Keith Shults

Immunocytochemical analysis of MNDA in tissue sections and sorted normal bone marrow cells documents expression only in maturing normal and neoplastic myelomonocytic cells and a subset of normal and neoplastic B lymphocytes

The human myeloid cell nuclear differentiation antigen (MNDA) is a nuclear antigen known to be expressed in mature myelomonocytic cell lines. An extensive immunocytochemical evaluation of fixed tissues confirmed MNDA expression in normal maturing granulocytes and monocytes and in acute nonlymphocytic leukemias and chronic myelogenous leukemia. MNDA was not detected in normal tissue histiocytes but was found in activated macrophages and foreign body giant cells associated with inflammation. Flow cytometric cell sorting of normal bone marrow established that MNDA is initially expressed in myeloid blast cells. Examination of lymphoid tissues showed a low level of expression in a population of normal mande B lymphocytes but not in germinal center cells or plasma cells. A subset of B cell neoplasms expressing MNDA included hairy cell leukemia, parafollicular (monocytoid) B cell lymphoma, mantle cell lymphoma, and small lymphocytic lymphoma. Cell sorting of normal bone marrow showed MNDA expression in CD20+/CD10-/CD5- B cells. MNDA was not detected in other normal bone marrow or all other nonhematopoietic cells. The hematopoietic cell-specific pattern of MNDA expression was elucidated through a comprehensive analysis of normal and neoplastic tissues, and the results provide further evidence of the coexpression of B- and myeloid cell markers in neoplastic B cells and identify a normal B cell population that might be related to the cell of origin of a subset of B cell neoplasms.

Dysregulated Human Myeloid Nuclear Differentiation Antigen Expression in Myelodysplastic Syndromes: Evidence for a Role in Apoptosis

Reduced levels of human myeloid nuclear differentiation antigen (MNDA) gene transcripts have been detected in both familial and sporadic cases of myelodysplastic syndromes (MDS). Numerous reports implicate elevated apoptosis/programmed cell death and death ligands and their receptors in the pathogenesis of MDS. MNDA and related proteins contain the pyrin domain that functions in signaling associated with programmed cell death and inflammation. We tested the hypothesis that MNDA is involved in the regulation of programmed cell death in human myeloid hematopoietic cells. Clones of K562 cells (MNDA-null) that expressed ectopic MNDA protein were established using retroviral transduction. MNDA-expressing K562 clones were resistant to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis, but were not protected from programmed cell death induced with genotoxic agents or H(2)O(2). MNDA protein expression assessed in control and intermediate and high-grade MDS marrows showed several patterns of aberrant reduced MNDA. These variable patterns of dysregulated MNDA expression may relate to the variable pathophysiology of MDS. We propose that MNDA has a role regulating programmed cell death in myeloid progenitor cells, and that its down-regulation in MDS is related to granulocyte-macrophage progenitor cell sensitivity to TRAIL-induced programmed cell death.

A new biomarker for mitotic cells

Many epitopes are phosphorylated during mitosis. These epitopes are useful biomarkers for mitotic cells. The most commonly used are MPM‐2 and serine 10 of histone H3. Here we investigated the use of an antibody generated against a phospho peptide matching residues 774–788 of the human retinoblastoma protein 1 (Rb) to detect mitotic cells. Human cell lines were stained with DNA dyes and antibodies reactive with epitopes defined by antibody MPM‐2, phospho‐S10‐histone‐H3, and the phospho‐serine peptide, TRPPTLSPIPHIPRC (phospho‐S780‐Rb). Immunoreactivity and DNA content were measured by flow and image cytometry. Correlation and pattern recognition analyses were performed on list mode data. Western blots and immunoprecipitation were used to investigate the number of peptides reactive with phospho‐S780‐Rb and the relationship between reactivity with this antibody and MPM‐2. Costaining for bromodeoxyuridine (BrdU) was used to determine acid resistance of the phospho‐S780‐Rb epitope. Cell cycle related phospho‐S780‐Rb immunofluorescence correlated strongly with that of MPM‐2. Laser scanning cytometry showed that phospho‐S780‐Rb immunofluorescence is expressed at high levels on all stages of mitotic cells. Western blotting and immunoprecipitation showed that the epitope is expressed on several peptides including Rb protein. Costaining of BrdU showed that the epitope is stable to acid. Kinetic experiments showed utility in complex cell cycle analysis aimed at measuring cell cycle transition state timing. The phospho‐S780‐Rb epitope is a robust marker of mitosis that allows cytometric detection of mitotic cells beginning with chromatin condensation and ending after cytokinesis. Costaining of cells with DNA dyes allows discrimination and counting of mitotic cells and post‐cytokinetic (“newborn”) cells. To facilitate use without confusion about specificity, we suggest the trivial name, pS780 for this mitotic epitope.

A standardized ZAP-70 assay--lessons learned in the trenches.

The disease of chronic lymphocytic leukemia (CLL) has been shown to exhibit varying clinical outcomes based on reported laboratory parameters. One of these parameters involves the measurement of the protein levels of zeta‐associated protein (ZAP‐70) in CLL cells. A standardized assay has not yet reached consensus in the clinical cytometry community.

Bivariate analysis of the p53 pathway to evaluate Ad-p53 gene therapy efficacy

BACKGROUND Gene therapy of human tumors with adenovirus vectors presents a clinical research challenge and a potential opportunity in cancer therapy. One of the research challenges is that endpoints like tumor reduction, time to recurrence, and survival do not provide information about whether a potential therapeutic infects the targeted cells or whether the transferred gene functions or induces a cellular response. Therefore, a flow cytometric approach was developed for a wildtype, p53 encoding adenoviral vector (Ad-p53) that provides (1) the relative level of p53 transferred by p53 immunoreactivity, (2) mdm2 immunoreactivity as an assay of p53 activity, and (3) estimates of the percentage of infected cells by dual parameter analysis (p53 versus mdm2). METHODS Three prostate cancer cell lines (PC-3, LNCaP, DU 145) that are null, wild-type, and mutant for p53, respectively, and two ovarian cancer cell lines (PA1, MDAH 2774) that are wild-type and mutant for p53, respectively, were tested for immunoreactivity and lack of cross-reactivity with the monoclonal antibodies, DO-7 (anti-p53) and IF2 (anti-mdm2). Optimal dual staining conditions for a flow cytometric assay employing saturating levels of antibody were developed and tested by infection of PC-3, PA1, and MDAH 2774 with Ad-p53 or a control virus, Ad-luc. Dual staining with DO-7 and propidium iodide was used to determine any biological effect of the transferred gene. RESULTS Neither DO-7 nor IF2 showed appreciable cross-reactions by Western blot analysis of representative prostate or ovarian cell lines. By flow cytometric titration, DO-7 appears to be a high avidity antibody (saturation staining of 10(6) DU 145 cells with 0.5ug) whereas IF2 appears less so (optimum signal to noise ratio at 1ug/10(6) cells). Infection with Ad-p53 was detected at 6 to 48 hours post infection as a uniform relative increase in p53 levels over background p53 levels. Coincident increases in mdm2 immunoreactivity were also detected. DNA content measurements of PA1 and MDAH 2774 cells indicated that G1 arrest and/or apoptosis occurred subsequent to Ad-p53 infection. p53 and mdm2 levels and DNA content distributions for Ad-luc infected cells were equivalent to uninfected cells. CONCLUSIONS A flow cytometric approach to measure the efficacy of an Ad-p53 gene therapy vector was developed that detects not only the gene transferred but also the activity of the transferred gene product.

Overexpression of GSTA2 protects against cell cycle arrest and apoptosis induced by the DNA inter‐strand crosslinking nitrogen mustard, mechlorethamine

The effectiveness of bifunctional alkylating nitrogen mustard compounds in chemotherapy is related to their ability to form DNA inter‐strand crosslinks. Patients exposed to DNA inter‐strand crosslinking (ICL) agents subsequently experience an elevated incidence of myelodysplastic syndromes (MDS) and MDS related acute myeloid leukemia. Fanconis anemia (FA) patients are deficient in the repair of crosslink DNA damage and they experience a high incidence of MDS. These observations indicate that hematopoietic cells are specific target for the transforming effects of DNA crosslinking damage. Changes in transcript levels were characterized in human hematopoietic cells occurring in response to the nitrogen mustard, mechlorethamine (HN2), but not in response to monofunctional analogs. Only modest changes in a few gene transcripts were detected in HL60 cells exposed to levels of HN2 tittered to maximal dose that caused growth suppression with minimal cell death and allowed eventual resumption of normal cell growth. Under conditions of transient growth suppression, a subset of glutathione‐S‐transferase (GST) isoenzyme genes was consistently upregulated three to fourfold by HN2, but not by monofunctional analogs. Subsequent efforts to confirm the changes detected by microarray analyses revealed an unexpected dependence on treatment conditions. The GST alpha class A2 subfamily member transcripts were upregulated 24 h after a 1 h exposure to HN2 that caused an extensive, but transient block in late S/G2 cell cycle phase, but were minimally altered with continuous exposure. The 1‐h exposure to HN2 caused a transient late S/G2 cell cycle arrest in both the HL‐60 cell line and the Colo 320HSR human colon cancer cell line. Overexpression of GSTA2 by transient transfection protected Colo 320HSR cells against both cycle arrest and apoptosis following exposure to HN2. Overexpression of GSTA2 in Colo 320HSR cells induced after exposure to HN2 did not alter cycle arrest or apoptosis. The results indicate that human GSTA2 facilitates the protection of cells from HN2 damage and not repair. Our results are consistent with the possibility that GSTA2 polymorphisms, variable isoenzyme expression, and variable induced expression may be factors in the pathogenesis of MDS.

Quantitative Assessment of Myeloid Nuclear Differentiation Antigen Distinguishes Myelodysplastic Syndrome From Normal Bone Marrow

By using flow cytometry, we analyzed myeloid nuclear differentiation antigen (MNDA) expression in myeloid precursors in bone marrow from patients with myelodysplastic syndrome (MDS) and control samples from patients undergoing orthopedic procedures. The median percentage of MNDA-dim myeloid precursors in MDS cases was 67.4% (range, 0.7%-97.5%; interquartile range, 44.9%-82.7%) of myeloid cells, with bimodal MNDA expression in most MDS samples. Control samples demonstrated a median MNDA-dim percentage in myeloid precursors of 1.2% (range, 0.2%-13.7%; interquartile range, 0.6%-2.7%), with no bimodal pattern in most samples. The area under the receiver operating characteristic curve for MNDA-dim percentage in myeloid precursors was 0.96 (P = 9 × 10(-7)). Correlation of MNDA-dim levels with World Health Organization 2008 morphologic diagnoses was not significant (P = .21), but correlation with patient International Prognostic Scoring System scores suggested a trend (P = .07). Flow cytometric assessment of MNDA in myeloid precursors in bone marrow may be useful for the diagnosis of MDS.

Innovative Analyses Support a Role for DNA Damage and an Aberrant Cell Cycle in Myelodysplastic Syndrome Pathogenesis

We used flow cytometry to analyze the cell cycle, DNA damage, and apoptosis in hematopoietic subsets in MDS marrow. Subsets were assigned using CD45, side scatter, CD34, and CD71. Cell cycle fractions were analyzed using DRAQ 5 (DNA content) and MPM-2 (mitoses). DNA damage was assessed using p-H2A.X, and apoptosis using Annexin V. Compared to controls, MDS patients demonstrated no increased mitoses in erythroid, myeloid, or CD34+ cells. Myeloid progenitors demonstrated increased G2 cells, which with no increased mitoses suggested delayed passage through G2. Myeloid progenitors demonstrated increased p-H2A.X, consistent with DNA damage causing this delay. Annexin V reactivity was equivalent in MDS and controls. Results for each parameter varied among hematopoietic compartments, demonstrating the need to analyze compartments separately. Our results suggest that peripheral cytopenias in MDS are due to delayed cell cycle passage of marrow progenitors and that this delayed passage and leukemic progression derive from excessive DNA damage.

Abstract 1372: Quantification of PD-L1 and PD-1 expression on tumor cells in non-small cell lung cancer (NSCLC) using non-enzymatic tissue dissociation and flow cytometry

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Objective: The objective of this study was to develop a truly quantitative technology for PD-L1 expression in NSCLC. In addition, we also present a non-enzymatic technology that creates a tumor cell suspension from fresh tumor tissue so that either fine needle aspiration or fresh tissue can be used in this assay. Methods: 4 mm punches were taken from each tumor. Non-enzymatic tissue homogenization (IncellPREP; IncellDx, Menlo Park, CA) was performed. A French technique FNA was also taken from the same tumor to create matched sample sets. Cells were labeled with antibodies directed against CD45, PD-1, PD-L1, fixed and permeabilized then stained with DAPI to identify intact, single cells, and to analyze cell cycle. Results: Both FNA and IncellPREP generated greater than 1 million cells per mL with the IncellPREP yields being 2.5 times the number of cells per preparation compared to FNA (Mann-Whitney, p = 0.003). Comparing the IncellPREP homogenization and FNA, a strong correlation (r2-0.8) was demonstrated for expression of PD-L1. We compared PD-L1 expression by flow cytometry using a 1%a cut-off for positivity in the tumor cell population and a 1% cut-off of cells with at least 1+ intensity in immunohistochemically stained tissue sections as positive (Table 1). As demonstrated in the table, 10 of 12 lung tumor samples were concordant while 2 were discordant, one positive by flow and negative by IHC and one negative by flow and positive by IHC. PD-L1 expression by flow cytometry varied widely (1.2% to 89.4%) even in the positive concordant cases. In addition, PD-L1 expression in the aneuploid tumor population did not necessarily agree with the expression in the diploid tumor population. Summary: Fine, unequivocal, quantification of PD-L1 on tumor and immune cells in NSCLC may allow for better prediction of response to therapies. The present study also offers a technology that can create a universal sample type from either FNA or fresh tissue. View this table: Table 1 Citation Format: Amanda Chargin, Rian Morgan, Uma Sundram, Navneet Ratti, Keith Shults, Bruce Patterson. Quantification of PD-L1 and PD-1 expression on tumor cells in non-small cell lung cancer (NSCLC) using non-enzymatic tissue dissociation and flow cytometry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1372.