The reported reduction in cancer risk in those suffering from schizophrenia may be because antipsychotic medications have antineoplastic effects. In this study, 6 antipsychotic agents with a range of structural and pharmacological properties (reserpine, chlorpromazine, haloperidol, pimozide, risperidone and olanzapine), were screened for their effect on the viability of cell lines derived from lymphoblastoma, neuroblastoma, non-small cell lung cancer and breast adenocarcinoma. We aimed to determine if antipsychotic drugs in general possess cancer-specific cytotoxic potential, and whether it can be attributed to a common mode of action. With the exception of risperidone, all drugs tested displayed selective inhibition of the viability of cancer cell lines compared with normal cells. Using Affymetrix expression microarrays and quantitative real-time polymerase chain reaction, we found that for the antipsychotic drugs, olanzapine and pimozide, cytotoxicity appeared to be mediated via effects on cholesterol homeostasis. The role of cholesterol metabolism in the selective cytotoxicity of these drugs was supported by demonstration of their increased lethality when coadministered with a cholesterol synthesis inhibitor, mevastatin. Also, pimozide and olanzapine showed accelerating cytotoxic effects from 12 to 48 hr in time course studies, mirroring the time-dependent onset of cytotoxicity induced by the amphiphile, U18666A. On the basis of these results, we concluded that the Class II cationic amphiphilic properties of antipsychotic drugs contribute to their cytotoxic effects by acting on cholesterol homeostasis and altering the biophysical properties of cellular membranes, and that drugs affecting membrane-related cholesterol pathways warrant further investigation as potential augmentors of standard cancer chemotherapy.

Imaging flow cytometry is emerging as a diagnostic tool for the assessment of leukemia. It has the functionality of standard flow cytometry and generates high‐resolution digital images of each cell with quantifiable numerical data. We demonstrate the use of an automated high‐throughput method for performing fluorescence in situ hybridization (FISH) on immunophenotyped whole cells in suspension and analyzed by imaging flow cytometry, a technique called “Immuno‐flowFISH”. The aim of this study was to demonstrate the application of immuno‐flowFISH for the detection of chromosomal abnormalities in CLL, specifically trisomy 12 and del(17p). Mononuclear cells were isolated and immunophenotyped with fluorescently conjugated CD3, CD5, and CD19 monoclonal antibodies. Following fixation, cells were permeabilized, dsDNA denatured and hybridized with chromosome 12 or 17 enumeration (CEP 12 and CEP17) and 17p12 locus‐specific FISH probes. Cells were analyzed on the Amnis ImageStream®X Mark II to assess the number and percent FISH‐positive CLL cells and the ratio of FISH spot counts for CD5/CD19‐positive CLL cells to CD3/CD5‐positive T cells (FISH “mean spot ratio”). Deletion of 17p was detected in about 8% of cases to date, with del(17p) ranged from 3.5–22.8% and the FISH “mean spot ratio” 0.86–0.96. Immuno‐flowFISH also detected a minimal residual disease case with +12 with a limit of detection of 0.13% and a rare case that presented with atypical phenotype and cytogenetics. Immuno‐flowFISH could detect del(17p) in phenotypically identified CD5/CD19‐positive B‐cells. The 100‐fold increase in analyzed cells, as well as the addition of cell phenotype increased the sensitivity and specificity over current clinical FISH testing. Furthermore, immuno‐flowFISH analysis demonstrated specific utility in unique clinical scenarios such as residual disease and atypical biology cases which may be of significant benefit with regards to prognostication and MRD analysis. The method will assist in therapeutic decision making and disease monitoring for many hematological malignancies. © 2019 International Society for Advancement of Cytometry

AimsCytogenetic abnormalities involving the IGH gene are seen in up to 55% of patients with multiple myeloma. Current testing is performed manually by fluorescence in situ hybridisation (FISH) on purified plasma cells. We aimed to assess whether an automated imaging flow cytometric method that uses immunophenotypic cell identification, and does not require cell isolation, can identify IGH abnormalities.MethodsAspirated bone marrow from 10 patients with multiple myeloma were studied. Plasma cells were identified by CD38 and CD138 coexpression and assessed with FISH probes for numerical or structural abnormalities of IGH. Thousands of cells were acquired on an imaging flow cytometer and numerical data and digital images were analysed.ResultsUp to 30 000 cells were acquired and IGH chromosomal abnormalities were detected in 5 of the 10 marrow samples. FISH signal patterns seen included fused IGH signals for IGH/FGFR3 and IGH/MYEOV, indicating t(4;14) and t(11;14), respectively. In addition, three IGH signals were identified, indicating trisomy 14 or translocation with an alternate chromosome. The lowest limit of detection of an IGH abnormality was in 0.05% of all cells.ConclusionsThis automated high-throughput immuno-flowFISH method was able to identify translocations and trisomy involving the IGH gene in plasma cells in multiple myeloma. Thousands of cells were analysed and without prior cell isolation. The inclusion of positive plasma cell identification based on immunophenotype led to a lowest detection level of 0.05% marrow cells. This imaging flow cytometric FISH method offers the prospect of increased precision of detection of critical genetic lesions involving IGH and other chromosomal defects in multiple myeloma.

Del(17p) in chronic lymphocytic leukemia (CLL) and plasma cell myeloma has a unique genomic profile leading to refractoriness to conventional therapies and poor overall survival. Detection is generally by fluorescence in situ hybridization (FISH) on a slide with analysis of up to 200 nuclei, not necessarily all of being neoplastic cells. The small cell sample analyzed, and high threshold for a positive result (>5% positive cells) makes FISH a low precision test. This means false negative results are inevitable, especially if del(17p) is only in a minor clone, potentially leading to suboptimal treatment. To address this, we developed an automated FISH method with analysis on an imaging flow cytometer, an instrument with the functionality of a standard flow cytometer which generates high-resolution digital images of each cell. By combining immunophenotyping with FISH, on whole cells in a single test, we can detect chromosomal defects in cells with a specific phenotype. Aims: Our aim was to determine the capability of this automated integrated immunophenotyping-FISH imaging flow cytometry method to detect del(17p) in CLL and myeloma, the two commonest hematological malignancies, and in which this genetic defect encodes poor prognosis. We hypothesized that it would be more sensitive and specific than standard FISH. Methods: Bone marrow and/or blood samples in EDTA anticoagulant from 19 cases of CLL or myeloma, at diagnosis or on therapy, were studied. After red cell lysis, cells were incubated with fluorochrome-conjugated antibodies to CD3, CD5, CD19, CD38, and CD138 antigens (fluorochromes: BV480, BV605, AF647). Following fixation, cell membranes were permeabilized and double-stranded DNA denatured (78oC for 5 mins). FISH probes to the centromere of chromosome 17 (CEP17, Spectrum Green) and 17p12 locus (Orange-Red) were added and hybridized for 24 hours at 37oC. Nuclei were stained with SYTOX AADvanced. Data for up to 200,000 cells was collected on the Amnis® ImageStream®XMk II imaging flow cytometer. Digital images (x60) and quantitative data derived from computational algorithms (IDEAS software) were used to assess FISH signals overlying the nuclei of CD5/CD19-positive CLL cells or CD38/CD138-positive plasma cells for each probe. Digital images and quantitative data were assessed for FISH signals within immunophenotyped cells. Results: Between 10,000 and 200,000 (mean 60,000) cells were analyzed per sample. The FISH signals were seen on the digital images and confirmed by quantitative mean channel fluorescence intensity of the probes. There were 12 CLL cases with one 17p FISH signal in the CD5/CD19-positive population, with the number of del(17p) CLL cells ranging from 2 - 35% (or 0.4 - 23% of all cells analyzed) (Fig 1). This amounted to between 270 and 35,441 cells in the analysed sample with del(17p). The lowest del(17p) burden was in a patient on cytoreductive therapy. All CLL cells had normal diploid spots for the control CEP17 probe, and the CD3/CD5-positive T cells had dual signals for both CEP17 and 17p12 probes. There were 5 myeloma cases with 1 FISH signal for 17p overlying the nucleus of the CD38/CD138-positive plasma cells (Fig 2). In these cases, 52-90% of cells (15,600-18,000 cells) had a plasma cell phenotype and 13-19% of these (or 2,340-2,964 CD38/CD138-positive cells) showed del(17p). This represented 1-5% of all cells in the sample. All gated plasma cells had 2 FISH spots for CEP17. Conclusion: This imaging flow cytometry method that integrates FISH with immunophenotyping could detect del(17p) in CLL and myeloma with a lowest limit of detection of 0.4% and 1% respectively. The high sensitivity was achieved as many thousands of cells were analyzed, and 17p was only assessed in gated cells with the phenotype of interest (i.e. CD5/CD19 or CD38/CD138). This method, that includes positive cell identification by phenotype, precludes the need for prior cell sorting or purification. Imaging flow cytometry for del(17p) by "immuno-flowFISH" brings a new dimension to FISH analysis at diagnosis and for disease monitoring. Its high precision and specificity will enable detection of del(17p), even when only present in minor sub-clones, for therapeutic decision making and prognostic stratification. This technique has a real place in clinical assessment of del(17p) in CLL and myeloma and could be applied for other significant chromosomal defects of therapeutic and prognostic significance. Figure 1 Disclosures Augustson: Roche: Other: Support of parent study and funding of editorial support. Cheah:Celgene, F. Hoffmann-La Roche, Abbvie, MSD: Research Funding; Celgene, F. Hoffmann-La Roche, MSD, Janssen, Gilead, Ascentage Pharma, Acerta, Loxo Oncology, TG therapeutics: Honoraria.

No abstract