefficient was used for evaluating interobserver and intraobserver variability. Results: In this retrospective study, 397 (248 male and 149 female) cases were evaluated. Ages ranged between 7.1 and 30.9. The mean age was 19.83 ± 6.49. We determined a positive significant correlation between the age and the ossification stages of ossification centers analyzed in both sexes. In each ossification center, except inferior angle, all of the stage 1 and 2 cases in both sexes were under 18 years old. Intraobserver and interobserver evaluations showed that reproducibility and consistency of the method was relatively good. Conclusions: The present study indicated that CT analysis of scapula ossification centers might be helpful in forensic age assessment of living individuals and dry bones.

Mesenchymal stromal cells with chimaeric antigen receptors for enhanced immunosuppression.

Allogeneic mesenchymal stromal cells (MSCs) are a safe treatment option for many disorders of the immune system. However, clinical trials using MSCs have shown inconsistent therapeutic efficacy, mostly owing to MSCs providing insufficient immunosuppression in target tissues. Here we show that antigen-specific immunosuppression can be enhanced by genetically modifying MSCs with chimaeric antigen receptors (CARs), as we show for E-cadherin-targeted CAR-MSCs for the treatment of graft-versus-host disease in mice. CAR-MSCs led to superior T-cell suppression and localization to E-cadherin+ colonic cells, ameliorating the animals' symptoms and survival rates. On antigen-specific stimulation, CAR-MSCs upregulated the expression of immunosuppressive genes and receptors for T-cell inhibition as well as the production of immunosuppressive cytokines while maintaining their stem cell phenotype and safety profile in the animal models. CAR-MSCs may represent a widely applicable therapeutic technology for enhancing immunosuppression.

CD19 occupancy with tafasitamab increases therapeutic index of CART19 cell therapy and diminishes severity of CRS.

ABSTRACT: In the development of various strategies of anti-CD19 immunotherapy for the treatment of B-cell malignancies, it remains unclear whether CD19 monoclonal antibody therapy impairs subsequent CD19-targeted chimeric antigen receptor T-cell (CART19) therapy. We evaluated the potential interference between the CD19-targeting monoclonal antibody tafasitamab and CART19 treatment in preclinical models. Concomitant treatment with tafasitamab and CART19 showed major CD19 binding competition, which led to CART19 functional impairment. However, when CD19+ cell lines were pretreated with tafasitamab overnight and the unbound antibody was subsequently removed from the culture, CART19 function was not affected. In preclinical in vivo models, tafasitamab pretreatment demonstrated reduced incidence and severity of cytokine release syndrome and exhibited superior antitumor effects and overall survival compared with CART19 alone. This was associated with transient CD19 occupancy with tafasitamab, which in turn resulted in the inhibition of CART19 overactivation, leading to diminished CAR T apoptosis and pyroptosis of tumor cells.

AXL Inhibition Improves the Antitumor Activity of Chimeric Antigen Receptor T Cells.

The receptor tyrosine kinase AXL is a member of the TYRO3, AXL, and proto-oncogene tyrosine-protein kinase MER family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CAR T)-cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CAR T (CART19)-cell functions. Our results demonstrate that T cells and CAR T cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CAR T cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CAR T cells, reduction of Th2 cytokines, reversal of CAR T-cell inhibition, and promotion of CAR T-cell effector functions. AXL inhibition is a novel strategy to enhance CAR T-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CAR T-cell inhibition through selective targeting of M2-polarized macrophages.

Hyperphosphorylated PTEN exerts oncogenic properties.

PTEN is a multifaceted tumor suppressor that is highly sensitive to alterations in expression or function. The PTEN C-tail domain, which is rich in phosphorylation sites, has been implicated in PTEN stability, localization, catalytic activity, and protein interactions, but its role in tumorigenesis remains unclear. To address this, we utilized several mouse strains with nonlethal C-tail mutations. Mice homozygous for a deletion that includes S370, S380, T382 and T383 contain low PTEN levels and hyperactive AKT but are not tumor prone. Analysis of mice containing nonphosphorylatable or phosphomimetic versions of S380, a residue hyperphosphorylated in human gastric cancers, reveal that PTEN stability and ability to inhibit PI3K-AKT depends on dynamic phosphorylation-dephosphorylation of this residue. While phosphomimetic S380 drives neoplastic growth in prostate by promoting nuclear accumulation of ß-catenin, nonphosphorylatable S380 is not tumorigenic. These data suggest that C-tail hyperphosphorylation creates oncogenic PTEN and is a potential target for anti-cancer therapy.

GM-CSF disruption in CART cells modulates T cell activation and enhances CART cell anti-tumor activity.

Inhibitory myeloid cells and their cytokines play critical roles in limiting chimeric antigen receptor T (CART) cell therapy by contributing to the development of toxicities and resistance following infusion. We have previously shown that neutralization of granulocyte-macrophage colony-stimulating factor (GM-CSF) prevents these toxicities and enhances CART cell functions by inhibiting myeloid cell activation. In this report, we study the direct impact of GM-CSF disruption during the production of CD19-directed CART cells on their effector functions, independent of GM-CSF modulation of myeloid cells. In this study, we show that antigen-specific activation of GM-CSFKO CART19 cells consistently displayed reduced early activation, enhanced proliferation, and improved anti-tumor activity in a xenograft model for relapsed B cell malignancies. Activated CART19 cells significantly upregulate GM-CSF receptors. However, the interaction between GM-CSF and its upregulated receptors on CART cells was not the predominant mechanism of this activation phenotype. GM-CSFKO CART19 cell had reduced BH3 interacting-domain death agonist (Bid), suggesting an interaction between GM-CSF and intrinsic apoptosis pathways. In conclusion, our study demonstrates that CRISPR/Cas9-mediated GM-CSF knockout in CART cells directly ameliorates CART cell early activation and enhances anti-tumor activity in preclinical models.

Challenges of chimeric antigen receptor T-cell therapy in chronic lymphocytic leukemia: lessons learned.

Development of chimeric antigen receptor T cell (CART) therapy has led to an unprecedented success against B-cell leukemia and lymphoma and resulted in U.S. Food and Drug Administration-approved treatment protocols. Despite the initial clinical response in B cell-related malignancies, high relapse rates suggest that much work is needed to uncover mechanisms of resistance. In chronic lymphocytic leukemia (CLL), the durable activity of CAR T-cells is limited, and CAR T-cell therapy success is lower than in other malignancies. T cells from these patients are vulnerable to a state of dysfunction because of stresses including chronic infection, rapid cell cycle on antigen recognition, immunosuppressive tumor microenvironment, and cancer-related treatments. T cells are also introduced to additional stresses when cultured ex vivo during the CAR T-cell manufacturing process. All these factors contribute to the limited regenerative capacity of T cells, which can lead to CAR T-cell treatment failure. In this article, we review the challenges of CAR T-cell therapy in patients with CLL and discuss potential strategies to overcome these challenges.

Targeting cancer-associated fibroblasts in the bone marrow prevents resistance to CART-cell therapy in multiple myeloma.

Pivotal clinical trials of B-cell maturation antigen-targeted chimeric antigen receptor T (CART)-cell therapy in patients with relapsed/refractory multiple myeloma (MM) resulted in remarkable initial responses, which led to a recent US Food and Drug Administration approval. Despite the success of this therapy, durable remissions continue to be low, and the predominant mechanism of resistance is loss of CART cells and inhibition by the tumor microenvironment (TME). MM is characterized by an immunosuppressive TME with an abundance of cancer-associated fibroblasts (CAFs). Using MM models, we studied the impact of CAFs on CART-cell efficacy and developed strategies to overcome CART-cell inhibition. We showed that CAFs inhibit CART-cell antitumor activity and promote MM progression. CAFs express molecules such as fibroblast activation protein and signaling lymphocyte activation molecule family-7, which are attractive immunotherapy targets. To overcome CAF-induced CART-cell inhibition, CART cells were generated targeting both MM cells and CAFs. This dual-targeting CART-cell strategy significantly improved the effector functions of CART cells. We show for the first time that dual targeting of both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART-cell therapy in MM.

p21 produces a bioactive secretome that places stressed cells under immunosurveillance.

Immune cells identify and destroy damaged cells to prevent them from causing cancer or other pathologies by mechanisms that remain poorly understood. Here, we report that the cell-cycle inhibitor p21 places cells under immunosurveillance to establish a biological timer mechanism that controls cell fate. p21 activates retinoblastoma protein (Rb)­dependent transcription at select gene promoters to generate a complex bioactive secretome, termed p21-activated secretory phenotype (PASP). The PASP includes the chemokine CXCL14, which promptly attracts macrophages. These macrophages disengage if cells normalize p21 within 4 days, but if p21 induction persists, they polarize toward an M1 phenotype and lymphocytes mount a cytotoxic T cell response to eliminate target cells, including preneoplastic cells. Thus, p21 concurrently induces proliferative arrest and immunosurveillance of cells under duress.

Development of a Clinically Relevant Reporter for Chimeric Antigen Receptor T-cell Expansion, Trafficking, and Toxicity.

Although chimeric antigen receptor T (CART)-cell therapy has been successful in treating certain hematologic malignancies, wider adoption of CART-cell therapy is limited because of minimal activity in solid tumors and development of life-threatening toxicities, including cytokine release syndrome (CRS). There is a lack of a robust, clinically relevant imaging platform to monitor in vivo expansion and trafficking to tumor sites. To address this, we utilized the sodium iodide symporter (NIS) as a platform to image and track CART cells. We engineered CD19-directed and B-cell maturation antigen (BCMA)-directed CART cells to express NIS (NIS+CART19 and NIS+BCMA-CART, respectively) and tested the sensitivity of 18F-TFB-PET to detect trafficking and expansion in systemic and localized tumor models and in a CART-cell toxicity model. NIS+CART19 and NIS+BCMA-CART cells were generated through dual transduction with two vectors and demonstrated exclusive 125I uptake in vitro. 18F-TFB-PET detected NIS+CART cells in vivo to a sensitivity level of 40,000 cells. 18F-TFB-PET confirmed NIS+BCMA-CART-cell trafficking to the tumor sites in localized and systemic tumor models. In a xenograft model for CART-cell toxicity, 18F-TFB-PET revealed significant systemic uptake, correlating with CART-cell in vivo expansion, cytokine production, and development of CRS-associated clinical symptoms. NIS provides a sensitive, clinically applicable platform for CART-cell imaging with PET scan. 18F-TFB-PET detected CART-cell trafficking to tumor sites and in vivo expansion, correlating with the development of clinical and laboratory markers of CRS. These studies demonstrate a noninvasive, clinically relevant method to assess CART-cell functions in vivo.

Medicolegal Evaluation of Long-Term Respiratory Functions in Patients Injured Due to Traffic Accidents.

Aim Blunt chest trauma is a frequent injury in developing countries, with motor vehicle accidents being the most common cause. Most studies about the effects of post-traumatic injuries on pulmonary functions are related to the acute phase. The aim of this study is to compare the effect of injury type on pulmonary function tests as a long-term disability in patients with severe chest trauma due to traffic accidents. Methods In our study, 53 patients were admitted to the Forensic Expert Council with the aim of determining the disability ratio at least six months after the traffic accident. All patients who had a respiratory function test because of respiratory symptoms and whose reporting period was completed were appreciated. A retrospective examination of the forensic committee reports, types of injuries, and current pulmonary function test results were analyzed and the data were evaluated by using the Statistical Package for the Social Sciences (SPSS) 22.0 program (IBM Corp, Armonk, NY). Results Thirty-two (32) of the patients were male while 21 were female. Their average age was 39.88 ± 15.29. Sixty-six percent (66%; n: 35) of the cases were injured due to in-vehicle traffic accidents, 18.9% (n: 10) due to motorcycle accidents, 15.1% (n: 8) due to non-vehicle traffic accidents. The number of cases with costa fractures was 47 and 74.4% of these cases had three or more rib fractures. The mean forced expiratory volume in 1 second/forced vital capacity ratio (FEV1/FVC-Tiffeneau-Pinelli index) was calculated as 85.3% ± 9.45, and the average FVC was 84.3 ± 14.98%. The average number of rib fractures in all patients was 3.41 ± 2.24. It was observed that tube thoracostomy was performed in seven of 12 cases with FEV1/FVC below 80%, and the average number of rib fractures was 3.75. In 20 cases where the FVC average was below 80%, the mean number of rib fractures was 3.8, and tube thoracostomy was performed in 10 of these cases. The highest FEV1 value was 116%, and the lowest FEV1 value was 35%. The FEV1 value of 23 cases was between 75% and 95%. The highest FEV1/FVC value was 113% and the lowest FEV1/FVC value was 50%. The FEV1/FVC values of 38 cases were between 80% and 100%.  Conclusions In our study, most patients achieve near-complete recovery in pulmonary function tests; the impact of pre-existing pulmonary compromise on recovery is less known. The number of rib fractures can reflect the severity of the blunt trauma but it would not necessarily predict the resulting pulmonary function. These results are consistent with the previous studies. Further larger prospective studies are required to investigate different factors affecting prognosis.

In vivo CART cell imaging: Paving the way for success in CART cell therapy.

Chimeric antigen receptor T (CART) cells are a promising immunotherapy that has induced dramatic anti-tumor responses in certain B cell malignancies. However, CART cell expansion and trafficking are often insufficient to yield long-term remissions, and serious toxicities can arise after CART cell administration. Visualizing CART cell expansion and trafficking in patients can detect an inadequate CART cell response or serve as an early warning for toxicity development, allowing CART cell treatment to be tailored accordingly to maximize therapeutic benefits. To this end, various imaging platforms are being developed to track CART cells in vivo, including nonspecific strategies to image activated T cells and reporter systems to specifically detect engineered T cells. Many of these platforms are clinically applicable and hold promise to provide valuable information and guide improved CART cell treatment.

Prognostic evidence of LEF1 isoforms in childhood acute lymphoblastic leukemia.

INTRODUCTION: The lymphoid enhancer factor 1 (LEF1) is a DNA-binding transcription factor that functions in the Wnt signaling pathway. Increased LEF1 activity is associated with progression of several types of cancer including leukemia. Here, we investigated LEF1 isoform expression and genomic variations in acute lymphoblastic leukemia (ALL). METHODS: LEF1 isoform expression was evaluated by quantitative real-time PCR in 87 newly diagnosed childhood ALL patients and controls. Moreover, Western blot analysis was performed for detection of LEF1 expression and the hotspot region of LEF1 was screened by deep sequencing. RESULTS: The LEF1 mRNA expression of B cell ALL patients was higher than the controls (LEF1-total P = .011, LEF1-long P = .026). Moreover, B-ALL samples showing higher total LEF1 expression had significantly shorter relapse-free survival (P = .008) and overall survival (P = .011). Although full-length LEF1 expression was similar to the controls in T-ALL, 50% (n = 15) of the ALL patients had increased full-length LEF1 protein expression. Imbalance between short- and full-length LEF1 isoforms may lead to cell survival in ALL. Beside the LEF1 activation, LEF1 gene variations were rarely observed in our cohort. CONCLUSION: The results indicate that the Wnt pathway may have a pathogenic function in a group of ALL patients and high LEF1-total expression might be a marker for shorter relapse-free survival time in B cell ALL.

Leukemic extracellular vesicles induce chimeric antigen receptor T cell dysfunction in chronic lymphocytic leukemia.

Chimeric antigen receptor (CAR) T cell therapy has yielded unprecedented outcomes in some patients with hematological malignancies; however, inhibition by the tumor microenvironment has prevented the broader success of CART cell therapy. We used chronic lymphocytic leukemia (CLL) as a model to investigate the interactions between the tumor microenvironment and CART cells. CLL is characterized by an immunosuppressive microenvironment, an abundance of systemic extracellular vesicles (EVs), and a relatively lower durable response rate to CART cell therapy. In this study, we characterized plasma EVs from untreated CLL patients and identified their leukemic cell origin. CLL-derived EVs were able to induce a state of CART cell dysfunction characterized by phenotypical, functional, and transcriptional changes of exhaustion. We demonstrate that, specifically, PD-L1+ CLL-derived EVs induce CART cell exhaustion. In conclusion, we identify an important mechanism of CART cell exhaustion induced by EVs from CLL patients.

Pyoderma gangrenosum with thymoma and myasthenia gravis: A case report.

Pyoderma gangrenosum (PG) is a rare, neutrophilic dermatosis which is characterized by painful, necrotic ulcer with violaceous border that heals with cribriform scar. Although the etiopathogenesis of PG is not known exactly, it can be triggered by many factors such as genetics, autoimmune, pathergy phenomenon, drugs, and paraneoplastic. It is frequently associated with autoimmune pathogenesis such as inflammatory bowel disease and rheumatologic disease. It can also be associated with hematological or solid organ malignancies, and then, it is called paraneoplastic PG. The association of PG with myasthenia gravis and thymoma has not been previously reported. In our case, these three diseases with a common paraneoplastic pathogenesis were seen together and the coexistence of the three diseases is rare. Treatment of PG should be decided according to the severity, spread of the lesions, concomitant disease, medical condition, and tolerance of the patient. The purpose of treatment is to control the lesions and related diseases for a long time with minimal side effects. Mycophenolate mofetil treatment was used safely and successfully for both generalized MG and PG in our case.

Revision of the medicolegal assessment criteria in hypovolemic patients.

Trauma scoring systems are often used for the determination of the severity level of the lesion and the clinical status in medico-legal assessment of the trauma patient. Trauma scoring systems are used also for the determination of the life-threatening conditions. Blood loss of more than 20% was reported as the only criterion for life-threatening conditions in the acute hypovolemia. The objective of this study was to revise the medico-legal assessment criteria in the patients with acute hypovolemia and to discuss other parameters, which might be used in the determination of the severity level of the clinical status.The medical reports of the patients with acute hypovolemia due to the trauma, which were sent by the judicial authorities and by other departments of our medical faculty to the department of the forensic medicine between 1999 and 2009, were evaluated. The characteristics such as age, gender, severity of the injury, type of the trauma, history of liquid replacement or blood transfusion, vital signs, type of the physical injury, injured region of the body, presence of any chronic disease were assessed and recorded.The mean age of the included 155 patients was 34.70 ±â€Š16.08 years (3-87 years). 118 (76%) of patients were males and 37 females (24%). Regarding the event types, road accidents were the most common cause (60.0%) and it was followed by sharp object injuries (18.7%) and firearm injuries (11.6%). 27.7% of the subjects received 2 units blood and blood products transfusion and 21.3% only 1 unit transfusion. According to the results of the medico-legal assessment, 84.5% of the patients had life-threatening conditions.While evaluating the severity of the clinical conditions in the hypovolemic patients, to report only the losses in percentage causes problems and limitations. Therefore, in respect of the medico-legal assessment of the hypovolemic patients, we believe that it would be more appropriate to use the physiological trauma scoring systems (like Revised Trauma Score) instead of the anatomic scoring systems.

Applicability of T1-weighted MRI in the assessment of forensic age based on the epiphyseal closure of the humeral head.

This work investigates the value of magnetic resonance imaging analysis of proximal epiphyseal fusion in research examining the growth and development of the humerus and its potential utility in establishing forensic age estimation. In this study, 428 proximal humeral epiphyses (patient age, 12-30 years) were evaluated with T1-weighted turbo spin echo (T1 TSE) sequences in coronal oblique orientation on shoulder MRI images. A scoring system was created following a combination of the Schmeling and Kellinghaus methods. Spearman's rank correlation analysis revealed a significant positive relationship between age and ossification stage of the proximal humeral epiphysis (all subjects: rho = 0.664, p < 0.001; males: 0.631, p < 0.001; females: rho = 0.651, p < 0.001). The intra- and inter-observer reliability assessed using Cohen's kappa statistic was κ = 0.898 and κ = 0.828, respectively. The earliest age of epiphysis closure was 17 years for females and 18 years for males. MRI of the proximal humeral epiphysis can be considered advantageous for forensic age estimation of living individuals in a variety of situations, ranging from monitoring public health to estimating the age of illegal immigrants/asylum seekers, minors engaged in criminal activities, and illegal participants in competitive sports, without the danger of radiation exposure.

Forensic age diagnostics by magnetic resonance imaging of the proximal humeral epiphysis.

The most commonly used radiological method for age estimation of living individuals is X-ray. Computed tomography is not commonly used due to high radiation exposure, which raises ethical concerns. This problem can be solved with the use of magnetic resonance imaging (MRI), which avoids the use of ionizing radiation. The purpose of the present study was to evaluate the utility of MRI analysis of the proximal humeral epiphyses for forensic age estimations of living individuals. In this study, 395 left proximal humeral epiphyses (patient age 12-30 years) were evaluated with fast-spin-echo proton density-weighted image (FSE PD) sequences in a coronal oblique orientation on shoulder MRI images. A five-stage scoring system was used following the method of Dedouit et al. The intra- and interobserver reliabilities assessed using Cohen's kappa statistic were κ = 0.818 and κ = 0.798, respectively. According to this study, stage five first appeared at 20 and 21 years of age in males and females, respectively. These results are not directly comparable to any other published study due to the lack of MRI data on proximal humeral head development. These findings may provide valuable information for legally important age thresholds using shoulder MRI. The current study demonstrates that MRI of the proximal humerus can support forensic age estimation. Further research is needed to establish a standardized protocol that can be applied worldwide.

Recurrent WNT pathway alterations are frequent in relapsed small cell lung cancer.

Nearly all patients with small cell lung cancer (SCLC) eventually relapse with chemoresistant disease. The molecular mechanisms driving chemoresistance in SCLC remain un-characterized. Here, we describe whole-exome sequencing of paired SCLC tumor samples procured at diagnosis and relapse from 12 patients, and unpaired relapse samples from 18 additional patients. Multiple somatic copy number alterations, including gains in ABCC1 and deletions in MYCL, MSH2, and MSH6, are identifiable in relapsed samples. Relapse samples also exhibit recurrent mutations and loss of heterozygosity in regulators of WNT signaling, including CHD8 and APC. Analysis of RNA-sequencing data shows enrichment for an ASCL1-low expression subtype and WNT activation in relapse samples. Activation of WNT signaling in chemosensitive human SCLC cell lines through APC knockdown induces chemoresistance. Additionally, in vitro-derived chemoresistant cell lines demonstrate increased WNT activity. Overall, our results suggest WNT signaling activation as a mechanism of chemoresistance in relapsed SCLC.

Ancestry estimation of three Mediterranean populations based on cranial metrics.

The estimation of ancestry is an essential benchmark for positive identification of heavily decomposed bodies that are recovered in a variety of death and crime scenes. This is especially true when reconstructing the biological profile of the deceased as most methods for sex, age and stature estimation are population-specific. Ancestry estimation methods vary from traditional morphological assessment of cranial features and biometric quantification to computer-aided shape analysis and classification with specialised software. The current paper aims to explore population differences between three neighbouring countries (Greece, Cyprus and Turkey) that have been in constant interaction through conflicts and population movements from the ancient past to the present day, through cranial measurements. The sample consists of 160 dry crania of Greek origin, 137 dry crania of Greek-Cypriot of origin Cyprus and 380 CT scans from Turks individuals. Twelve measurements were taken in both dry and virtual skulls. Data were submitted to principal component analysis and discriminant function analysis. Intra- and inter-observer error as well as the measurement error between virtual and physical measurements were quantified using TEM, rTEM and R. Measurement error was very low in all cases. Classification accuracy for cross-validated data ranged from 74.1 to 97.9%. The highest accuracy was obtained for the Turks sample both in males and females. The results are in accordance with genetic data on the three populations. These results create great confidence in the application of the produced functions in forensic cases requiring ancestry estimation in Cyprus, specifically to unidentified individuals from the 1974 conflict. In addition, these standards can be applied in other forensic situations where ethnicity is an issue but the geographic area of origin is limited to the area encompassing Turkey, Cyprus and Greece.