Intrahepatic hepatitis C virus RNA quantification in microdissected hepatocytes

BACKGROUND/AIMS: Debate continues on whether serum and intrahepatic HCV viral loads are correlated and if HCV viral load correlates with the severity of liver disease. These difficulties may at least in part be linked to liver cell heterogeneity, when total liver extracts from HCV-infected individuals are tested for HCV RNA quantification. We have therefore investigated the feasibility of quantifying HCV replication using a laser-based microdissection technique. METHODS: We compared the results with those obtained for serum HCV RNA quantification and immunochemistry in the case of HCV antigen detection in the liver. Twenty-one HCV-positive patients with chronic active hepatitis (n=10) or cirrhosis (n=11) were analyzed. RESULTS: A positive correlation (P=0.0019) was observed between HCV RNA quantifications in sera and microdissected cells. Immunohistochemistry demonstrated that HCV antigen hepatocytes were randomly distributed within liver lobules. Their percentage varied in different patients (0-40%), but did not correlate with the HCV viral load. CONCLUSIONS: We have designed a sensitive methodology to evaluate the intrahepatic HCV viral load by combining a standardized RNA quantification method with microdissected hepatocytes from frozen liver needle biopsies. Our results directly demonstrate a positive correlation between serum and intrahepatic viral loads, which therefore provides a reliable reflection of intrahepatic HCV replication.     

Overexpression of dominant-negative Ikaros 6 protein is restricted to a subset of B common adult acute lymphoblastic leukemias that express high levels of the CD34 antigen

Ikaros is a critical regulator of hematopoiesis. Its effects result in part from the balance between the isoforms that are produced by differential splicing of the pre-mRNA. Short isoforms that lack the DNA-binding domain act as dominant negatives by binding long isoforms through the C-terminal zinc-finger domain, which allows for the homo- or heterodimerization of the proteins. There are a number of evidences that different subsets of murine hematopoietic progenitors - as defined by phenotype - have different patterns of Ikaros expression. Forced expression of short isoforms (Ik5, Ik6 or Ik7) in murine or human hematopoietic progenitors alters the differentiation capacities of these cells. Human leukemias provide additional information: because of the blockade in differentiation, leukemias represent an equivalent of a particular stage of human hematopoietic hierarchy. We and others have shown that human acute leukemias are heterogeneous for the pattern of Ikaros isoform expression. The present study focused on adult de novo B ALLs and the Ikaros 6 isoform. ProB (BI, n=3), common B (BII, n=15) and preB (BIII, n=3) ALL were identified by their phenotype. RT-PCR and Western blot analyses of blast cell protein lysates suggest that approximately 50% of BII leukemias overexpress Ikaros 6 RNA and protein. Comparison of BII cells with high or normal levels of Ik6 shows a higher level of expression for the membrane stem cell antigen CD34 in the former, as detected with flow cytometry and confirmed with DNA arrays.     

From the Cover: Radiation-induced neoantigens broaden the immunotherapeutic window of cancers with low mutational loads

Immunotherapies are a promising advance in cancer treatment. However, because only a subset of cancer patients benefits from these treatments it is important to find mechanisms that will broaden the responding patient population. Generally, tumors with high mutational burdens have the potential to express greater numbers of mutant neoantigens. As neoantigens can be targets of protective adaptive immunity, highly mutated tumors are more responsive to immunotherapy. Given that external beam radiation 1) is a standard-of-care cancer therapy, 2) induces expression of mutant proteins and potentially mutant neoantigens in treated cells, and 3) has been shown to synergize clinically with immune checkpoint therapy (ICT), we hypothesized that at least one mechanism of this synergy was the generation of de novo mutant neoantigen targets in irradiated cells. Herein, we use Kras^G12D x p53^−/− sarcoma cell lines (KP sarcomas) that we and others have shown to be nearly devoid of mutations, are poorly antigenic, are not controlled by ICT, and do not induce a protective antitumor memory response. However, following one in vitro dose of 4- or 9-Gy irradiation, KP sarcoma cells acquire mutational neoantigens and become sensitive to ICT in vivo in a T cell-dependent manner. We further demonstrate that some of the radiation-induced mutations generate cytotoxic CD8⁺ T cell responses, are protective in a vaccine model, and are sufficient to make the parental KP sarcoma line susceptible to ICT. These results provide a proof of concept that induction of new antigenic targets in irradiated tumor cells represents an additional mechanism explaining the clinical findings of the synergy between radiation and immunotherapy.

Immune checkpoint therapy (ICT) can lead to durable responses in subsets of cancer patients (1–8). On the basis of computational analyses, the patients who most benefit from ICT are those with cancers that have high mutational burden (9–18). For example, patients bearing tumors with high mutational burden caused by environmental exposure (such as ultraviolet-induced melanoma) or deficiencies in DNA repair (such as microsatellite instability-high colorectal cancers) tend to respond well to immunotherapy (18–26). Presumably the sensitivity of such cancers reflects the increased likelihood of formation of immunogenic, tumor-specific mutant neoantigens (27). We and others previously showed that certain tumor-specific neoantigens are major targets of natural and therapeutically induced antitumor responses in both mice and humans (28–41). Therefore, the presence of immunogenic tumor neoantigens is currently thought to contribute to tumor sensitivity to immunotherapy.However, many cancer patients do not respond to ICT, suggesting that their neoantigen burden is either of insufficient magnitude or immunogenicity to function as targets for T cell-dependent antitumor mechanisms. Indeed, there are many tumor types, such as acute myeloid leukemia, estrogen receptor-positive breast, and prostate cancers, that have limited mutational burdens and display low response rates to ICT (9, 13, 42, 43). Additionally, tumor cell clones expressing immunogenic neoantigens that develop during tumor evolution may be eliminated from tumors with high mutational burden by the process of cancer immunoediting, resulting in outgrowth of tumor cell clones with reduced immunogenicity that can then grow progressively in the presence of the unmanipulated immune system (33, 44, 45). Therefore, a process by which tumors with low neoantigen burden can acquire immunogenic mutations has the potential to expand the number of patients able to benefit from ICT.Ionizing radiation has been shown to elicit DNA damage in tumor cells, leading to an increase in overall mutational load (46–52). This damage is thought to occur primarily through generation of reactive oxygen species which induce base pair substitutions by mechanisms involving transitions, transversions, and/or faulty DNA repair (53). Multiple preclinical studies have demonstrated antitumor responses when focal radiation is combined with ICT in tumors that do not respond to ICT alone (54–60) and several clinical studies have demonstrated that human tumor patients have improved responsiveness to ICT following focal radiation (e.g., {"type":"clinical-trial","attrs":{"text":"NCT02303990","term_id":"NCT02303990"}}NCT02303990, {"type":"clinical-trial","attrs":{"text":"NCT02298946","term_id":"NCT02298946"}}NCT02298946, {"type":"clinical-trial","attrs":{"text":"NCT02383212","term_id":"NCT02383212"}}NCT02383212) (61–67). Radiation has been demonstrated to function as an in vivo tumor vaccine by inducing damage-associated molecular patterns (DAMP)-dependent immunogenic cell death (68), inducing DNA damage sensed by pattern recognition receptors (69, 70), enhancing access of immune effector cells to their cognate targets through tumor cell debulking and vasculature changes (71, 72), up-regulating major histocompatibility complex class I (MHC-I) receptors (73), up-regulating cell-surface molecules such as Fas (74), and augmenting tumor antigen cross-presentation by specific subsets of dendritic cells through up-regulation of type I interferon (IFN), which results in increased numbers and action of tumor-specific CD8⁺ T cells (75–77). However, none of these explanations take into account that following irradiation, tumor cells acquire novel mutations that may function as effective tumor neoantigens. In fact, two groups have demonstrated broadening of the T cell repertoire following radiation treatment of mouse 4T1 mammary tumors and B16F10 melanoma tumors (56, 78). Radiation-induced neoantigens may partially explain the broadening of the T cell repertoire reported during noncurative doses of irradiation.Given the above observations, we specifically explored whether one dose of in vitro irradiation could increase the immunogenicity of poorly immunogenic tumor cell lines through mechanisms involving the de novo generation of tumor-specific mutant neoantigens. For this purpose, we used a mouse Kras^G12D x p53^−/− sarcoma cell line as a model system since the R.D.S. and T.J. laboratories have previously shown that these tumor cells express a very limited number of somatic mutations, are essentially devoid of mutational neoantigens, and are nonimmunogenic and grow progressively in syngeneic wild-type (WT) mice either following treatment with control antibody or the combination of anti–PD-1/anti–CTLA-4 (34, 41). We find that treating these cell lines with noncurative doses of irradiation induces expression of somatic mutations, some of which function as neoantigens and render the sarcoma cells susceptible to ICT in vivo. These data support the concept that an additional mechanism underlying the synergy between radiation therapy and immunotherapy is that the former induces immunogenic mutations in tumors that now function as targets for the latter.     

Thalidomide in advanced mastocytosis

Mastocytosis is an acquired orphan disease characterized by the abnormal accumulation of mast cells responsible for organ failure and systemic symptoms. Cytoreductive drugs have been shown to be effective, but have rarely resulted in complete or long-term remission. We report two patients with advanced systemic mastocytosis (SM) who were treated successfully with thalidomide, given at the maximal tolerated dosage. B and C-findings as well as clinical symptoms rapidly improved. After a follow-up of more than 1 year, the patients remained in partial remission. Thalidomide seems to be an active drug in advanced SM. However, clinical trials are warranted to define its efficacy and safety profiles.     

Primary Care Tasks Associated with Provider Burnout: Findings from a Veterans Health Administration Survey

Background

The patient-centered medical home (PCMH) is a primary care delivery model predicated on shared responsibility for patient care among members of an interprofessional team. Effective task sharing may reduce burnout among primary care providers (PCPs). However, little is known about the extent to which PCPs share these responsibilities, and which, if any, of the primary care tasks performed independently by the PCPs (vs. shared with the team) are particularly associated with PCP burnout. A better understanding of the relationship between these tasks and their effects on PCP burnout may help guide focused efforts aimed at reducing burnout.

Objective

To investigate (1) the extent to which PCPs share responsibility for 14 discrete primary care tasks with other team members, and (2) which, if any, of the primary care tasks performed by the PCPs (without reliance on team members) are associated with PCP burnout.

Design

Secondary data analysis of Veterans Health Administration (VHA) survey data from two time periods.

Participants

327 providers from 23 VA primary care practices within one VHA regional network.

Main Measures

The dependent variable was PCP report of burnout. Independent variables included PCP report of the extent to which they performed 14 discrete primary care tasks without reliance on team members; team functioning; and PCP-, clinic-, and system-level variables.

Key Results

In adjusted models, PCP reports of intervening on patient lifestyle factors and educating patients about disease-specific self-care activities, without reliance on their teams, were significantly associated with burnout (intervening on lifestyle: b = 4.11, 95% CI = 0.39, 7.83, p = 0.03; educating patients: b = 3.83, 95% CI = 0.33, 7.32, p = 0.03).

Conclusions

Performing behavioral counseling and self-management education tasks without relying on other team members for assistance was associated with PCP burnout. Expanding the roles of nurses and other healthcare professionals to assume responsibility for these tasks may ease PCP burden and reduce burnout.

     

Prospective gene expression analysis accurately subtypes acute leukaemia in children and establishes a commonality between hyperdiploidy and t(12;21) in acute lymphoblastic leukaemia

We have prospectively analysed and correlated the gene expression profiles of children presenting with acute leukaemia to the Royal London and Great Ormond Street Hospitals with morphological diagnosis, immunophenotype and karyotype. Total RNA extracted from freshly sorted blast cells was obtained from 84 lymphoblastic [acute lymphoblastic leukaemia (ALL)], 20 myeloid [acute myeloid leukaemia (AML)] and three unclassified acute leukaemias and hybridised to the high density Affymetrix U133A oligonucleotide array. Analysis of variance and significance analysis of microarrays was used to identify discriminatory genes. A novel 50-gene set accurately identified all patients with ALL and AML and predicted for a diagnosis of AML in three patients with unclassified acute leukaemia. A unique gene set was derived for each of eight subtypes of acute leukaemia within our data set. A common profile for children with ALL with an ETV6-RUNX1 fusion, amplification or deletion of ETV6, amplification of RUNX1 or hyperdiploidy with an additional chromosome 21 was identified. This suggests that these rearrangements share a commonality in biological pathways that maintains the leukaemic state. The gene TERF2 was most highly expressed in this group of patients. Our analyses demonstrate that not only is microarray analysis the single most effective tool for the diagnosis of acute leukaemias of childhood but it has the ability to identify unique biological pathways. To further evaluate its prognostic value it needs to be incorporated into the routine diagnostic analysis for large-scale clinical trials in childhood acute leukaemias.