Exploring the impact of tertiary lymphoid structures maturity in NSCLC: insights from TLS scoring.

Tertiary Lymphoid Structures (TLS) are lymphoid structures commonly associated with improved survival of cancer patients and response to immunotherapies. However, conflicting reports underscore the need to consider TLS heterogeneity and multiple features such as TLS size, composition, and maturation status, when assessing their functional impact. With the aim of gaining insights into TLS biology and evaluating the prognostic impact of TLS maturity in Non-Small Cell Lung Carcinoma (NSCLC), we developed a multiplex immunofluorescent (mIF) panel including T cell (CD3, CD8), B cell (CD20), Follicular Dendritic cell (FDC) (CD21, CD23) and mature dendritic cell (DC-LAMP) markers. We deployed this panel across a cohort of primary tumor resections from NSCLC patients (N=406) and established a mIF image analysis workstream to specifically detect TLS structures and evaluate the density of each cell phenotype. We assessed the prognostic significance of TLS size, number, and composition, to develop a TLS scoring system representative of TLS biology within a tumor. TLS relative area, (total TLS area divided by the total tumor area), was the most prognostic TLS feature (C-index: 0.54, p = 0.04). CD21 positivity was a marker driving the favorable prognostic impact, where CD21+ CD23- B cells (C-index: 0.57, p = 0.04) and CD21+ CD23- FDC (C-index: 0.58, p = 0.01) were the only prognostic cell phenotypes in TLS. Combining the three most robust prognostic TLS features: TLS relative area, the density of B cells, and FDC CD21+ CD23- we generated a TLS scoring system that demonstrated strong prognostic value in NSCLC when considering the effect of age, sex, histology, and smoking status. This TLS Score also demonstrated significant association with Immunoscore, EGFR mutational status and gene expression-based B-cell and TLS signature scores. It was not correlated with PD-L1 status in tumor cells or immune cells. In conclusion, we generated a prognostic TLS Score representative of the TLS heterogeneity and maturity undergoing within NSCLC tissues. This score could be used as a tool to explore how TLS presence and maturity impact the organization of the tumor microenvironment and support the discovery of spatial biomarker surrogates of TLS maturity, that could be used in the clinic.

Abnormal subcortical deep-gray matter susceptibility-weighted imaging filtered phase measurements in patients with multiple sclerosis: a case-control study.

OBJECTIVE: To investigate abnormal phase on susceptibility-weighted imaging (SWI)-filtered phase images indicative of iron content, in subcortical deep-gray matter (SDGM) of multiple sclerosis (MS) patients and healthy controls (HC), and to explore its relationship with MRI outcomes. METHODS: 169 relapsing-remitting (RR) and 64 secondary-progressive (SP) MS patients, and 126 age- and sex-matched HC were imaged on a 3T scanner. Mean phase of the abnormal phase tissue (MP-APT), normal phase tissue volume (NPTV) and normalized volume were determined for total SDGM, caudate, putamen, globus pallidus, thalamus, pulvinar nucleus of thalamus (PVN), hippocampus, amygdala, nucleus accumbens, red nucleus and substantia nigra. 63 HC were used for establishment of normal reference phase values, while additional 63 HC were used for blinded comparisons with MS patients. RESULTS: Increased MP-APT, decreased normalized volume and decreased NPTV were detected in total SDGM, caudate, putamen, globus pallidus, thalamus and PVN in MS patients compared to HC (p<.0004). MS patients also showed decreased volume in hippocampus (<.0001) and decreased NPTV in the hippocampus, amygdala and accumbens (<.0004). SPMS patients had increased MP-APT, decreased volume and decreased NPTV in total SDGM, caudate and amygdala compared to RRMS (p<.005), while individual measure differences were also detected in putamen, thalamus, hippocampus and accumbens (p<.006). RRMS patients showed a significant relationship between increased MP-APT and increased lesion burden and more advanced brain atrophy (p<.004). CONCLUSIONS: Abnormal phase, indicative of higher iron content was significantly increased in MS patients compared to HC, and was related to more severe lesion burden and brain atrophy.

Iron deposition in multiple sclerosis lesions measured by susceptibility-weighted imaging filtered phase: a case control study.

PURPOSE: To investigate phase lesions identified on susceptibility-weighted imaging (SWI)-filtered phase images in patients with multiple sclerosis (MS), clinically isolated syndrome (CIS) and healthy controls (HC). To relate phase lesion characteristics to other clinical and MRI outcomes. MATERIALS AND METHODS: 95 relapsing-remitting (RR), 40 secondary-progressive (SP) MS patients, as well as 19 CIS patients and 49 age- and sex-matched HC, were scanned on a 3T scanner. Phase-, T1-, and T2-lesion characteristics were determined. Overlap of T1- and T2-weighted imaging (WI) lesions with phase lesions (T1P and T2P), as well as brain atrophy outcomes, was assessed. RESULTS: MS patients showed significantly greater numbers and larger volume of phase lesions, compared with HC (P < 0.001). 23.6% of T2 lesions overlapped with phase lesions, whereas the same figure for T1 lesions was 37.3%. Conversely, 33.4% and 69.7% of phase lesions were not visible on T2- or T1-WI, respectively. Phase, T1P and T2P lesions were not related to clinical outcomes, but phase lesions were related to ventricular enlargement. CONCLUSION: Phase lesions were present in both MS and CIS patients, and showed partial overlap with lesions observed using conventional MRI. The role of phase lesions in clinical progression remains unclear and should be further explored.

Vitamin D metabolites are associated with clinical and MRI outcomes in multiple sclerosis patients.

PURPOSE: The associations between vitamin D and MRI measures of brain tissue injury have not been previously investigated in multiple sclerosis (MS). This research evaluates the significance of vitamin D and its active metabolites in brain tissue injury and clinical disability in MS patients. METHODS: The study population consisted of 193 MS patients (152 women and 41 men; mean age 46.1 (SD 8.4) years; disease duration 13.8 (SD 8.4) years). Serum levels of 25-hydroxyvitamin D(3) (25(OH)VD(3)), 25-hydroxyvitamin D(2) (25(OH)VD(2)), 1α, 25-dihydroxyvitamin D(3) (1, 25(OH)(2)VD(3)) and 24(R), 25-dihydroxyvitamin D(3) (24, 25(OH)(2)VD(3)) were measured using a novel capillary liquid-chromatography-mass spectrometry method. Disability was assessed with the Expanded Disability Status Scale (EDSS) and the MS Severity Scale (MSSS). MRI measures included T2 lesion volume (LV), T1-LV and brain parenchymal fraction. The associations between deseasonalised levels of vitamin D metabolites and clinical and MRI measurements were assessed using regression analyses. RESULTS: Lower deseasonalised levels of total 25(OH)VD (p=0.029), 25(OH)VD(3) (p=0.032) and 24, 25(OH)(2)VD(3) (p=0.005) were associated with higher MSSS. Similarly, lower deseasonalised levels of 24, 25(OH)(2)VD(3) (p=0.012) were associated with higher EDSS. Higher values of the 25(OH)VD(3) to 24, 25(OH)(2)VD(3) ratio were associated with higher MSSS (p=0.041) and lower brain parenchymal fraction (p=0.008). CONCLUSIONS: Vitamin D metabolites have protective associations with disability and brain atrophy in MS. In particular, the results indicate strong associations for the 24, 25(OH)(2)VD(3) metabolite, which has not been extensively investigated in MS patients.

Associations between changes in ferritin levels and susceptibility-weighted imaging filtered phase in patients with relapsing-remitting multiple sclerosis over 24 weeks of therapy with subcutaneous interferon beta-1a three times weekly.

Subanalysis of a pilot study (NCT01085318) assessed correlations between serum ferritin and imaging assessments in relapsing-remitting multiple sclerosis patients (n = 23) receiving 44 µg interferon beta-1a subcutaneously three times weekly. At baseline, 12, and 24 weeks, mean ferritin was 75, 127 (p < 0.001 vs baseline), and 101 (p=0.020 vs baseline) ng/mL. No relationship between ferritin and susceptibility-weighted imaging (SWI)-filtered phase of subcortical deep gray matter was found. Increasing ferritin correlated with decreasing lesion numbers on both fluid attenuated inversion recovery and SWI phase at 12 weeks (r = -0.62; p = 0.003; n = 21), and with decreasing gadolinium-enhancing lesion volume at 24 weeks (r = -0.71; p = 0.050; n = 8).

A pilot, longitudinal, 24-week study to evaluate the effect of interferon beta-1a subcutaneous on changes in susceptibility-weighted imaging-filtered phase assessment of lesions and subcortical deep-gray matter in relapsing-remitting multiple sclerosis.

BACKGROUND: Studies have shown a relationship between increased iron content and clinical progression, cognitive impairment, and brain atrophy in patients with multiple sclerosis. Altered phase, as determined by susceptibility-weighted imaging (SWI), can potentially capture iron content changes. OBJECTIVE: The objective of this study was to investigate phase changes in white matter (WM) lesions and subcortical deep-gray matter (SDGM) of patients with relapsing-remitting (RR) MS treated with interferon beta-1a administered subcutaneously versus untreated healthy controls (HCs). METHODS: We conducted a 24-week, nonrandomized, open-label pilot study of 23 patients with RRMS receiving interferon beta-1a administered subcutaneously and 15 HCs. Patients were imaged on a 3T scanner at baseline, 12, and 24 weeks; changes in phase behavior in WM lesions and regional SDGM [mean phase of low-phase voxels (MP-LPV)], and in SDGM volumes, were measured. Between- and within-group changes were tested using nonparametric statistics adjusted for multiple comparisons. RESULTS: The number (p = 0.003) and volume (p < 0.001) of phase WM lesions both significantly decreased among RRMS patients over 24 weeks. At baseline, MP-LPV was lower (suggestive of greater iron content) in total SDGM among RRMS patients versus HCs (p = 0.002). Week 24 MP-LPV changes from baseline were not significantly different between groups in total SDGM or any region except the putamen (-0.0025 radians in RRMS patients versus 0.0035 radians in HCs; p = 0.041). CONCLUSIONS: Over 24 weeks, phase lesions were reduced significantly in the RRMS group. These preliminary results suggest that SWI-filtered phase may become a useful tool for monitoring RRMS disease activity.

Genome activation in bovine embryos: review of the literature and new insights from RNA sequencing experiments.

Maternal-to-embryonic transition (MET) is the period in early embryonic development when maternal RNAs and proteins stored in the oocyte are gradually degraded and transcription of the embryonic genome is activated. First insights into the timing of embryonic genome activation (EGA) came from autoradiographic analyses of embryos following incorporation of [(3)H]uridine. These studies identified the eight- to 16-cell stage of bovine embryos as the period of major EGA, but detected first transcriptional activity already in one-cell embryos. Subsequent studies compared the transcriptome profiles of untreated embryos and of embryos incubated with the transcription inhibitor α-amanitin to reveal transcripts of embryonic origin. In addition, candidate gene-based and global gene expression studies over several stages of early development were performed and characteristic profiles were revealed. However, the onset of embryonic transcription was obscured by the presence of maternal transcripts and could only be determined for genes which are not expressed in oocytes. Using RNA sequencing of bovine germinal vesicle and metaphase II oocytes, and of four-cell, eight-cell, 16-cell and blastocyst stage embryos, we established the most comprehensive transcriptome data set of bovine oocyte maturation and early development. EGA was analyzed by (i) detection of embryonic transcripts which are not present in oocytes; (ii) detection of transcripts from the paternal allele; and (iii) detection of primary transcripts with intronic sequences. Using these three approaches we were able to map the onset of embryonic transcription for almost 7400 genes. Genes activated at the four-cell stage or before were functionally related to RNA processing, translation, and transport, preparing the embryo for major EGA at the eight-cell stage, when genes from a broad range of functional categories were found to be activated. These included transcriptional and translational functions as well as protein ubiquitination. The functions of the genes activated at the 16-cell stage were consistent with ongoing transcription and translation, while the genes activated in blastocysts included regulators of early lineage specification. Fine mapping of EGA provides a new layer of information for detecting disturbances of early development due to genetic, epigenetic, and environmental factors.

Gray matter SWI-filtered phase and atrophy are linked to disability in MS.

The association between clinical outcomes and abnormal susceptibility-weighted imaging (SWI)-filtered phase, indicative of increased iron content, as well as atrophy, was investigated in the subcortical deep-gray matter (SDGM) of multiple sclerosis (MS) patients. 149 relapsing-remitting (RR) and 61 secondary-progressive (SP) MS patients underwent SWI on a 3T scanner. Mean phase of the abnormal phase tissue (MP-APT) and normalized volumes were determined for the total and region-specific SDGM structures. In an age- and gender-adjusted regression model, total SDGM volume was the strongest predictor of Expanded Disability Status Scale (EDSS) (beta = -.224, p<.001), followed by total SDGM MP-APT (beta = -.168, p <.019). This model accounted for 30.4% of the variance in EDSS. Only SDGM MP-APT added additional variance in predicting EDSS, compared to conventional MRI metrics. Caudate and red nucleus MP-APT and amygdala volume were associated with EDSS. Our findings suggest that disability in MS patients is associated better with SDGM pathology, as indicated by increased iron content and atrophy, than with lesion burden or white matter and cortical volumes.

Basal ganglia, thalamus and neocortical atrophy predicting slowed cognitive processing in multiple sclerosis.

Information-processing speed (IPS) slowing is a primary cognitive deficit in multiple sclerosis (MS). Basal ganglia, thalamus and neocortex are thought to have a key role for efficient information-processing, yet the specific relative contribution of these structures for MS-related IPS impairment is poorly understood. To determine if basal ganglia and thalamus atrophy independently contribute to visual and auditory IPS impairment in MS, after controlling for the influence of neocortical volume, we enrolled 86 consecutive MS patients and 25 normal controls undergoing 3T brain MRI and neuropsychological testing. Using Sienax and FIRST software, neocortical and deep gray matter (DGM) volumes were calculated. Neuropsychological testing contributed measures of auditory and visual IPS using the Paced Auditory Serial Addition Test (PASAT) and the Symbol Digit Modalities Test (SDMT), respectively. MS patients exhibited significantly slower IPS relative to controls and showed reduction in neocortex, caudate, putamen, globus pallidus, thalamus and nucleus accumbens volume. SDMT and PASAT were significantly correlated with all DGM regions. These effects were mitigated by controlling for the effects of neocortical volume, but all DGM volumes remained significantly correlated with SDMT, putamen (r = 0.409, p < 0.001) and thalamus (r = 0.362, p < 0.001) having the strongest effects, whereas for PASAT, the correlation was significant for putamen (r = 0.313, p < 0.01) but not for thalamus. We confirm the significant role of thalamus atrophy in MS-related IPS slowing and find that putamen atrophy is also a significant contributor to this disorder. These DGM structures have independent, significant roles, after controlling for the influence of neocortex atrophy.