Arjun N. Jeganathan

Alloreactive CD4 T cell activation in vivo: an autonomous function of the indirect pathway of alloantigen presentation.

Activation of alloreactive CD4 T cells occurs via the direct and indirect pathways of alloantigen presentation. A novel TCR/alloantigen transgenic system was designed that permitted in vivo visualization of CD4 T cell priming through these pathways. When both pathways of alloantigen presentation were intact, CD4 T cell activation in response to cardiac allografts was rapid and systemic by day 4 after transplantation, in contrast to that seen in response to skin allografts, which was delayed until 10–12 days after transplantation. Despite this systemic CD4 T cell activation in response to cardiac allografts, there was a paucity of activated graft-infiltrating CD4 T cells at 4 days posttransplantation. This finding suggests that the initial priming of alloimmune CD4 T cell responses occurs within draining lymphoid organs. Furthermore, alloantigens derived from cardiac allografts failed to promote thymic negative selection of developing thymocytes expressing the alloreactive TCR clonotype. In the absence of a functional direct pathway, the kinetics of activation, anatomic localization, and effector function of alloreactive CD4 T cells remained unchanged. Overall, the present study defines the anatomic and temporal characteristics of CD4 T cell alloimmune responses and demonstrates that CD4 T cell priming via the indirect pathway proceeds optimally in the absence of the direct pathway of alloantigen presentation.

Let-7 Represses Carcinogenesis and a Stem Cell Phenotype in the Intestine via Regulation of Hmga2.

Let-7 miRNAs comprise one of the largest and most highly expressed family of miRNAs among vertebrates, and is critical for promoting differentiation, regulating metabolism, inhibiting cellular proliferation, and repressing carcinogenesis in a variety of tissues. The large size of the Let-7 family of miRNAs has complicated the development of mutant animal models. Here we describe the comprehensive repression of all Let-7 miRNAs in the intestinal epithelium via low-level tissue-specific expression of the Lin28b RNA-binding protein and a conditional knockout of the MirLet7c-2/Mirlet7b locus. This ablation of Let-7 triggers the development of intestinal adenocarcinomas concomitant with reduced survival. Analysis of both mouse and human intestinal cancer specimens reveals that stem cell markers were significantly associated with loss of Let-7 miRNA expression, and that a number of Let-7 targets were elevated, including Hmga1 and Hmga2. Functional studies in 3-D enteroids revealed that Hmga2 is necessary and sufficient to mediate many characteristics of Let-7 depletion, namely accelerating cell cycle progression and enhancing a stem cell phenotype. In addition, inactivation of a single Hmga2 allele in the mouse intestine epithelium significantly represses tumorigenesis driven by Lin28b. In aggregate, we conclude that Let-7 depletion drives a stem cell phenotype and the development of intestinal cancer, primarily via Hmga2.

A Nomogram to Predict Lymph Node Positivity Following Neoadjuvant Chemoradiation in Locally Advanced Rectal Cancer.

BACKGROUND: Patients with locally advanced rectal cancer typically receive neoadjuvant chemoradiation followed by total mesorectal excision. Other treatment approaches, including transanal techniques and close surveillance, are becoming increasingly common following positive responses to chemoradiation. Lack of pathologic lymph node staging is one major disadvantage of these novel strategies. OBJECTIVE: The purposes of this study were to determine clinicopathologic factors associated with positive lymph nodes following neoadjuvant chemoradiation for rectal cancer and to create a nomogram using these factors to predict rates of lymph node positivity. DESIGN: This is a retrospective cohort analysis. SETTINGS: This study used the National Cancer Database. PATIENTS: Patients aged 18 to 90 with clinical stage T3/T4, N0, M0 or Tany, N1-2, M0 adenocarcinoma of the rectum who underwent neoadjuvant chemoradiation before total mesorectal excision from 2010 to 2012 were identified. MAIN OUTCOME MEASURES: The primary outcome measure was lymph node positivity after neoadjuvant chemoradiation for locally advanced rectal cancer. Bivariate and multivariate analyses were used to determine the associations of clinicopathologic variables with lymph node positivity. RESULTS: Eight thousand nine hundred eighty-four patients were included. Young age, lower Charlson score, mucinous histology, poorly differentiated and undifferentiated tumors, the presence of lymphovascular invasion, elevated CEA level, and clinical lymph node positivity were significantly predictive of pathologic lymph node positivity following neoadjuvant chemoradiation. The predictive accuracy of the nomogram is 70.9%, with a c index of 0.71. There was minimal deviation between the predicted and observed outcomes. LIMITATIONS: This study is retrospective, and it cannot be determined when in the course of treatment the data were collected. CONCLUSIONS: We created a nomogram to predict lymph node positivity following neoadjuvant chemoradiation for locally advanced rectal cancer that can serve as a valuable complement to imaging to aid clinicians and patients in determining the best treatment strategy.

Differential Regulation of LET-7 by LIN28B Isoform–Specific Functions

The RNA-binding protein LIN28B plays an important role in development, stem cell biology, and tumorigenesis. LIN28B has two isoforms: the LIN28B-long and -short isoforms. Although studies have revealed the functions of the LIN28B-long isoform in tumorigenesis, the role of the LIN28B-short isoform remains unclear and represents a major gap in the field. The LIN28B-long and -short isoforms are expressed in a subset of human colorectal cancers and adjacent normal colonic mucosa, respectively. To elucidate the functional and mechanistic aspects of these isoforms, colorectal cancer cells (Caco-2 and LoVo) were generated to either express no LIN28B or the -short or -long isoform. Interestingly, the long isoform suppressed LET-7 expression and activated canonical RAS/ERK signaling, whereas the short isoform did not. The LIN28B-long isoform–expressing cells demonstrated increased drug resistance to 5-fluorouracil and cisplatin through the upregulation of ERCC1, a DNA repair gene, in a LET-7–dependent manner. The LIN28B-short isoform preserved its ability to bind pre-let-7, without inhibiting the maturation of LET-7, and competed with the LIN28B-long isoform for binding to pre-let-7. Coexpression of the short isoform in the LIN28B-long isoform–expressing cells rescued the phenotypes induced by the LIN28B-long isoform. Implications: This study demonstrates the differential antagonistic functions of the LIN28B-short isoform against the LIN28B-long isoform through an inability to degrade LET-7, which leads to the novel premise that the short isoform may serve to counterbalance the long isoform during normal colonic epithelial homeostasis, but its downregulation during colonic carcinogenesis may reveal the protumorigenic effects of the long isoform. Mol Cancer Res; 16(3); 403–16. ©2018 AACR.

Colorectal specialization and survival in colorectal cancer

It is recognized that higher surgeon volume is associated with improved survival in colorectal cancer. However, there is a paucity of national studies that have evaluated the relationship between surgical specialization and survival.

Lymph node identification following neoadjuvant therapy in rectal cancer: A stage-stratified analysis using the surveillance, epidemiology, and end results (SEER)-medicare database

Neoadjuvant chemoradiation (nCRT) for rectal adenocarcinoma reduces lymph node (LN) identification following surgical resection. We sought to evaluate the relationship between LN identification following nCRT and disease‐specific survival (DSS), stratified by pathologic stage.

Anal and Perineal Injuries

Abstract With increased use of explosive devices in warfare, anal trauma is often seen coupled with more complex pelviperineal injury. While the associated mortality is high, casualties that survive are often left with disabling fecal incontinence from damage to the anosphincteric complex. After resolution of the acute insult, the initial evaluation mandates a thorough physical exam, including endoscopic evaluation with rigid proctoscopy and flexible sigmoidoscopy, as well as adjunctive testing, specifically anal manometry and endoanal ultrasound. First‐line therapy favors bulking agents and antidiarrheals, in conjunction with biofeedback, due to a minimal risk profile. Surgical options range from direct sphincter repairs to complex anosphincteric reconstruction with widely variable results. Most recently, burgeoning therapies in the treatment of fecal incontinence, including sacral nerve stimulation and magnetic anal sphincters, offer excellent alternatives with promising long‐term outcomes. In summation, the goal of all interventions is the re‐establishment of bowel continence, but, in its absence, permanent fecal diversion for devastating fecal incontinence is a reasonable option with excellent patient satisfaction scores.

The frequency of double‐positive thymocytes expressing an αβ TCR clonotype regulates peripheral CD4 T cell compartment homeostasis

The present study aimed to determine whether the frequency of double positive (DP) thymocytes expressing αβ T‐cell receptor (TCR) clonotypes at the time of selection regulates peripheral CD4 T‐cell compartment size. Scid recipients were inoculated with various ratios of TCR Cα0/0 and wild‐type bone marrow (BM) stem cells. Increasing the frequency of TCR Cα0/0 thymocytes at steady‐state introduced a graded decrease in the maturation probability of the total DP thymocyte pool. At 12–14 weeks following BM inoculation, the frequency of TCR Cα0/0 DP thymocytes was inversely correlated with that of CD4 single positive (SP) thymocytes. Notwithstanding, a decreased frequency of wild‐type DP thymocytes led to a marked increase in their transit efficiency from the DP to SP compartments. The frequency‐dependent increase in thymocyte transit efficiency was associated with a CD4 SP cell surface phenotype indicative of increased antigenic experience. Importantly, the frequency of DP thymocytes capable of expressing TCR clonotypes dictated the steady‐state size of the peripheral CD4 T cell compartment and its potential for homeostatic proliferation. Collectively, these results indicate that the efficiency of DP to CD4 SP transit is a frequency dependent process, which determines (1) the steady‐state size of the peripheral T cell compartment and (2) the threshold for homeostatic expansion of peripheral CD4 T cells.

Abstract 1136: Cooperative functional roles of RNA binding proteins LIN28B and IMP1 in the pathogenesis of colorectal cancer

Introduction: RNA binding proteins and miRNAs have emerged as crucial regulators of intestinal homeostasis by controlling the stability and translation of target mRNAs. LIN28B, an mRNA binding protein, plays a critical role in regulating growth and proliferation in the intestinal epithelium. Previous work in our lab revealed that LIN28B promotes growth and tumorigenesis of the intestinal epithelium via suppression of mature let-7 miRNAs.LIN28B suppression of let-7 promotes upregulation of let-7 targets, including IMP1 (Insulin-like growth factor II mRNA-binding protein 1). Indeed, previous studies from our lab have shown that transgenic mice expressing LIN28B from the mouse Vil1 promoter (Vil-Lin28b mice) have an increase in IMP1 protein levels that is increased further with conditional knockout of let-7. Mechanistically, Let-7 isoforms have been known to physically and functionally interact with IMP1; however, the specific role of IMP1 in Lin28b-let 7-mediated tumorigenesis remains unknown. The current study tested the hypothesis that IMP1 maybe required for LIN28B-mediated tumorigenesis and that LIN28B and IMP1 may cooperatively promote a tumor-initiating phenotype. Methods: We evaluated LIN28B and IMP1 expression and localization in colorectal cancer patient samples using tissue microarrays and clinical outcomes. We used intestinal epithelial cell lines with LIN28B overexpression and CRISPR-Cas9 mediated knockout of IMP1 to study the functional consequences on migration, invasion and proliferation. Results: LIN28B expression correlates with expression of IMP1 in colorectal cancer patient samples. Individually, LIN28B and IMP1 expression intensity each was associated with worse prognosis in stage II colon cancer. Knock-down of IMP1 in Lin28B overexpressing cells decreased migration of colorectal cancer cell lines, suggesting a relationship of IMP1 for the tumorigenic effects of LIN28B. Furthermore, our RNA target analyses show that Lin28b and IMP1 both bind to target mRNAs in the WNT and adherens junction pathways. Conclusions: These data implicate a novel role for the Lin28b-let7-IMP1 axis in colorectal cancer and support an emerging paradigm for a critical and cooperative role of RNA-binding proteins in intestinal homeostasis and cancer. We are using the cell lines generated for orthotopic xenograft studies to see the effects of IMP1 loss and Lin28b overexpression on tumor growth and dissemination. Furthermore, we are generating mice with intestinal epithelium specific Lin28b overexpression-IMP1 loss to study the effects in vivo. Citation Format: Priya Chatterji, Kathryn Hamilton, Sarah Andres, Rei Mizuno, Philip Hicks, Arjun Jeganathan, Monte M. Winslow, Antoni Castells, Miriam Cuatrecasas, Blair Madison, Anil Rustgi. Cooperative functional roles of RNA binding proteins LIN28B and IMP1 in the pathogenesis of colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1136.