Analytical assay validation for acute myeloid leukemia measurable residual disease assessment by multiparametric flow cytometry.

BACKGROUND: Measurable residual disease (MRD) assessed by multiparametric flow cytometry (MFC) has gained importance in clinical decision-making for acute myeloid leukemia (AML) patients. However, complying with the recent In Vitro Diagnostic Regulations (IVDR) in Europe and Food and Drug Administration (FDA) guidance in the United States requires rigorous validation prior to their use in investigational clinical trials and diagnostics. Validating AML MRD-MFC assays poses challenges due to the unique underlying disease biology and paucity of patient specimens. In this study, we describe an experimental framework for validation that meets regulatory expectations. METHODS: Our validation efforts focused on evaluating assay accuracy, analytical specificity, analytical and functional sensitivity (limit of blank (LoB), detection (LLoD) and quantitation (LLoQ)), precision, linearity, sample/reagent stability and establishing the assay background frequencies. RESULTS: Correlation between different MFC methods was highly significant (r = 0.99 for %blasts and r = 0.93 for %LAIPs). The analysis of LAIP specificity accurately discriminated from negative control cells. The assay demonstrated a LoB of 0.03, LLoD of 0.04, and LLoQ of 0.1%. Precision experiments yielded highly reproducible results (Coefficient of Variation <20%). Stability experiments demonstrated reliable measurement of samples up to 96 h from collection. Furthermore, the reference range of LAIP frequencies in non-AML patients was below 0.1%, ranging from 0.0% to 0.04%. CONCLUSION: In this manuscript, we present the validation of an AML MFC-MRD assay using BM/PB patient specimens, adhering to best practices. Our approach is expected to assist other laboratories in expediting their validation activities to fulfill recent health authority guidelines.

Human breast cancer histoid: an in vitro 3-dimensional co-culture model that mimics breast cancer tissue.

Progress in our understanding of heterotypic cellular interaction in the tumor microenvironment, which is recognized to play major roles in cancer progression, has been hampered due to unavailability of an appropriate in vitro co-culture model. The aim of this study was to generate an in vitro 3-dimensional human breast cancer model, which consists of cancer cells and fibroblasts. Breast cancer cells (UACC-893) and fibroblasts at various densities were co-cultured in a rotating suspension culture system to establish co-culture parameters. Subsequently, UACC-893, BT.20, or MDA.MB.453 were co-cultured with fibroblasts for 9 days. Co-cultures resulted in the generation of breast cancer histoid (BCH) with cancer cells showing the invasion of fibroblast spheroids, which were visualized by immunohistochemical (IHC) staining of sections (4 µm thick) of BCH. A reproducible quantitative expression of C-erbB.2 was detected in UACC-893 cancer cells in BCH sections by IHC staining and the Automated Cellular Imaging System. BCH sections also consistently exhibited qualitative expression of pancytokeratins, p53, Ki-67, or E-cadherin in cancer cells and that of vimentin or GSTPi in fibroblasts, fibronectin in the basement membrane and collagen IV in the extracellular matrix. The expression of the protein analytes and cellular architecture of BCH were markedly similar to those of breast cancer tissue.

Formamide as a denaturant for bisulfite conversion of genomic DNA: Bisulfite sequencing of the GSTPi and RARbeta2 genes of 43 formalin-fixed paraffin-embedded prostate cancer specimens.

Analysis of methylated DNA, which refers to 5-methycytosine (5mC) versus cytosine (C) at specific loci in genomic DNA (gDNA), has received increased attention in epigenomics, particularly in the area of cancer biomarkers. Many different methods for analysis of methylated DNA rely on initial reaction of gDNA with concentrated acidic sodium bisulfite to quantitatively convert C to uracil (U) via sulfonation of denatured, single-stranded gDNA under conditions where 5mC is resistant to analogous sulfonation leading to thymine (T). These methods typically employ polymerase chain reaction (PCR) amplification after bisulfite conversion, thereby leading to readily detectable amounts of amplicons where T and C are measured as surrogates for C and 5mC in the original unconverted gDNA. However, incomplete bisulfite conversion of C in gDNA has been reported to be a common source of error in analysis of methylated DNA. Incomplete conversion can be revealed during the course of bisulfite sequencing, which is the generally accepted "gold standard" for analysis of methylated DNA. Previous bisulfite sequencing investigations of conventional predenaturation of gDNA with NaOH followed by the use of bisulfite containing added urea to maintain denaturation and thus mitigate incomplete conversion of C have been reported to give conflicting results. The current study describes a new approach where conventional predenaturation of gDNA with NaOH is instead achieved with formamide and maintains denaturation during subsequent sample handling and sulfonation. This formamide-based method was applied to 46 formalin-fixed/paraffin-embedded (FFPE) biopsy tissue specimens from well-characterized patients with primary prostate cancer. These specimens were representative of difficult-to-analyze samples due to the chemically compromised nature of the gDNA, which was recovered by modifying the protocol for a commercially available total RNA/DNA extraction kit (RecoverALL). An additional novel aspect of this study was analysis of CpG-rich promoter regions of two prostate cancer-related genes: glutathione S-transferase pi (GSTPi) and retinoic acid receptor beta2 (RARbeta2). High-quality bisulfite sequencing results were obtained for both genes in 43 of 46 (93%) specimens. Detection of methylated GSTPi and RARbeta2 genes was significantly associated with primary prostate cancer as compared with the benign prostate (Fisher's exact test, P < 0.001). The sensitivity and specificity of detection of methylated GSTPi and RARbeta2 genes were 86% and 100% and 91% and 100%, respectively. Moreover, the presence of either methylated gene was detected in primary prostate cancer with sensitivity and specificity of 100% and 100%, respectively. The results demonstrated a high degree of reliability of formamide-based denaturation and bisulfite conversion that should extend, generally, to FFPE and other types of samples intended for any analytical method predicated on bisulfite conversion. This pilot study also demonstrated the efficacy of determining methylation of these two genes with high sensitivity and specificity in FFPE biopsy tissue specimens. Moreover, the results showed a highly significant association of methylated GSTPi and RARbeta2 genes with primary prostate cancer. Finally, this improved procedure for determining these two methylated genes may allow the detection of prostate cancer cells in core biopsy specimens with insufficient numbers of cells and poor morphology.

Unmethylated E-cadherin gene expression is significantly associated with metastatic human prostate cancer cells in bone.

BACKGROUND: The concurrent determination of methylation status of E-cadherin gene and E-cadherin protein expression remains scant in metastatic prostate cancer cells in bone, the most prevalent site for metastatic growth. Therefore, the study was undertaken to ascertain the methylation status of E-cadherin gene, a most frequent and known epigenetic mechanism of its regulation, and the protein expression in prostate tissue biopsy specimen. METHODS: The methylation of E-cadherin gene was determined by methylation specific-PCR and the protein expression by immunohistochemical method in the consecutive sections of each prostate tissue biopsy specimen. RESULTS: The unmethylated E-cadherin gene and homogeneous E-cadherin protein expression was significantly associated with BPH as compared to the primary prostate carcinoma (Fisher's Exact P < 0.001). A significant association was observed between the concurrent methylated gene and markedly reduced expression of the protein in the primary prostate cancer cells as compared to the BPH cells, suggesting methylation-dependent regulation of the gene expression in these cases. In contrast to the primary cancer, a highly significant increase in the frequency of metastatic prostate cancer cells in bone exhibited the concurrent expression of unmethylated gene and homogeneous protein (Fisher's Exact P < 0.001). CONCLUSIONS: The study clearly demonstrated a significant association of the concurrent expression of unmethylated E-cadherin gene and E-cadherin protein with metastatic prostate cancer cells in bone, and that its expression may have a role in the intercellular adhesion in the formation of metastatic lesions in bone.

Nilotinib treatment in mouse models of P190 Bcr/Abl lymphoblastic leukemia.

BACKGROUND: Ph-positive leukemias are caused by the aberrant fusion of the BCR and ABL genes. Nilotinib is a selective Bcr/Abl tyrosine kinase inhibitor related to imatinib, which is widely used to treat chronic myelogenous leukemia. Because Ph-positive acute lymphoblastic leukemia only responds transiently to imatinib therapy, we have used mouse models to test the efficacy of nilotinib against lymphoblastic leukemia caused by the P190 form of Bcr/Abl. RESULTS: After transplant of 10,000 highly malignant leukemic cells into compatible recipients, untreated mice succumbed to leukemia within 21 days, whereas mice treated with 75 mg/kg nilotinib survived significantly longer. We examined cells from mice that developed leukemia while under treatment for Bcr/Abl kinase domain point mutations but these were not detected. In addition, culture of such cells ex vivo showed that they were as sensitive as the parental cell line to nilotinib but that the presence of stromal support allowed resistant cells to grow out. Nilotinib also exhibited impressive anti-leukemia activity in P190 Bcr/Abl transgenic mice that had developed overt leukemia/lymphoma masses and that otherwise would have been expected to die within 7 days. Visible lymphoma masses disappeared within six days of treatment and leukemic cell numbers in peripheral blood were significantly reduced. Treated mice survived more than 30 days. CONCLUSION: These results show that nilotinib has very impressive anti-leukemia activity but that lymphoblastic leukemia cells can become unresponsive to it both in vitro and in vivo through mechanisms that appear to be Bcr/Abl independent.

Use of bone marrow-derived macrophages to model murine innate immune responses.

The innate immune system is composed of neutrophils and monocyte/macrophages. As a cell type, bone marrow-derived macrophage (BMM) are easier to study than neutrophils since they are still capable of cell division and have a longer life span. However, in comparison with neutrophils, few methodological studies on the production of reactive oxygen species (ROS) by such macrophages have been reported. Here we present studies on ROS production of this cell type under various conditions including the use of different priming and stimulating agents. In addition, we report that the de novo adhesion of BMM to tissue culture plates induces superoxide anion production and this can be further enhanced by stimulation with PMA. BMM are able to adhere to endothelial cells that have been activated by TNF-alpha exposure, and under these circumstances also generate ROS. We explored different methods to introduce gene products into BMM without activating them to avoid complicating subsequent studies of ROS production. Infection with lentiviral vectors was very efficient, allowed long-term expression and did not activate the BMM. We conclude that BMM are very suitable for the biochemical study of the oxidative burst.