Performance of the OncomineTM Lung cfDNA Assay for Liquid Biopsy by NGS of NSCLC Patients in Routine Laboratory Practice

Targeted next-generation sequencing (NGS) based on molecular tagging technology allowed considerable improvement in the approaches of cell-free DNA (cfDNA) analysis. Previously, we demonstrated the feasibility of the OncomineTM Lung cell-free DNA Assay (OLcfA) NGS panel when applied on plasma samples of post-tyrosine kinase inhibitors (TKIs) non-small cell lung cancer (NSCLC) patients. Here, we explored in detail the coverage metrics and variant calling of the assay and highlighted strengths and challenges by analyzing 92 plasma samples collected from a routine cohort of 76 NSCLC patients. First, performance of OLcfA was assessed using Horizon HD780 reference standards and sensitivity and specificity of 92.5% and 100% reported, respectively. The OLcfA was consequently evaluated in our plasma cohort and NGS technically successful in all 92 sequenced libraries. We demonstrated that initial cfDNA amount correlated positively with library yields (p < 0.0001) and sequencing performance (p < 0.0001). In addition, 0.1% limit of detection could be achieved even when < 10 ng cfDNA was employed. In contrast, the cfDNA amount seems to not affect the EGFR mutational status (p = 0.16). This study demonstrated an optimal performance of the OLcfA on routine plasma samples from NSCLC patients and supports its application in the liquid biopsy practice for cfDNA investigation in precision medicine laboratories

Motoric Cognitive Risk Syndrome, Subtypes and 8-Year All-Cause Mortality in Aging Phenotypes: The Salus in Apulia Study

BackgroundThis study aims to establish the key clinical features of different motoric cognitive risk (MCR) subtypes based on individual quantitative measures of cognitive impairment and to compare their predictive power on survival over an 8-year observation time.MethodsWe analyzed data from a population-based study of 1138 subjects aged 65 years and older in south Italy. These individuals were targeted and allocated to subtypes of the MCR phenotype according to the slowness criterion plus one other different cognitive domain for each characterized phenotype (Subjective Cognitive Complaint [SCC]; Global Function [Mini Mental State Examination (MMSE) &lt; 24]; or a combination of both). Clinical evaluation and laboratory assays, along with a comprehensive battery of neuropsychological and physical tests, completed the sample investigation.ResultsMCR prevalence was found to be 9.8% (n = 112), 3.6% (n = 41), 3.4% (n = 39) and 1.8% (n = 21) for the MCR, MCR-GlobalFunction, MCR-StructuredSCC and MCR-SCC and GlobalFunction, respectively. Univariate Cox survival analysis showed an association only of the MCR-GlobalFunction subtype with an almost three-fold increased risk of overall death as compared to the other counterparts (HR 2.53, 95%CI 1.28 to 4.99) over an 8-year observation period. Using Generalized Estimating Equations (GEE) for clustered survival data, we found that MCR males had an increased and significant mortality risk with respect to MCR female subjects.ConclusionsMCR phenotypes assigned to the MMSE cognitive domain are more likely to have an increased risk of overall mortality, and gender showed a huge effect on the risk of death for MCR subjects over the 8-year observation

Role of Dietary Carotenoids in Frailty Syndrome: A Systematic Review

Unbalanced diets and altered micronutrient intake are prevalent in the aging adult population. We conducted a systematic review to appraise the evidence regarding the association between single (α-carotene, β-carotene, lutein, lycopene, β-cryptoxanthin) or total carotenoids and frailty syndrome in the adult population. The literature was screened from study inception to December 2021, using six different electronic databases. After establishing inclusion criteria, two independent researchers assessed the eligibility of 180 retrieved articles. Only 11 fit the eligibility requirements, reporting five carotenoid entries. No exclusion criteria were applied to outcomes, assessment tools, i.e., frailty constructs or surrogates, recruitment setting, general health status, country, and study type (cohort or cross-sectional). Carotenoid exposure was taken as either dietary intake or serum concentrations. Cross-sectional design was more common than longitudinal design (n = 8). Higher dietary and plasma levels of carotenoids, taken individually or cumulatively, were found to reduce the odds of physical frailty markedly, and the evidence showed consistency in the direction of association across all selected studies. Overall, the methodological quality was rated from moderate (27%) to high (73%). Prevention of micronutrient deficiencies has some potential to counteract physical decline. Considering carotenoids as biological markers, when monitoring micronutrient status, stressing increased fruit and vegetable intake may be part of potential multilevel interventions to prevent or better manage disability

Microenvironmental regulation of the IL-23R/IL-23 axis overrides chronic lymphocytic leukemia indolence

Although the progression of chronic lymphocytic leukemia (CLL) requires the cooperation of the microenvironment, the exact cellular and molecular mechanisms involved are still unclear. We investigated the interleukin (IL)-23 receptor (IL-23R)/IL-23 axis and found that circulating cells from early-stage CLL patients with shorter time-to-treatment, but not of those with a more benign course, expressed a defective form of the IL-23R complex lacking the IL-12R beta 1 chain. However, cells from both patient groups expressed the complete IL-23R complex in tissue infiltrates and could be induced to express the IL-12R. 1 chain when cocultured with activated T cells or CD40L(+) cells. CLL cells activated in vitro in this context produced IL-23, a finding that, together with the presence of IL-23 in CLL lymphoid tissues, suggests the existence of an autocrine/paracrine loop inducing CLL cell proliferation. Interference with the IL-23R/IL-23 axis using an anti-IL-23p19 antibody proved effective in controlling disease onset and expansion in xenografted mice, suggesting potential therapeutic strategies

Dietary Patterns Associated with Diabetes in an Older Population from Southern Italy Using an Unsupervised Learning Approach

Dietary behaviour is a core element in diabetes self-management. There are no remarkable differences between nutritional guidelines for people with type 2 diabetes and healthy eating recommendations for the general public. This study aimed to evaluate dietary differences between subjects with and without diabetes and to describe any emerging dietary patterns characterizing diabetic subjects. In this cross-sectional study conducted on older adults from Southern Italy, eating habits in the “Diabetic” and “Not Diabetic” groups were assessed with FFQ, and dietary patterns were derived using an unsupervised learning algorithm: principal component analysis. Diabetic subjects (n = 187) were more likely to be male, slightly older, and with a slightly lower level of education than subjects without diabetes. The diet of diabetic subjects reflected a high-frequency intake of dairy products, eggs, vegetables and greens, fresh fruit and nuts, and olive oil. On the other hand, the consumption of sweets and sugary foods was reduced compared to non-diabetics (23.74 ± 35.81 vs. 16.52 ± 22.87; 11.08 ± 21.85 vs. 7.22 ± 15.96). The subjects without diabetes had a higher consumption of red meat, processed meat, ready-to-eat dishes, alcoholic drinks, and lower vegetable consumption. The present study demonstrated that, in areas around the Mediterranean Sea, older subjects with diabetes had a healthier diet than their non-diabetic counterparts

Analysis of 13 cell types reveals evidence for the expression of numerous novel primate- and tissue-specific microRNAs

MicroRNAs (miRNAs) are small ∼22-nt RNAs that are important regulators of posttranscriptional gene expression. Since their initial discovery, they have been shown to be involved in many cellular processes, and their misexpression is associated with disease etiology. Currently, nearly 2,800 human miRNAs are annotated in public repositories. A key question in miRNA research is how many miRNAs are harbored by the human genome. To answer this question, we examined 1,323 short RNA sequence samples and identified 3,707 novel miRNAs, many of which are human-specific and tissue-specific. Our findings suggest that the human genome expresses a greater number of miRNAs than has previously been appreciated and that many more miRNA molecules may play key roles in disease etiology

T- Lymphoblastic lymphoma in adults Linfoma linfoblástico T dos adultos

Adult T-lymphoblastic lymphoma is rare and has a poor prognosis. In the 80s, following the introduction of sequential, intensified chemotherapy, complete remissions in the order of 75%-95% of treated patients, were achieved. However, several patients, namely those with advanced disease, continued to relapse either in remission or during maintenance therapy. Moreover, all these early studies were not able to detect any valuable prognostic index to predict the outcome. In an attempt to reduce the relapse rate, upfront autologous stem cell transplantation in patients in complete remission was introduced. The results obtained with this approach were quite homogeneous, indicating a probability of disease-free survival of about 65%-75% and an overall survival rate of 60%. Successive therapies designed since 2000 were able to obtain complete remissions of above 90%, with a relapse rate in the order of 30% and an overall survival comparable to that obtained with the transplant procedure. Yet, these studies were also unable to detect valuable prognostic factors predictive of the outcome. Moreover, no study on the biologic profile of the disease has been developed. To improve the prognosis of T-lymphoblastic lymphoma it seems necessary to create national registries to collect both clinical and biological data of all lymphoblastic lymphoma patients. In this way it will be possible to reach critical numbers of data with which valid statistical analysis may be performed that is able to detect factors influencing the outcome. Moreover, subsets of patients needing intensified procedures such as stem cell transplant may be detected at diagnosis.O linfoma linfoblástico de célula T é raro e com prognóstico ruim. Após introdução de terapêutica quimioterápica seqüencial e intensificada, remissões completas passaram a ser obtidas em 75%-95% dos pacientes. Entretanto, muitos pacientes, particularmente aqueles com a chamada doença avançada, continuaram a recair tanto durante a terapia de indução como na manutenção. Além disso, todos estes estudos iniciais não foram capazes de detectar qualquer índice prognóstico capaz de prever a evolução dos pacientes. No sentido de reduzir as taxas de recidiva, o transplante autólogo de célula progenitora hematopoética em pacientes em remissão completa foi introduzido. Os resultados obtidos com esta abordagem foram bastante homogêneos, indicando uma probabilidade de sobrevida livre de doença de 65%-75% e uma sobrevida global de 60%. Sucessivos tratamentos desenhados já nos anos 2000, foram capazes de obter remissões completas acima de 90%, com taxas de recidivas da ordem de 30% e uma sobrevida global comparável à obtida com o transplante. Ainda, estes estudos também não foram capazes de detectar fatores prognósticos relacionados à evolução válidos. Mais ainda, qualquer estudo com perfil biológico foi desenvolvido. Para melhorar o prognóstico do LLB-T parece ser necessário esforço multicêntrico, de caráter nacional ou internacional, para coletar dados clínicos e biológicos. Nesta linha, é possível alcançar número crítico de dados com valor estatístico que poderiam ser capazes de detectar fatores com influência prognóstica. Finalmente, grupos de pacientes necessitam ser identificados para selecionar aqueles que poderiam se beneficiar do transplante de célula progenitora hematopoética detectados ao diagnóstico

2003-07

Background and Objectives. B-cell chronic lymphocytic leukemia (B-CLL) results from the accumulation of monoclonal CD5 + B cells. Despite its homogeneity at cellular level, B-CLL is clinically heterogeneous. Clinical studies indicate that CD38 + B-CLL are characterized by a more aggressive clinical course than are CD38 − B-CLL. On the basis of these studies and considering the established correlation between specific chromosome aberrations and the clinical course of B-CLL, it is possible that CD38 + B-CLL cases are also characterized by specific subsets of chromosomal alterations. Design and Methods. Comparative genomic hybridization (CGH) was performed on purified B-cells from peripheral blood of 52 patients with B-CLL in order to detect chromosome imbalance. The immunophenotype of the patients, including CD38 expression, was also determined by flow cytometry. The results of CGH experiments were then compared with CD38 expression. Results. We found a clear correlation between the presence of chromosomal imbalances and CD38 expression: 13/16 CD38 + cases had chromosome imbalances, most of them (12/13) correlated with a poor prognosis. Among the CD38 − B-CLL patients, only 8/36 displayed chromosome imbalances; the only three cases with loss in 13q as a single aberration, considered a good prognostic marker, were in this group. Moreover, we found that cytogenetic alterations were also more complex in the CD38 + B-CLL subset, since 9/10 with two or more aberrations were in the CD38 + group. Interpretation and Conclusions. Collectively, the data reinforce the value of CD38 as a prognostic factor and indicate that genotypic/phenotypic features distinguish B-CLL subsets. Key words: B-CLL, molecular cytogenetics, chromosome aberrations, immunophenotype, CD38. from the progressive accumulation of monoclonal CD5-positive B cells. Despite its homogeneity at cellular level, B-CLL is clinically heterogeneous since some patients survive for a long time without therapy, while others progress towards more advanced stages and die despite aggressive treatment. Design and Methods Clinical features of patients Fifty-two patients with B-CLL (32 males and 20 females) were studied. Their characteristics are described in n.a. data not available; *from diagnosis. © F e r r a t a S t o r t i F o u n d a t i o n haematologica/journal of hematology vol. 88 CGH To increase the sensitivity of the CGH, B cells were purified from PBMC by removing monocytes and CD3 + T cells by adherence to plastic surfaces and magnetic beads, respectively. DNA extraction and CGH were performed as described elsewhere. Digital image analysis Image acquisition, processing, and evaluation were performed as described elsewhere. Results Identification of two groups of B-CLL according to CD38 expression The purified malignant B-CLL cells from 52 patients were double stained with CD38 and CD19 monoclonal antibodies. Two groups of B-CLL, i.e. CD38 + and CD38 − B-CLL, were identified using the cut-off limit of 30%, already utilized in previous studies. CGH analysis Chromosome imbalances were detected by CGH. Two examples of merged CGH images and the relative mean profiles of ten metaphases with chromosomal imbalances are shown in Discussion Samples from 52 patients with typical B-CLL, diagnosed according to morphology and surface phenotype, were subjected to CGH analysis in order to obtain a comprehensive view of chromosomal gains and losses and to identify copy number aberrations specific for this pathology. To increase the sensitivity of CGH, we purified B-cells from the peripheral blood of B-CLL patients when there were less than 90% B cells. Twenty-one out of 52 (40%) patients showed chromosome imbalances; 11/21 had single imbalances, whereas the remaining 10 patients had two or more chromosome alterations. Thirty-three per cent of patients had received chemotherapy before cytogenetic analysis. The presence of patients subjected to therapy in the cohort can hardly be avoided in this kind of study. As an example, a recent study based on a large cohort of patients included a similar percentage of treated patients as did our study. Imbalances involving chromosomes 11, 12, 13 and 17 are among the most important factors in predicting survival: patients with 17p deletions are those with the worst prognosis, followed by patients with 11q deletion, those with 12q trisomy, and those with normal karyotypes, whereas patients with 13q deletions as the sole abnormality have the longest estimated survival times. This finding is possibly explained by the fact that our cohort included only patients with typical morphology/immunophenotype and that there were fewer patients with advanced stage disease. It has been demonstrated that atypical morphology and advanced stage disease correlate with loss in 17p. The B-CLL cases in this study could be subdivided into two groups according to the surface expression of CD38 by the malignant cells. This confirms previous findings from our laboratory and is also in line with data reported by others. The striking finding of this study was the clear correlation existing between the presence of chromosomal abnormalities and CD38 expression by the malignant cells. Thirteen out of 16 CD38 + patients also had chromosomal abnormalities, whereas, among the 36 CD38 − patients only 8 displayed chromosomal imbalances. These differences are highly significant (p=0.0001). Three out of the 8 CD38 − patients with chromosomal alterations had a loss in 13q as a single aberration, which generally correlates with a good prognosis, 3 patients had rare alterations, the prognostic value of which remains to be determined, while the remaining 2 patients had aberrations correlated with a poor prognosis (-11q; +12). Twelve of 13 CD38 + patients with chromosomal alterations displayed aberrations that are correlated with a poor clinical outcome (-11q; +12; -17p), whereas one patient had a gain in © F e r r a t a S t o r t i F o u n d a t i o n Chromosome aberrations and CD38 expression in B-CLL haematologica/journal of hematology vol. 88(07):july 2003 775 chromosome 3q, which is rarely found in B-CLL and is hence of undetermined prognostic value. Moreover, of the 10 patients with two or more chromosome imbalances (another marker of poor prognosis) detected in this study, 9 were within the CD38 + group. Remarkably, among the cases with the highest values of CD38 expression, 3 cases had simultaneous gain of chromosomes 12 and 18. Gain of chromosome 18 never appeared alone, but was always associated with gain of chromosome 12. Although this association has already been described in B-CLL by classical cytogenetic studies, 24,40 its significance and real frequency are not well documented. This is, in part, because most of the studies on chromosomal aberrations in large cohorts of B-CLL patients were performed with FISH using a panel of probes not including the chromosome 18 probe. Our data are in keeping with the recent observations that the unbalanced distribution of genomic aberrations in IgVH high mutation and low mutation subgroups might point toward a distinct biological background in such B-CLL subgroups and may in part, explain their different behaviors. 14 In the study by Kröber et al., 14 genomic aberrations and VH mutation status appeared to have a complementary role in estimating prognosis. Although CD38 expression has been proposed as an easily performed surrogate of VH mutational status analysis 4 its prognostic value is not completely clarified. Moreover, the relationship between CD38 expression and chromosomal aberrations has not been extensively studied. In a recent paper, Chevallier et al. Concerning the prognostic significance of CD38 expression in multivariate analysis, the authors suggested that a much larger group of patients was needed. Oscier et al. 36 showed that the mean survival of patients with loss in 17p was the shortest (47 months). In the present study the groups of patients with and without chromosomal alterations do not differ in terms of survival (not shown) probably since all but 2 patients are still alive. However, the simple patient in our cohort with loss in 17p at diagnosis was followed for only 1 year and was experiencing a poor clinical course. The different biological properties showed by B-CLL cells, including the expression of CD38, can help to explain the differences in the patients&apos; outcomes. Recent studies, including those from our laboratory, 7,41 demonstrated that CD38 + B-CLL cells with unmutated VH/VL region genes have a viable IgM initiated signal transduction pathway. This pathway can lead to proliferation/differentiation or apoptosis depending on co-signals received by the cells in vitro. In contrast, most of the CD38 − mutated B-CLL cells do not respond to signals delivered to surface Ig. Therefore, the interaction between the cells and the environment via B-cell receptor is much less marked in CD38 − mutated cases than in CD38 + unmutated cases. These data suggest that CD38 + B-CLL cells are likely to be continuously stimulated via surface Ig. This is related to the fact that surface Ig, encoded by unmutated VH/VL genes, retain natural antibody activity and hence can react continuously with autoantigens in vivo. In the case of surface CD38-negative, mutated B-CLL cells, it is unlikely that the B-cell receptor can exert a promoting role on cell expansion since there is a not a viable IgM signal transducing pathway. Moreover, Ig encoded by mutated VH/VL genes rarely have natural antibody activity and, therefore, can rarely encounter the appropriate foreign antigen. Collectively, these considerations raise the issue of whether antigenic stimulation in B-CLL continues to exert a promoting effect on the growth of the malignant cells following transformation, and whether this is the reason for the clinical differences in B-CLL. Finally, it is unlikely that CD38 is solely a marker of cellular differentiation and clinical course. It is more probable that it also functions as a signaling molecule and, therefore may be directly involved in differences in disease severity. CD38 is known to play a role as an accessory molecule in B-cell receptor mediated signal transduction, 42,43 as well as regulating cell apoptosis in certain normal B-cell subsets. A number of conclusions can be drawn from this study. First, considering the increasing recognition of the importance of chromosome alterations in predicting the clinical outcome of B-CLL, the observation that chromosomal alterations are significantly more frequent within the CD38-positive cases lends further support to the prognostic value of the surface marker, CD38. Second, the finding that CD38 + cases can be subdivided into two groups (i.e. with and without chromosomal alterations) may lead to the delineation of further prognostic subsets of B-CLL. Third, the paucity of chromosomal alter- © F e r r a t a S t o r t i F o u n d a t i o