Novel Kras-mutant murine models of non-small cell lung cancer possessing co-occurring oncogenic mutations and increased tumor mutational burden.

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 [KrasG12D (K), KrasG12DTp53-/-(KP), KrasG12DTp53+/-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.

Pediatric necrobiosis lipoidica: case report and review of the literature.

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.

Therapeutic plasma exchange for hyperviscosity syndrome secondary to high rheumatoid factor.

Hyperviscosity syndrome (HVS) is most commonly associated with Waldenstrom's macroglobulinemia, where it may be life-threatening. HVS may also occur in autoimmune diseases; data pertaining to efficacy of therapeutic plasma exchange (TPE) in HVS arising in non-malignant gammopathy are limited. We report a case of 71-year-old female with erosive rheumatoid arthritis with profoundly elevated rheumatoid factor (57,400 IU/ml; normal <35) who presented with findings consistent with HVS: profound weakness, headache, epistaxis and plasma viscosity (8.5 centipoise). She was successfully treated with pulsed high-dose steroids and TPE. Her symptoms of HVS have not recurred and the plasma viscosity has remained less than 3 centipoise. Given a slow onset of non-specific symptoms, HVS may be missed, incurring high risk of adverse effect. In symptomatic patients with high RF activity, a high index of suspicion for HVS is necessary to ensure timely identification and treatment with TPE, a safe and effective therapy.

Incidence, survival, pathology, and genetics of adult Latino Americans with glioblastoma.

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.

Early experience with formalin-fixed paraffin-embedded (FFPE) based commercial clinical genomic profiling of gliomas-robust and informative with caveats.

BACKGROUND: Commercial targeted genomic profiling with next generation sequencing using formalin-fixed paraffin embedded (FFPE) tissue has recently entered into clinical use for diagnosis and for the guiding of therapy. However, there is limited independent data regarding the accuracy or robustness of commercial genomic profiling in gliomas. METHODS: As part of patient care, FFPE samples of gliomas from 71 patients were submitted for targeted genomic profiling to one commonly used commercial vendor, Foundation Medicine. Genomic alterations were determined for the following grades or groups of gliomas; Grade I/II, Grade III, primary glioblastomas (GBMs), recurrent primary GBMs, and secondary GBMs. In addition, FFPE samples from the same patients were independently assessed with conventional methods such as immunohistochemistry (IHC), Quantitative real-time PCR (qRT-PCR), or Fluorescence in situ hybridization (FISH) for three genetic alterations: IDH1 mutations, EGFR amplification, and EGFRvIII expression. RESULTS: A total of 100 altered genes were detected by the aforementioned targeted genomic profiling assay. The number of different genomic alterations was significantly different between the five groups of gliomas and consistent with the literature. CDKN2A/B, TP53, and TERT were the most common genomic alterations seen in primary GBMs, whereas IDH1, TP53, and PIK3CA were the most common in secondary GBMs. Targeted genomic profiling demonstrated 92.3%-100% concordance with conventional methods. The targeted genomic profiling report provided an average of 5.5 drugs, and listed an average of 8.4 clinical trials for the 71 glioma patients studied but only a third of the trials were appropriate for glioma patients. CONCLUSIONS: In this limited comparison study, this commercial next generation sequencing based-targeted genomic profiling showed a high concordance rate with conventional methods for the 3 genetic alterations and identified mutations expected for the type of glioma. While it may not be feasible to exhaustively independently validate a commercial genomic profiling assay, examination of a few markers provides some reassurance of its robustness. While potential targeted drugs are recommended based on genetic alterations, to date most targeted therapies have failed in glioblasomas so the usefulness of such recommendations will increase with development of novel and efficacious drugs.

Systemic inflammation, intestine, and paraoxonase-1.

Serum paraoxonase 1 (PON1) has been shown to act as an important guardian against cellular damage from oxidized lipids in low-density lipoprotein (LDL), plasma membrane, against toxic agents such as pesticide residues including organophosphates and against bacterial endotoxin. PON1 associated with circulating high-density lipoprotein (HDL) has the ability to prevent the generation of pro inflammatory oxidized phospholipids by reactive oxygen species. The activities of the HDL-associated PON1 and several other anti-inflammatory factors in HDL are in turn negatively regulated by these oxidized lipids. In rabbits, mice, and humans there appears to be an increase in the formation of these oxidized lipids during the acute phase response. This results in the association of acute phase proteins with HDL and inhibition of the HDL-associated PON1 that renders HDL pro inflammatory.In populations, low serum HDL-cholesterol is a risk factor for atherosclerosis and efforts are directed toward therapies to improve the quality and the relative concentrations of LDL and HDL. Apolipoprotein A-I (apoA-I) has been shown to reduce atherosclerotic lesions in laboratory animals. ApoA-I, however, is a large protein that is costly and needs to be administered parenterally. Our group has developed apoA-I mimetic peptides that are much smaller than apoA-I (18 amino acids long vs 243 in ApoA-I itself). These HDL mimetic peptides are much more effective in removing the oxidized phospholipids and other oxidized lipids. They improve LDL and HDL composition and function and reduce lesion formation in animal models of atherogenesis. Following is a brief description of some of the HDL mimetic peptides that can improve HDL and the effect of the peptide on PON1 activity.

CCL21-DC in situ vaccination in murine NSCLC overcomes resistance to immunotherapy and generates systemic tumor-specific immunity.

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 (KrasG12D/P53+/-/Lkb1-/- (KPL); KrasG12D/P53+/- (KP); and KrasG12D (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.

Artifactual Displacement of Ductal Carcinoma In Situ (ADDCIS) (Toothpaste Effect): A Mimicker of Invasive Ductal Carcinoma.

Needle tract displacement is a recognized mimicker of invasive ductal carcinoma (IDC). Artifactual displacement of ductal carcinoma in situ (ADDCIS) unassociated with needle tracts may occur secondary to mechanical compression of breast specimens but has not been systematically studied. We identified 16 cases of ADDCIS unassociated with needle tract changes; the majority (75%) were internal referrals to the breast pathology service to rule out IDC, 19% were received as external diagnostic consultations to rule out IDC, and 6% were routine second review cases originally diagnosed as IDC at an outside hospital. The majority (62.5%) of ADDCIS occurred in lumpectomies, whereas 25% occurred in mastectomies and 12.5% in core biopsies. ADDCIS foci ranged from <1 to 5 mm; however, all ADDCIS spanning >4 mm demonstrated a linear pattern of displacement. In all cases, ADDCIS involved mammary stroma in a nonlobular distribution; in half, ADDCIS extended between benign lobules. Immunohistochemistry revealed no myoepithelial cells around the ADDCIS (n=7), adding to the concern for IDC. However, in contrast to most IDC, ADDCIS lacked stromal reaction and showed degenerative, smudged chromatin. None of the 9 patients with significant follow-up (mean, 7 y) developed metastasis. All received further local therapy for DCIS (5 radiation, 4 completion mastectomy); 1 received adjuvant systemic therapy (hormone therapy for contralateral IDC). In conclusion, ADDCIS mimics IDC, particularly given its permeative pattern and absence of myoepithelial cells. ADDCIS is most common in lumpectomies but can occur in mastectomies or core biopsies. Diagnostic clues include smudged nuclear chromatin, lack of stromal response, and linear pattern of displacement in larger lesions. The benign follow-up without systemic therapy supports our view that ADDCIS does not represent true IDC.

Extramammary Paget Disease of the Scrotum: A Contemporary Clinicopathologic Analysis of 20 Cases in the United States.

Extramammary Paget disease (EMPD) often involves apocrine gland-bearing locations including vulva and perianal area. EMPD of the scrotum is rare. Twenty patients were identified from the pathology files of 4 institutions between 2000 and 2018. Patients were 63- to 87-year-old (mean: 73 y) with a history of symptoms of between 4 months and 10 years. Two patients had a history of prostate cancer. Follow-up was available in 11 patients for a median of 71 months (range: 8 to 126 mo). Nine of 11 patients (82%) had positive margins, and 73% required reexcisions. Three patients had a focal dermal invasion, 1 of whom reportedly died of another etiology 25 months post diagnosis and 2 were disease-free at 24 and 68 months. No patient had inguinal lymphadenopathy. Two patients were alive with disease. Immunohistochemically, GATA3 and GCDFP15 were expressed in 6/6 cases, CK7 in 8/8 cases, and androgen receptor in 13/13 cases. HER2 was positive in 5/12 cases. PSA was positive in 1 patient who had a history of prostate cancer, whereas other prostate markers (NKX3.1 and prostein) were negative, and CK7 and GCDFP15 were positive, rendering primary EMPD diagnosis. Twelve other cases were negative for PSA and NKX3.1. In conclusion, EMPD of the scrotum has an insidious onset and its nonspecific symptoms can be misdiagnosed as dermatitis or fungal infection. Although localized EMPD has a favorable prognosis, the invasive disease is rare and did not predict metastasis or progression. Margins are frequently positive requiring reexcision. Occasionally, cases can be positive for PSA leading to diagnostic pitfalls.

Study on the correlations among disease activity index and salivary transforming growth factor-beta 1 and nitric oxide in ulcerative colitis patients.

Growth factors and nitric oxide (NO) play a major role in dysregulated immune response in ulcerative colitis (UC). Recent evidence has shown increased levels of transforming growth factor-beta(1) (TGF-beta(1)) in UC and suggested an anti-inflammatory effect for this factor. Based on our recent study, dysfunctional immunoregulation is present in saliva of UC patients, we hypothesized that salivary level of NO and TGF-beta(1) may differ by severity of UC and be useful to determine the activity of the disease. Thirty-seven UC patients and 15 healthy controls were enrolled and saliva samples were obtained. Truelove-Witts severity index and modified Truelove-Witts severity index were used to determine the severity of the disease. NO and TGF-beta(1) levels were detected in saliva of all patients and control subjects using enzyme-linked immunosorbent assay. A total of 21 patients had mild disease while 8 had moderate and 8 had severe colitis. Adjusted for baseline characteristics, the levels of NO and TGF-beta(1) in different groups were compared. Salivary NO and TGF-beta(1) levels were higher in UC patients comparing to controls (P < 0.00005 and P = 0.005, respectively). The levels of NO and TGF-beta(1) showed no significant differences among the severity groups (P = 0.46 and P = 0.23, respectively). NO levels linearly increased by age (Coeff = 1.5, r = 0.38, P = 0.02). Gender, extension of disease, and medical treatment did not affect NO and TGF-beta(1) levels. Although UC patients have abnormal amounts of NO and TGF-beta(1) in their saliva, their disease activity cannot be predicted by these factors, which may indicate a pathophysiologic role rather than being nonspecific inflammatory markers for TGF-beta(1) and NO.

Molecular Alterations in Patients with Pulmonary Adenocarcinoma Presenting with Malignant Pleural Effusion at the First Diagnosis.

OBJECTIVES: The aim of this study was to report cytologic and molecular features of pulmonary adenocarcinoma patients presenting with a malignant pleural effusion at the first diagnosis. STUDY DESIGN: Patients who had a cytopathologic diagnosis conclusive for lung adenocarcinoma for the first time on their pleural fluid specimen, and molecular testing done, were studied. The control group consisted of patients with a malignant pleural effusion that developed during disease progression. RESULTS: We identified 18 patients (9 males and 9 females). Micropapillary and/or solid adenocarcinoma type features predominated among cytologic specimens (n = 15), while acinar patterns predominated in controls. Survival was not significantly different from that of the control group (mean 13.8 vs. 13.9 months, respectively; p = 0.61). Ten (55%) cases had mutations in EGFR (n = 6; 60%), KRAS (n = 3; 30%), or ALK translocation (n = 1; 10%). No mutations were identified in BRAF, AKT, ERBB2, NRAS, or PIK3CA (tested in 7 patients). Patients positive for the tested mutations had a better overall survival than patients negative for the mutations (mean survival 16.2 vs. 6.05 months, respectively; p = 0.006, log-rank test). Ten (84%) control patients were positive for mutations in EGFR (n = 5; 42%), KRAS (n = 4; 34%), or ALK translocation (n = 1; 8.4%). CONCLUSION: In our series, a micropapillary-like and solid-like morphology, common in cytologic specimens, and alterations in EGFR were the most frequent identifiable molecular changes.

Clinicopathologic study of calcifying fibrous tumor of the gastrointestinal tract: a case series.

Calcifying fibrous tumor (CFT) is a rare benign mesenchymal lesion known to arise at multiple body sites that may clinically mimic other more aggressive lesions in the gastrointestinal (GI) tract. In this study we describe the clinicopathologic findings of 28 GI tract CFTs. Tumors predominantly arose in middle-aged adults with a slight female predominance. The most commonly involved sites were small bowel and colon, followed by stomach and appendix. Tumors ranged from 0.3 to 9.3 cm (median 1.4 cm), and submucosa was the most commonly involved layer. All tumors were well circumscribed and unencapsulated. Microscopically, tumors were hypocellular and composed of spindle cells with abundant, haphazardly arranged hyalinized collagen. No necrosis and less than one mitosis per 10 HPF were identified in all cases. Calcification was present in most (81%) of the cases. All cases had lymphoplasmacytic inflammatory infiltrates either scattered throughout the lesion with occasional perivascular conglomeration or in the form of lymphoid aggregates. A lymphoplasmacytic cuff was usually present (81%). Immunostains showed variable CD34 immunoreactivity and variable numbers of IgG4-positive plasma cells. The lesional cells were negative for DOG-1, ALK-1, S100, C-kit, Sox10, Melan A, HMB45, desmin, CK7, and CK20, and showed cytoplasmic staining for ß-catenin. Follow-up information was available in 5 cases with no recurrences reported to date (mean follow-up, 3 years). CFT is a rare benign tumor that can occur in part of the GI tract and should be distinguished from other mesenchymal tumors due to its low risk of recurrence.

Metastatic Colorectal Cancer in Hispanics: Treatment Outcomes in a Treated Population.

BACKGROUND: In United States Hispanics have disparities in the presentation and outcome of colorectal cancer (CRC) largely attributed to their late presentation and lower socioeconomic status. Impact of treatment, especially in the metastatic setting, in the observed outcome is an unexplored area. We explored the role of treatment in the outcome of metastatic CRC we performed a retrospective analysis to assess the contribution of demographics, tumor characteristics, and health care setting on survival differences. PATIENTS AND METHODS: We conducted a retrospective study of patients who were treated with metastatic CRC at Los Angeles County Hospital-University of Southern California (LAC-USC, a public hospital) and Norris Comprehensive Cancer Center (NCCC, private hospital) between 2002 and 2012. Both these institutions are staffed by the same providers and therefore treatment algorithms and access to drugs were similar. We identified metastatic CRC patients who received chemotherapy from administrative records. Demographics, tumor, and treatment related factors were collected. The primary end point was time to progression (TTP: time from the first day of chemotherapy to the date of progression). Overall survival (OS) was measured from the first day of chemotherapy to death or last follow-up. Descriptive statistics were used to describe the population and chi-square, Wilcoxon, and log-rank tests were used for comparison between the groups. RESULTS: A total of 242 patients, 44% Hispanic, 26% non-Hispanic whites (NHWs), 21% Asian and 9% black were included. Median TTP was 9.2 months (95% confidence interval [CI], 7.6-11.6) in Hispanics, and 20.7 months (95% CI, 9.6-27.5; P < .05) in NHWs. Median OS in Hispanics was 16.3 months (95% CI, 13.3-18.5), and in NHWs was 33.5 months (95% CI, 22.1-63.6; P < .001). Hispanics who were treated at LAC-USC had longer TTP in comparison to Hispanics at NCCC (P = .04). CONCLUSION: Hispanics with metastatic CRC have shorter TTP and OS on first line therapy when adjusted for health care setting, demographics, disease characteristics, and treatment factors.

Relationship Between [18F]FDOPA PET Uptake, Apparent Diffusion Coefficient (ADC), and Proliferation Rate in Recurrent Malignant Gliomas.

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.

Ribosomal Proteins RPS11 and RPS20, Two Stress-Response Markers of Glioblastoma Stem Cells, Are Novel Predictors of Poor Prognosis in Glioblastoma Patients.

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.

A practical approach to clinical and research biobanking.

Powerful technologies critical to personalized medicine and targeted therapeutics require the analysis of carefully validated, procured, stored, and managed biospecimens. Reflecting advancements in biospecimen science, the National Cancer Institute and the International Society for Biological and Environmental Repositories are periodically publishing best practices that can guide the biobanker. The modern biobank will operate more like a clinical laboratory with formal accreditation, standard operating procedures, and quality assurance protocols. This chapter highlights practical issues of consent, procurement, storage, quality assurance, disbursement, funding, and space. Common topics of concern are discussed including the differences between clinical and research biospecimens, stabilization of biospecimens during procurement, optimal storage temperatures, and technical validation of biospecimen content and quality. With quickly expanding biospecimen needs and limited healthcare budgets, biobanks may need to be selective as to what is stored. Furthermore, a shift to room-temperature storage modalities where possible can reduce long-term space and fiscal requirements.

The procurement, storage, and quality assurance of frozen blood and tissue biospecimens in pathology, biorepository, and biobank settings.

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.

A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories.

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.

Timeliness of adjuvant chemotherapy for stage III adenocarcinoma of the colon: a measure of quality of care.

BACKGROUND: Findings from multiple clinical trials established AC as a standard of care for stage III colon cancer. However, there is no recommended standard time for delivery of AC. We explored the timeliness of AC with FOLFOX as a predictor of recurrence and its role as a quality indicator in patients with stage III colon cancer. PATIENTS AND METHODS: We conducted a retrospective analysis of patients with colon cancer who received AC at Los Angeles County Hospital and Norris Cancer Center between 2003 and 2011. Time to recurrence (TTR) was the primary end point of the study, Kaplan-Meier curves and log-rank tests were used to assess the association between timing of the AC and TTR. RESULTS: We identified 102 patients with stage III colon cancer who had received AC. With a median follow-up of 3.2 years, time from surgery to AC was not a predictor of recurrence (P = .19). However, there was a nonsignificant trend toward higher risk of systemic recurrence when the delay of AC was more than 12 weeks (P = .068). Additionally, a significant association was found between age, race, type of hospital, and timeliness of AC. CONCLUSION: To date, our study is the largest data set to assess the timeliness of FOLFOX as a predictor of outcome in stage III colon cancer. Because FOLFOX is the current standard for AC for colon cancer, we report a trend toward worse outcome when FOLFOX is delayed more than 12 weeks. This result, thus supports quality measures to assess the timeliness of AC in stage III colon cancer and might have a meaningful effect on the care of patients with colon cancer.

Comparison of highly purified urinary versus recombinant FSH: Effect on ART outcomes in polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common cause of ovulatory disorders and infertility with high LH to FSH ratio. In order to prevent further increase of LH and follicle atresia, different regimens for ovulation induction have been recommended using FSH alone. OBJECTIVE: This study was performed in PCOS patients to compare ART outcomes in cycles induced by FSH alone, using either recombinant or urinary products. MATERIALS AND METHODS: In a randomized trial, from 623 patients who underwent down regulation with GnRH analogue in a long protocol, 160 PCOS patients were randomly divided into two groups of 80. Group A received 150 IU/d recombinant FSH (Gonal-F) and group B 150 IU/d urinary FSH (Fostimon). RESULTS: 33 cases (41.2%) in group A and 36 (45%) in group B achieved clinical pregnancy, which was not significantly different (p=0.67). Total number of oocytes retrieved (13.03±5.56 vs. 14.17±4.89, p=0.17), quality and number of embryos (7.42±3.35 vs. 7.63±3.28, p=0.68) and OHSS rate were similar in group A compared to group B. Endometrial thickness which was 9.66±1.67 mm in group A and 10.36±1.35 mm in group B, showed a significant difference (p=0.004). CONCLUSION: It seems that in PCOS patients, both pure FSH products used for controlled ovarian hyperstimulation have similar effects on ART outcome and can be used according to availability and patient acceptance without significant difference.