Expansion of KRAS hotspot mutations reactive T cells from human pancreatic tumors using autologous T cells as the antigen-presenting cells.

Adoptive cell therapy (ACT) with expanded tumor-infiltrating lymphocytes (TIL) or TCR gene-modified T cells (TCR-T) that recognize mutant KRAS neo-antigens can mediate tumor regression in patients with advanced pancreatic ductal adenocarcinoma (PDAC) (Tran et al in N Engl J Med, 375:2255-2262, 2016; Leidner et al in N Engl J Med, 386:2112-2119, 2022). The mutant KRAS-targeted ACT holds great potential to achieve durable clinical responses for PDAC, which has had no meaningful improvement over 40 years. However, the wide application of mutant KRAS-centric ACT is currently limited by the rarity of TIL that recognize the mutant KRAS. In addition, PDAC is generally recognized as a poorly immunogenic tumor, and TILs in PDAC are less abundant than in immunogenic tumors such as melanoma. To increase the success rate of TIL production, we adopted a well-utilized K562-based artificial APC (aAPC) that expresses 4-1BBL as the costimulatory molecules to enhance the TIL production from PDCA. However, stimulation with K562-based aAPC led to a rapid loss of specificity to mutant KRAS. To selectively expand neo-antigen-specific T cells, particularly mKRAS, from the TILs, we used tandem mini gene-modified autologous T cells (TMG-T) as the novel aAPC. Using this modified IVS protocol, we successfully generated TIL cultures specifically reactive to mKRAS (G12V). We believe that autologous TMG-T cells provide a reliable source of autologous APC to expand a rare population of neoantigen-specific T cells in TILs.

Single-cell RNA-seq reveals the genesis and heterogeneity of tumor microenvironment in pancreatic undifferentiated carcinoma with osteoclast-like giant-cells.

BACKGROUND: Undifferentiated carcinoma with osteoclast-like giant cells (OGCs) of pancreas (UCOGCP) is a rare subtype of pancreatic ductal adenocarcinoma (PDAC), which had poorly described histopathological and clinical features. METHODS: In this study, single-cell RNA sequencing (scRNA-seq) was used to profile the distinct tumor microenvironment of UCOGCP using samples obtained from one UCOGCP patient and three PDAC patients. Bioinformatic analysis was carried out and immunohistochemical (IHC) staining was used to support the findings of bioinformatic analysis. After quality control of the raw data, a total of 18,376 cells were obtained from these four samples for subsequent analysis. These cells were divided into ten main cell types following the Seurat analysis pipeline. Among them, the UCOGCP sample displayed distinct distribution patterns from the rest samples in the epithelial cell, myeloid cell, fibroblast, and endothelial cell clusters. Further analysis supported that the OGCs were generated from stem-cell-like mesenchymal epithelial cells (SMECs). RESULTS: Functional analysis showed that the OGCs cluster was enriched in antigen presentation, immune response, and stem cell differentiation. Gene markers such as LOX, SPERINE1, CD44, and TGFBI were highly expressed in this SMECs cluster which signified poor prognosis. Interestingly, in myeloid cell, fibroblasts, and endothelial cell clusters, UCOGCP contained higher percentage of these cells and unique subclusters, compared with the rest of PDAC samples. CONCLUSIONS: Analysis of cell communication depicted that CD74 plays important roles in the formation of the microenvironment of UCOGCP. Our findings illustrated the genesis and function of OGCs, and the tumor microenvironment (TME) of UCOGCP, providing insights for prognosis and treatment strategy for this rare type of pancreatic cancer.

Prognostic and biological significance of microRNA-127 expression in human breast cancer.

The purpose of this study was to determine the expression of miR-127 and analyze its prognostic and biological significance in breast cancer (BC). A quantitative reverse transcription PCR assay was performed to detect the expression of miR-127 in 15 pairs of BC and corresponding noncancerous tissues. The expression of miR-127 was detected in another 110 BC tissues and its correlations with clinicopathological factors of patients were examined. Univariate and multivariate analyses were performed to analyze the prognostic significance of miR-127 expression. The effects of miR-127 expression on malignant phenotypes of BC cells and its possible molecular mechanisms were further determined. miR-127 was significantly downregulated in BC tissues, and low miR-127 expression was significantly correlated with lymph node metastasis and advanced clinical stage. Patients with low miR-127 showed poorer overall survival than those with high miR-127. Multivariate analyses indicated that status of miR-127 was an independent prognostic factor for patients. Functional analyses showed that upregulation of miR-127 significantly inhibited growth, enhanced apoptosis, and reduced migration and invasion in BC cells by targeting the protooncogene BCL-6. Therefore, miR-127 may be a potential biomarker for predicting the survival of BC patients and might be a molecular target for treatment of human BCs.

[Outcomes after surgery for refractory constipation patients complicated with megacolon].

OBJECTIVE: To explore the efficacy of different procedures for refractory constipation complicated with megacolon. METHODS: Clinical data of 112 patients of refractory constipation complicated with megacolon undergoing surgery in our institute from June 2007 to January 2013 were retrospectively analyzed. Of these 112 patients, the duration of constipation ranged from 4 to 22 years. Seventy-four patients had previous abdominal operations. Surgical procedures: (1)Jinling procedure (subtotal colectomy plus ascending colorectal posterior wall side-to-side anastomosis, n=81), including 24 laparoscopy-assisted procedures, 18 terminal ileostomies. (2)total colectomy plus ileorectal side-to-side anastomosis(n=18). (3)total colectomy plus end ileostomy, and ileorectal posterior wall side-to-side anastomosis 6 months later(n=13). The end ileostomy was reversed 6 months after operation. RESULTS: The successful rate was 100%, and no surgery-related deaths were found. Postoperative complications included early diarrhea (90 cases, 80.4%), anal pain and incomplete evacuation (22 cases, 19.6%), urinary retention within 24-48 h after catheter removal (16 cases, 14.2%), anastomosis bleeding (9 cases, 8.0%), anastomosis leakage (6 cases, 5.4%), and intestinal obstruction (15 cases, 13.4%). Six patients with intestinal obstruction underwent adhesiolysis, and others were managed by conservative therapy. At the postoperative follow-up at 6 months, the Wexner constipation score was significantly reduced (5.8-8.3 vs. 21.4-28.7, P<0.01), and malnutrition improved as well. CONCLUSION: Surgical intervention results in good efficacy for refractory constipation complicated with megacolon.

Novel surgery for refractory mixed constipation: Jinling procedure - technical notes and early outcome.

INTRODUCTION: The treatment of slow-transit constipation combined with outlet obstruction is controversial. This study introduced a new surgical strategy, subtotal colectomy combined with a modified Duhamel procedure (Jinling procedure), of which the safety and satisfactory rate were examined. MATERIAL AND METHODS: Ninety patients with refractory slow-transit constipation associated with outlet obstruction were consecutively included between Jan 2010 and Dec 2010. All underwent the laparoscopic-assisted Jinling procedure, which added a new side-to-side anastomosis to the colorectal posterior anastomosis after subtotal colectomy. The pre- and post-operative data were collected. RESULTS: There was no surgery-related death. A total of 39 complications and adverse events were reported in 22 patients (morbidity rate of 24.4%). Most complications were managed conservatively without significant events. The satisfactory rate was 93.1% at 6-month follow-up. CONCLUSIONS: The Jinling procedure is safe for refractory slow-transit constipation associated with outlet obstruction, with minimal major complications and a high satisfaction rate. However, this procedure requires rigorous preoperative examination, exquisite surgical and laparoscopic techniques and excellent perioperative management. The pelvic floor, especially the presacral space, is damaged, and therefore it may be unsalvageable if severe complications, such as anastomosis leakage or ischemia, occur.

[Novel surgery for refractory mixed constipation: analysis of the technical notes and outcome to Jinling procedure].

OBJECTIVE: To discuss a new surgical strategy: Jinling procedure (subtotal colectomy combined with modified Duhamel procedure), of which the indications, technical notes and outcomes were analyzed. METHODS: The 590 patients with refractory slow-transit constipation associated with outlet obstruction was strictly included between February 2000 and December 2011. The patients included 103 males and 487 females. Their age were 14-75 years (average 42 ± 13). The 412 patients received laparoscopic-assistant Jinling procedure, and 178 patients with open Jinling procedure. The pre- and post-operation data were collected. The follow up rate were 100%, 98.1%, 95.8% and 92.7% at 3, 6, 12 and 24 months. RESULTS: There was no surgery-related death. Mean hospital day was (12 ± 9) days. Most complications were managed conservatively without significant events. The common complications after surgery were adhesive intestinal obstruction (9.2%), anastomosis bleeding (8.1%) and anastomosis leakage (2.9%). The gastrointestinal quality of life index score was 72 ± 9 preoperatively and increased to 68 ± 11, 99 ± 6, 105 ± 9, 106 ± 9 at 3, 6, 12 and 24 month follow-up, respectively (t = 62.1, -25.1, -126.5, -143.2, P < 0.01). The Wexner constipation scale was 21.9 ± 4.5 preoperatively and decreased to 9.6 ± 2.4, 5.9 ± 2.1, 4.6 ± 1.9, 4.5 ± 1.8 at 3, 6, 12 and 24 month follow-up, respectively (t = 48.6, 61.8, 58.2, 45.9, P < 0.01). The satisfactory rate was 77.5%, 92.1%, 93.0% and 94.1% at 3, 6, 12, and 24 month follow-up. CONCLUSIONS: Jinling procedure provides a good surgical option for refractory slow-transit constipation associated with outlet obstruction.

Berberine ameliorates pro-inflammatory cytokine-induced endoplasmic reticulum stress in human intestinal epithelial cells in vitro.

Endoplasmic reticulum stress (ER stress) in intestinal epithelial cells (IECs) plays an important role in the pathogenesis and perpetuation of inflammatory bowel disease (IBD). The aim of this study is to explore the potential of berberine (BBR) in regulating pro-inflammatory cytokine-induced ER stress and apoptosis in IECs. ER stress in cultured Caco-2 cells was induced by IFN-γ/TNF-α and tunicamycin, respectively. The experimental groups were pretreated with BBR. Cell viability was determined by MTT assay. In vitro apoptosis was examined by flow cytometry using annexin V-FITC labeling. The molecular markers of ER stress, including GRP-78, p-JNK, caspase-12, and cleaved caspase-3 were analyzed by Western blot. Xbp-1 mRNA splicing was detected by RT-PCR. Pretreatment of BBR helped to survive in cytokine-induced Caco-2 cells. BBR significantly attenuated cytokine-induced Caco-2 apoptosis. GRP78 expression and xbp-1 mRNA splicing were enhanced significantly in the presence of IFN-γ/TNF-α and tunicamycin, and they could be dampened by BBR. Further study revealed BBR could downregulate JNK phosphorylation, and the level of caspase-12 and cleaved caspase-3. Berberine can ameliorate pro-inflammatory cytokines induced ER stress in vitro, and it might be one of the targeted therapeutic agents for IBD.