Mutation Profile Assessed by Next-Generation Sequencing (NGS) of Circulating Tumor DNA (ctDNA) in Chinese Lung Adenocarcinoma Patients: Analysis of Real-World Data

Background. Genomic testing gives guidance to the treatment options in lung adenocarcinoma patients, but some patients are unable to obtain tissue samples due to lesion location or intolerance. Cell-free circulating tumor DNA (ctDNA) tested in plasma or pleural effusion is an advanced access to solve the problem. Our study descriptively identified the genetic variations of advanced Chinese lung adenocarcinoma patients and analyzed the overall survival of patients with EGFR mutations. Methods. A total of 152 patients’ plasma samples were included, and gene mutations were detected by NGS using an Illumina Miseq tabletop sequencer. Results. Frequencies of altered were EGFR 46.05%, ALK 7.24%, KRAS 6.58%, PIK3CA 6.58%, PTEN 2.63%, HER2 1.97%, MET 1.97%, BRAF 1.32%, NF1 1.32%, and ROS1 0.66%. We identified 48 cases with double or triple driver gene mutations. Multiple mutations were more frequently observed in EGFR and PIK3CA genes. Patients harboring coexistent mutations with an EGFR mutation tended to have a shorter overall survival than those with exclusively EGFR mutations. Conclusion. EGFR, ALK, and KRAS were common driver gene in Chinese patients with stage IV lung adenocarcinoma. Multiple mutations were detected in the ctDNA samples and involve more EGFR and PIK3CA mutations. The existence of coexisting gene mutations may have adverse effects on the prognosis of patients with EGFR mutation. The unknown mutations discovered by NGS may provide new targets for gene targeting therapy, and ctDNA test by NGS is an effective method for making appropriate treatment choices

Circulating CCR7+ICOS+ Memory T Follicular Helper Cells in Patients with Multiple Sclerosis.

This study is aimed at examining the potential roles of circulating memory T follicular helper (Tfh) cells in patients with multiple sclerosis (MS).The numbers of different subsets of circulating memory Tfh cells in 25 patients with relapsed MS before and after treatment as well as 14 healthy controls (HC) were examined by flow cytometry. The levels of plasma IL-21 in all patients and cerebrospinal fluid (CSF) IL-21 in some MS patients and controls with non-inflammatory neuronal diseases (NND) were measured by ELISA.In comparison with that in the HC, the numbers of circulating CD3+CD4+CXCR5+CD45RA-, ICOS+, CCR7+ and CCR7+ICOS+ memory Tfh cells and the levels of plasma IL-21 significantly increased in MS patients, but significantly decreased in the patients with complete remission (CR). The levels of CSF IL-21 were significantly higher in the MS patients than that in the NND patients. The numbers of CCR7+ICOS+ memory Tfh cells were positively correlated with the EDSS scores, the levels of plasma and CSF IL-21, IgG, MBP-Ab or MOG-Ab.Our findings indicated that circulating memory Tfh cells, especially CCR7+ICOS+ memory Tfh cells, may be associated with the relapse of MS and may serve as a new therapeutic target

Analysis of plasma and CSF IL-21 in MS patients before and after treatment.

<p>The levels of plasma and CSF IL-21 in individual subjects were tested by ELISA. (A) The levels of plasma IL-21 in the HC, MS patients pre- and post-treatment. (B) The levels of plasma IL-21 in the MS-CR patients pre- and post- treatment. (C) The levels of plasma IL-21 in the MS-PR patients pre- and post- treatment. (D) The levels of CSF IL-21 in the MS patients pre-treatment and NND patients. Data are expressed as the mean values of individual samples. The horizontal lines represented the median values.</p

Correlation analysis of the numbers of different subsets of circulating memory Tfh cells and the values of clinical measures in MS patients.

<p>(A-B) The numbers of ICOS+ and CCR7+ICOS+ memory Tfh cells were positively associated with the EDSS scores in MS patients. (C-D) The numbers of ICOS+ and CCR7+ICOS+ memory Tfh cells were positively associated with the levels of plasma IL-21. (E-F) The numbers of CCR7+ICOS+ memory Tfh cells were positively correlated with the levels of CSF IL-21 and CSF IgG. (G-H) The numbers of CCR7+ICOS+ memory Tfh cells were positively correlated with the levels of CSF MBP-Ab and CSF MOG-Ab.</p

A Lower Proportion of Regulatory B Cells in Patients with Henoch–Schoenlein Purpura Nephritis

<div><p>Background</p><p>Henoch—Schoenlein purpura is the one of most common types of systemic vasculitis that involves impaired renal function and Henoch-Schoenlein purpura nephritis (HSPN). The diagnosis of this condition is largely based on immunohistologic detection of immunoglobulin A1-containing immune complex in the glomerular deposits of mesangium. Despite clinical advances, the etiopathogenesis of HSPN is still largely unknown.</p><p>Methods</p><p>In this study, we enrolled 25 newly diagnosed HSPN patients and 14 healthy controls. Then, fractions of B cell subtypes were determined in venous blood using flow cytometry. The serum interleukin (IL)-10 concentration was determined by enzyme-linked immunosorbent assay.</p><p>Results</p><p>Compared to those in healthy controls, the numbers of CD38⁺CD19⁺, CD86⁺CD19⁺, CD38⁺CD86⁺CD19⁺, and CD95⁺CD19⁺ B cells per microliter of blood were significantly higher in HSPN patients. In contrast, the numbers of CD5⁺CD19⁺, IL-10⁺CD19⁺, CD5⁺CD1d⁺CD19⁺, and IL-10⁺CD5⁺CD1d⁺CD19⁺ B cells per microliter of blood and the serum IL-10 concentration were significantly lower in HSPN patients. Following treatment, the numbers of CD38⁺CD19⁺ and CD86⁺CD19⁺ B cells per microliter of blood were significantly reduced in HSPN patients. However, the numbers of CD5⁺CD1d⁺CD19⁺, CD5⁺CD1d⁺IL-10⁺CD19⁺, and IL-10⁺CD19⁺ B cells per microliter of blood and the serum IL-10 concentration were significantly increased in HSPN patients following treatment. The estimated glomerular filtration rate (eGFR) was negatively correlated with the number of CD38⁺CD19⁺ B cells but positively correlated with the numbers of IL-10⁺CD19⁺, CD1d⁺CD5⁺CD19⁺, and IL-10⁺CD1d⁺CD5⁺CD19⁺B cells per microliter of blood and the serum IL-10 concentration. The 24-h urinary protein concentration was positively correlated with the number of CD38⁺CD19⁺B cells but negatively correlated with the numbers of IL-10⁺CD19⁺, CD1d⁺CD5⁺CD19⁺, and IL-10⁺CD1d⁺CD5⁺CD19⁺B cells per microliter of blood and the serum IL-10 concentration.</p><p>Conclusion</p><p>Our results suggest that CD38⁺CD19⁺ and CD1d⁺CD5⁺CD19⁺ B cells (Bregs) contribute to the pathogenesis of HSPN.</p></div

FACS analysis of the numbers of circulating CCR7+ and CCR7+ICOS+ memory Tfh cells in individual subjects.

<p>After being stained with different fluorescent antibodies, the CD3+CD4+CXCR5+CD45RA- T cells were gated. Subsequently, memory Tfh cells were gated on CCR7 expression and the frequency of CCR7+, CCR7+ICOS+, CCR7- and CCR7-ICOS+ memory Tfh cells was analyzed by flow cytometry and the numbers of each type of cells were calculated. (A) Flow cytometry analysis. (B) The numbers of CCR7+ memory Tfh cells in the HC, MS patients pre- and post-treatment. (C) The numbers of CCR7+ memory Tfh cells in the MS-CR patients pre- and post- treatment. (D) The numbers of CCR7+ memory Tfh cells in the MS-PR patients pre- and post-treatment. (E) The numbers of CCR7+ICOS+ memory Tfh cells in the HC, MS patients pre- and post-treatment. (F) The numbers of CCR7+ICOS+ memory Tfh cells in the MS-CR patients pre- and post-treatment. (G) The numbers of CCR7+ICOS+ memory Tfh cells in the MS-PR patients pre- and post-treatment. (H) The numbers of CCR7- memory Tfh cells in the HC, MS patients pre- and post-treatment. (I) The numbers of CCR7- memory Tfh cells in the MS-CR patients pre- and post- treatment. (J) The numbers of CCR7- memory Tfh cells in the MS-PR patients pre- and post-treatment. (K)The numbers of CCR7-ICOS+ memory Tfh cells in the HC, MS patients pre- and post-treatment. (L) The numbers of CCR7-ICOS+ memory Tfh cells in the MS-CR patients pre- and post-treatment. (M) The numbers of CCR7-ICOS+ memory Tfh cells in the MS-PR patients pre- and post-treatment. There was no significant difference in the numbers of CCR7-PD-1+, CCR7-PD-1+ICOS+, CCR7-CD40L+, CCR7+PD-1+, CCR7+PD-1+ICOS+, CCR7+CD40L+ memory Tfh cells between the patients and HC as well as in MS patients before and after treatment (data not shown). The horizontal lines indicate the median values for each group.</p

The demographic and clinical features of MS patients and HC.

<p>Data shown are median and range, except specified. ARR: Annual relapse rate; EDSS: Expanded disability status scale; MS: Multiple sclerosis; WBC: White blood cells. Normal values: WBC, 3.5–9.5 ×10⁹/L; Lymphocytes, 1.1–3.2 ×10⁹/L.</p><p>The demographic and clinical features of MS patients and HC.</p

FACS analysis of the numbers of circulating memory Tfh cells and ICOS+ memory Tfh cells in individual subjects.

<p>PBMCs were isolated from individual subjects and stained with different fluorescent antibodies. The cells were gated sequentially on living lymphocytes, CD3+ and CD4+, and then on CXCR5+ and CD45RA- cells. The frequency of CD3+CD4+CXCR5+CD45RA- (memory) Tfh cells was determined. Subsequently, memory Tfh cells were gated on ICOS and the frequency of memory Tfh and ICOS+ memory Tfh cells in lymphocytes was analyzed and the numbers of each type of cells in total lymphocytes per liter were calculated. (A) Flow cytometry analysis. (B) The numbers of memory Tfh cells in the HC, MS patients pre- and post-treatment. (C) The numbers of memory Tfh cells in the MS-CR patients pre- and post-treatment. (D) The numbers of memory Tfh cells in the MS-PR patients pre- and post-treatment. (E) The numbers of ICOS+ memory Tfh cells in the HC, MS patients pre- and post-treatment. (F) The numbers of ICOS+ memory Tfh cells in the MS-CR patients pre- and post-treatment. (G) The numbers of ICOS+ memory Tfh cells in the MS-PR patients pre- and post-treatment. There was no significant difference in the numbers of PD-1+, PD-1+ICOS+ and CD40L+ memory Tfh cells between the patients and HC as well as in MS patients pre- and post-treatment (data not shown). The horizontal lines indicate the median values for each group.</p

The demographic and clinical features of 12 MS patients after treatment.

<p>Data shown are median and range, except specified. CR: Complete remission; CSF: Cerebrospinal fluid; PR: Partial remission; WBC: White blood cells. Normal values: CSF WBC, 0–8 ×10⁶/L; CSF proteins, 0.15–0.45 g/L; CSF IgG, 0–34 mg/l. *P<0.05 vs. the HC.</p><p>The demographic and clinical features of 12 MS patients after treatment.</p

The demographic and clinical features of MS patients and NND who received a lumbar puncture.

<p>Data shown are median and range, except specified. CSF: Cerebrospinal fluid; MBP-Ab: Antibodies against myelin basic protein; MOG-Ab: Antibodies against myelin oligodendrocyte glycopotein; NND: Non-inflammatory neurological diseases; OD: Optical density; WBC: White blood cell. Normal values: CSF WBC, 0–8 ×10⁶/L; CSF proteins, 0.15–0.45 g/L; CSF IgG, 0–34 mg/l; Plasma MBP-Ab < 0.75; CSF MBP-Ab < 0.65; Plasma MOG-Ab < 0.64; CSF MOG-Ab < 0.56. *P<0.05 vs. the HC.</p><p>The demographic and clinical features of MS patients and NND who received a lumbar puncture.</p