Microsatellite Instability with BRAF V600E Associated with Delayed Presentation but Poor Survival in Stage III Colorectal Cancer.

Colorectal cancer (CRC) has tremendous molecular and genetic heterogeneity, making it a difficult cancer to treat. Two of the key prognostic indicators of CRC include microsatellite instability (MSI) and BRAF V600E mutation. Here, we performed a retrospective survival analysis on 145 stage II and III CRC patients treated at the University of Kansas Cancer Center between 2009 and 2020. Of the 145 patients, BRAF V600E was observed in 15% patients and MSI in 28% patients. Median survival was not reached for stage II. For stage III, patients with BRAF V600E showed poor overall survival, which worsened with concurrent presence of MSI [χ2=6.4, p=0.01]. Eighty-five percent of this group was found to have right-sided CRC. For stage III, overall survival (OS) was 27 months, 37 months, 87 months and not reached for MSI-H/BRAF V600E, MSS/BRAF V600E, MSS/BRAF WT and MSI-H/BRAF WT, respectively. Although associated with poor prognosis, presence of MSI in BRAF V600E patients was associated with delayed disease presentation (mean age 77) compared to those with stable microsatellite (mean age 63) [p=0.01]. Although median survival between the groups could not be assessed for stage II due to very few deaths and/or inadequate length of study, comparison of survival trend suggests that BRAF V600E, rather than MSI, is what drives prognosis in stage II CRC. Our findings suggest that prognostic value of MSI is more relevant for stage III than stage II CRC. Patients with MSI-H and BRAF V600E have advantage of late presentation, although at the cost of poor overall prognosis.

Cryptococcal Neoformans and Varicella Zoster Meningitis in a Patient With Selective Innate Immunodeficiency: A Case Report.

Cryptococcal neoformans (C. neoformans) and varicella-zoster (VZV) meningitis are opportunistic infections that are primarily seen in immunocompromised patients, including those with HIV, cancer, or receiving transplants. Despite treatment, infection in immunocompromised patients can be lethal, including those with T-cell dysfunction or deficiency. Whether innate immunodeficiencies also predispose to these infections remains less clear. Here, we report a case of disseminated C. neoformans and VZV meningitis in a young male with idiopathic hypereosinophilic syndrome and hypocomplementemia and no history of HIV infection, malignancy, or transplant. The patient presented with a pulsating headache, myalgia, joint pain, insomnia, night sweats, and subjective fever, along with clusters of vesicular lesions on his neck and back. A lumbar puncture and an MRI of the brain confirmed C. neoformans and VZV meningitis. Vesicular skin lesions proved to be VZV, and blood culture confirmed fungemia, suggesting disseminated disease. We investigated his medical history further to determine the underlying cause of his prior hypereosinophilia and current meningitis. The patient had idiopathic hypereosinophilia with high IgE levels, low complement levels, high rheumatoid factor levels, and an intermittent rash dating back two years, which had been treated intermittently with prednisone and hydroxyurea, with the most recent admission three weeks prior to this admission. Prior to admission, the patient had a peak absolute eosinophil count of 18.6 x103/uL. The patient was discharged on a daily dose of 60 mg of prednisone without hydroxyurea. In further evaluating his immune status, we found he was HIV-negative, with a normal CD4 count and high IgE. We also tested lymphocyte subsets and proliferation, which showed a low CD16/56 level, suggesting possibly reduced natural killer (NK) cell quantity. The patient responded well to acyclovir, amphotericin, and flucytosine therapy. After follow-up cerebrospinal fluid (CSF) and blood cultures were negative, the patient was discharged with fluconazole as maintenance therapy.

Treating Colorectal Cancer with Immunotherapy: Implications for Single versus Combination Therapy.

PURPOSE OF REVIEW: Colorectal cancer (CRC) is the second leading cause of cancer-associated deaths in the United States, with most metastatic cases subsequently turning refractory to standard chemotherapy. One of the promising current interventions is immunotherapy that relies on harnessing the body's immune mechanisms to kill the cancer cells. The aim of this review is to highlight the implications of single versus combination immunotherapy and identify the molecular features and mutations that enhance or deter responsiveness. RECENT FINDINGS: Based on current findings, responsiveness is associated with deficiency of mismatch repair (dMMR) genes or presence of microsatellite instability (MSI-high), with high immunoscore and tumor-mutational burden contributing to better efficacy while BRAF mutation conferring no significant effect. Combination immunotherapy demonstrates better efficacy in treating MSI-high CRC compared to single agent immunotherapy or chemotherapy. SUMMARY: Given improved responsiveness and overall survival, there is potential for immunotherapy to change the standard of care for metastatic CRC. Furthermore, stratifying the patients by their molecular features and mutation status is critical for establishing care.

Transcription factories in Igκ allelic choice and diversity.

The vertebrate immune system is tasked with the challenge of responding to any pathogen the organism might encounter, and retaining memory of that pathogen in case of future infection. Recognition and memory of pathogens are encoded within the adaptive immune system and production of T and B lymphocytes with diverse antigen receptor repertoires. In B lymphocytes, diversity is generated by sequential recombination between Variable (V), Diversity (D) and Joining (J) gene segments in the immunoglobulin heavy chain gene (Igh) and subsequent V-J recombination in immunoglobulin light chain genes (Igκ followed by Igλ). However, the process by which particular V, D and J segments are selected during recombination, and stochasticity is maintained to ensure antibody repertoire diversity, is still unclear. In this review, we focus on Igκ and recent findings regarding the relationships between gene structure, the generation of diversity and allelic choice. Surprisingly, the nuclear environment in which each Igκ allele resides, including transcription factories assembled on the nuclear matrix, plays critical roles in both gene regulation and in shaping the diversity of Vκ genes accessible to recombination. These findings provide a new paradigm for understanding Igκ recombination and Vκ diversity in the context of B lymphopoiesis.

BRWD1 orchestrates epigenetic landscape of late B lymphopoiesis.

Transcription factor (TF) networks determine cell fate in hematopoiesis. However, how TFs cooperate with other regulatory mechanisms to instruct transcription remains poorly understood. Here we show that in small pre-B cells, the lineage restricted epigenetic reader BRWD1 closes early development enhancers and opens the enhancers of late B lymphopoiesis to TF binding. BRWD1 regulates over 7000 genes to repress proliferative and induce differentiation programs. However, BRWD1 does not regulate the expression of TFs required for B lymphopoiesis. Hypogammaglobulinemia patients with BRWD1 mutations have B-cell transcriptional profiles and enhancer landscapes similar to those observed in Brwd1-/- mice. These data indicate that, in both mice and humans, BRWD1 is a master orchestrator of enhancer accessibility that cooperates with TF networks to drive late B-cell development.

Regulated Capture of Vκ Gene Topologically Associating Domains by Transcription Factories.

Expression of vast repertoires of antigen receptors by lymphocytes, with each cell expressing a single receptor, requires stochastic activation of individual variable (V) genes for transcription and recombination. How this occurs remains unknown. Using single-cell RNA sequencing (scRNA-seq) and allelic variation, we show that individual pre-B cells monoallelically transcribe divergent arrays of Vκ genes, thereby opening stochastic repertoires for subsequent Vκ-Jκ recombination. Transcription occurs upon translocation of Vκ genes to RNA polymerase II arrayed on the nuclear matrix in transcription factories. Transcription is anchored by CTCF-bound sites or E2A-loaded Vκ promotors and continues over large genomic distances delimited only by topological associating domains (TADs). Prior to their monoallelic activation, Vκ loci are transcriptionally repressed by cyclin D3, which prevents capture of Vκ gene containing TADs by transcription factories. Cyclin D3 also represses protocadherin, olfactory, and other monoallelically expressed genes, suggesting a widely deployed mechanism for coupling monoallelic gene activation with cell cycle exit.

Balancing Proliferation with Igκ Recombination during B-lymphopoiesis.

The essential events of B-cell development are the stochastic and sequential rearrangement of immunoglobulin heavy (Igµ) and then light chain (Igκ followed by Igλ) loci. The counterpoint to recombination is proliferation, which both maintains populations of pro-B cells undergoing Igµ recombination and expands the pool of pre-B cells expressing the Igµ protein available for subsequent Igκ recombination. Proliferation and recombination must be segregated into distinct and mutually exclusive developmental stages. Failure to do so risks aberrant gene translocation and leukemic transformation. Recent studies have demonstrated that proliferation and recombination are each affected by different and antagonistic receptors. The IL-7 receptor drives proliferation while the pre-B-cell antigen receptor, which contains Igµ and surrogate light chain, enhances Igκ accessibility and recombination. Remarkably, the principal downstream proliferative effectors of the IL-7R, STAT5 and cyclin D3, directly repress Igκ accessibility through very divergent yet complementary mechanisms. Conversely, the pre-B-cell receptor represses cyclin D3 leading to cell cycle exit and enhanced Igκ accessibility. These studies reveal how cell fate decisions can be directed and reinforced at each developmental transition by single receptors. Furthermore, they identify novel mechanisms of Igκ repression that have implications for gene regulation in general.

Multiple interferon stimulated genes synergize with the zinc finger antiviral protein to mediate anti-alphavirus activity.

The zinc finger antiviral protein (ZAP) is a host factor that mediates inhibition of viruses in the Filoviridae, Retroviridae and Togaviridae families. We previously demonstrated that ZAP blocks replication of Sindbis virus (SINV), the prototype Alphavirus in the Togaviridae family at an early step prior to translation of the incoming genome and that synergy between ZAP and one or more interferon stimulated genes (ISGs) resulted in maximal inhibitory activity. The present study aimed to identify those ISGs that synergize with ZAP to mediate Alphavirus inhibition. Using a library of lentiviruses individually expressing more than 350 ISGs, we screened for inhibitory activity in interferon defective cells with or without ZAP overexpression. Confirmatory tests of the 23 ISGs demonstrating the largest infection reduction in combination with ZAP revealed that 16 were synergistic. Confirmatory tests of all potentially synergistic ISGs revealed 15 additional ISGs with a statistically significant synergistic effect in combination with ZAP. These 31 ISGs are candidates for further mechanistic studies. The number and diversity of the identified ZAP-synergistic ISGs lead us to speculate that ZAP may play an important role in priming the cell for optimal ISG function.

Host factors associated with the Sindbis virus RNA-dependent RNA polymerase: role for G3BP1 and G3BP2 in virus replication.

Sindbis virus (SINV) is the prototype member of the Alphavirus genus, whose members cause severe human diseases for which there is no specific treatment. To ascertain host factors important in the replication of the SINV RNA genome, we generated a SINV expressing nsP4, the viral RNA-dependent RNA polymerase, with an in-frame 3xFlag epitope tag. Proteomic analysis of nsP4-containing complexes isolated from cells infected with the tagged virus revealed 29 associated host proteins. Of these, 10 proteins were associated only at a later time of infection (12 h), 14 were associated both early and late, and five were isolated only at the earlier time (6 h postinfection). These results demonstrate the dynamic nature of the virus-host interaction that occurs over the course of infection and suggest that different host proteins may be required for the multiple functions carried out by nsP4. Two related proteins found in association with nsP4 at both times of infection, GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) and G3BP2 were also previously identified as associated with SINV nsP2 and nsP3. We demonstrate a likely overlapping role for these host factors in limiting SINV replication events. The present study also identifies 10 host factors associated with nsP4 6 h after infection that were not found to be associated with nsP2 or nsP3. These factors are candidates for playing important roles in the RNA replication process. Identifying host factors essential for replication should lead to new strategies to interrupt alphavirus replication.