Necrobiosis lipoidica (NL) is a rare, granulomatous disease considered to be associated with diabetes. It is frequently seen in female and middle-aged patients and is rarely observed in children. We present a 14-year-old boy with poorly controlled type 1 diabetes who developed biopsy-proven NL. He had improvement, but not resolution of the plaque with improved glycemic control. Pediatric NL may be associated with diabetes and could be related to poor glycemic control. However, further investigation is warranted in this young population.

Well preserved frozen biospecimens are ideal for evaluating the genome, transcriptome, and proteome. While papers reviewing individual aspects of frozen biospecimens are available, we present a current overview of experimental data regarding procurement, storage, and quality assurance that can inform the handling of frozen biospecimens. Frozen biospecimen degradation can be influenced by factors independent of the collection methodology including tissue type, premortem agonal changes, and warm ischemia time during surgery. Rapid stabilization of tissues by snap freezing immediately can mitigate artifactually altered gene expression and, less appreciated, protein phosphorylation profiles. Collection protocols may be adjusted for specific tissue types as cellular ischemia tolerance varies widely. If data is not available for a particular tissue type, a practical goal is snap freezing within 20min. Tolerance for freeze-thaw events is also tissue type dependent. Tissue storage at -80°C can preserve DNA and protein for years but RNA can show degradation at 5years. For -80°C freezers, aliquots frozen in RNAlater or similar RNA stabilizing solutions are a consideration. It remains unresolved as to whether storage at -150°C provides significant advantages relative to that at -80°C. Histologic quality assurance of tissue biospecimens is typically performed at the time of surgery but should also be conducted on the aliquot to be distributed because of tissue heterogeneity. Biobanking protocols for blood and its components are highly dependent on intended use and multiple collection tube types may be needed. Additional quality assurance testing should be dictated by the anticipated downstream applications.

Unlabelled

Frozen biospecimens are crucial for translational research and contain well-preserved nucleic acids and protein. However, the risks of freezer failure as well as space, cost, and environmental concerns of frozen biospecimens are substantial.

Objective

The purpose of the study was to review the current status of room temperature biospecimen storage.

Methods

We searched Pubmed and vendor websites to identify relevant information.

Results

Formalin-fixed paraffin embedded (FFPE) tissues have great value but their use is limited by cross-linking and fragmentation of nucleic acids, as well as loss of enzymatic activity. Stabilization solutions can now robustly preserve fresh tissue for up to 7days at room temperature. For longer term storage, commercial vendors of chemical matrices claim real time stability of nucleic acids of over 2 years and their accelerated aging studies to date suggest stability for 12years for RNA and 60years for DNA. However, anatomic pathology biorepositories store mostly frozen tissue rather than nucleic acids. Small quantities of tissue can be directly placed on some chemical matrices to stabilize DNA, however RNA and proteins are not preserved. Current lyophilization approaches can preserve histomorphology, DNA, RNA, and proteins though RNA shows moderate degradation after 1-2years. Formalin-free fixatives show improved but varying abilities to preserve nucleic acids and face validation as well as cost barriers in replacing FFPE specimens. The paraffin embedding process can degrade RNA.

Conclusion

Development of robust long-term room temperature biospecimen tissue storage technology can potentially reduce costs for the biomedical community in the face of growing targeted therapy needs and decreasing budgets.

Purpose

Diffusion magnetic resonance imaging (MRI) and 6-[(18)F]fluoro-L-dopa ([(18)F]FDOPA) positron emission tomography (PET) are used to interrogate malignant tumor microenvironment. It remains unclear whether there is a relationship between [(18)F]FDOPA uptake, diffusion MRI estimates of apparent diffusion coefficient (ADC), and mitotic activity in the context of recurrent malignant gliomas, where the tumor may be confounded by the effects of therapy. The purpose of the current study is to determine whether there is a correlation between these imaging techniques and mitotic activity in malignant gliomas.

Procedures

We retrospectively examined 29 patients with recurrent malignant gliomas who underwent structural MRI, diffusion MRI, and [(18)F]FDOPA PET prior to surgical resection. Qualitative associations were noted, and quantitative voxel-wise and median measurement correlations between [(18)F]FDOPA PET, ADC, and mitotic index were performed.

Results

Areas of high [(18)F]FDOPA uptake exhibited low ADC and areas of hyperintensity T2/fluid-attenuated inversion recovery (FLAIR) with low [(18)F]FDOPA uptake exhibited high ADC. There was a significant inverse voxel-wise correlation between [(18)F]FDOPA and ADC for all patients. Median [(18)F]FDOPA uptake and median ADC also showed a significant inverse correlation. Median [(18)F]FDOPA uptake was positively correlated, and median ADC was inversely correlated with mitotic index from resected tumor tissue.

Conclusions

A significant association may exist between [(18)F]FDOPA uptake, diffusion MRI, and mitotic activity in recurrent malignant gliomas.

Conditional genetically engineered mouse models (GEMMs) of non-small cell lung cancer (NSCLC) harbor common oncogenic driver mutations of the disease, but in contrast to human NSCLC these models possess low tumor mutational burden (TMB). As a result, these models often lack tumor antigens that can elicit host adaptive immune responses, which limits their utility in immunotherapy studies. Here, we establish Kras-mutant murine models of NSCLC bearing the common driver mutations associated with the disease and increased TMB, by in vitro exposure of cell lines derived from GEMMs of NSCLC [Kras^G12D (K), Kras^G12DTp53^-/-(KP), Kras^G12DTp53^+/-Lkb1^-/- (KPL)] to the alkylating agent N-methyl-N-nitrosourea (MNU). Increasing the TMB enhanced host anti-tumor T cell responses and improved anti-PD-1 efficacy in syngeneic models across all genetic backgrounds. However, limited anti-PD-1 efficacy was observed in the KPL cell lines with increased TMB, which possessed a distinct immunosuppressed tumor microenvironment (TME) primarily composed of granulocytic myeloid-derived suppressor cells (G-MDSCs). This KPL phenotype is consistent with findings in human KRAS-mutant NSCLC where LKB1 loss is a driver of primary resistance to PD-1 blockade. In summary, these novel Kras-mutant NSCLC murine models with known driver mutations and increased TMB have distinct TMEs and recapitulate the therapeutic vulnerabilities of human NSCLC. We anticipate that these immunogenic models will facilitate the development of innovative immunotherapies in NSCLC.

Background

Despite recent advances in immunotherapy, many patients with non-small cell lung cancer (NSCLC) do not respond to immune checkpoint inhibitors (ICI). Resistance to ICI may be driven by suboptimal priming of antitumor T lymphocytes due to poor antigen presentation as well as their exclusion and impairment by the immunosuppressive tumor microenvironment (TME). In a recent phase I trial in patients with NSCLC, in situ vaccination (ISV) with dendritic cells engineered to secrete CCL21 (CCL21-DC), a chemokine that facilitates the recruitment of T cells and DC, promoted T lymphocyte tumor infiltration and PD-L1 upregulation.

Methods

Murine models of NSCLC with distinct driver mutations (Kras^G12D/P53^+/-/Lkb1^-/- (KPL); Kras^G12D/P53^+/- (KP); and Kras^G12D (K)) and varying tumor mutational burden were used to evaluate the efficacy of combination therapy with CCL21-DC ISV plus ICI. Comprehensive analyses of longitudinal preclinical samples by flow cytometry, single cell RNA-sequencing (scRNA-seq) and whole-exome sequencing were performed to assess mechanisms of combination therapy.

Results

ISV with CCL21-DC sensitized immune-resistant murine NSCLCs to ICI and led to the establishment of tumor-specific immune memory. Immunophenotyping revealed that CCL21-DC obliterated tumor-promoting neutrophils, promoted sustained infiltration of CD8 cytolytic and CD4 Th1 lymphocytes and enriched progenitor T cells in the TME. Addition of ICI to CCL21-DC further enhanced the expansion and effector function of T cells both locally and systemically. Longitudinal evaluation of tumor mutation profiles revealed that CCL21-DC plus ICI induced immunoediting of tumor subclones, consistent with the broadening of tumor-specific T cell responses.

Conclusions

CCL21-DC ISV synergizes with anti-PD-1 to eradicate murine NSCLC. Our data support the clinical application of CCL21-DC ISV in combination with checkpoint inhibition for patients with NSCLC.

Glioblastoma stem cells (GSC) co-exhibiting a tumor-initiating capacity and a radio-chemoresistant phenotype, are a compelling cell model for explaining tumor recurrence. We have previously characterized patient-derived, treatment-resistant GSC clones (TRGC) that survived radiochemotherapy. Compared to glucose-dependent, treatment-sensitive GSC clones (TSGC), TRGC exhibited reduced glucose dependence that favor the fatty acid oxidation pathway as their energy source. Using comparative genome-wide transcriptome analysis, a series of defense signatures associated with TRGC survival were identified and verified by siRNA-based gene knockdown experiments that led to loss of cell integrity. In this study, we investigate the prognostic value of defense signatures in glioblastoma (GBM) patients using gene expression analysis with Probeset Analyzer (131 GBM) and The Cancer Genome Atlas (TCGA) data, and protein expression with a tissue microarray (50 GBM), yielding the first TRGC-derived prognostic biomarkers for GBM patients. Ribosomal protein S11 (RPS11), RPS20, individually and together, consistently predicted poor survival of newly diagnosed primary GBM tumors when overexpressed at the RNA or protein level [RPS11: Hazard Ratio (HR) = 11.5, p<0.001; RPS20: HR = 4.5, p = 0.03; RPS11+RPS20: HR = 17.99, p = 0.001]. The prognostic significance of RPS11 and RPS20 was further supported by whole tissue section RPS11 immunostaining (27 GBM; HR = 4.05, p = 0.01) and TCGA gene expression data (578 primary GBM; RPS11: HR = 1.19, p = 0.06; RPS20: HR = 1.25, p = 0.02; RPS11+RPS20: HR = 1.43, p = 0.01). Moreover, tumors that exhibited unmethylated O-6-methylguanine-DNA methyltransferase (MGMT) or wild-type isocitrate dehydrogenase 1 (IDH1) were associated with higher RPS11 expression levels [corr (IDH1, RPS11) = 0.64, p = 0.03); [corr (MGMT, RPS11) = 0.52, p = 0.04]. These data indicate that increased expression of RPS11 and RPS20 predicts shorter patient survival. The study also suggests that TRGC are clinically relevant cells that represent resistant tumorigenic clones from patient tumors and that their properties, at least in part, are reflected in poor-prognosis GBM. The screening of TRGC signatures may represent a novel alternative strategy for identifying new prognostic biomarkers.

Latino Americans are a rapidly growing ethnic group in the United States but studies of glioblastoma in this population are limited. We have evaluated characteristics of 21,184 glioblastoma patients from the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute. This SEER data from 2001 to 2011 draws from 28% of the U.S.

Population

Latinos have a lower incidence of GBM and present slightly younger than non-Latino Whites. Cubans present at an older age than other Latino sub-populations. Latinos have a higher incidence of giant cell glioblastoma than non-Latino Whites while the incidence of gliosarcoma is similar. Despite lower rates of radiation therapy and greater rates of sub-total resection than non-Latino Whites, Latinos have better 1 and 5 year survival rates. SEER does not record chemotherapy data. Survivals of Latino sub-populations are similar with each other. Age, extent of resection, and the use of radiation therapy are associated with improved survival but none of these variables are sufficient in a multivariate analysis to explain the improved survival of Latinos relative to non-Latino Whites. As molecular data is not available in SEER records, we studied the MGMT and IDH status of 571 patients from a UCLA database. MGMT methylation and IDH1 mutation rates are not statistically significantly different between non-Latino Whites and Latinos. For UCLA patients with available information, chemotherapy and radiation rates are similar for non-Latino White and Latino patients, but the latter have lower rates of gross total resection and present at a younger age.