Flow cytometric analysis of fine needle aspirates is affected by tumor subtype, but not by anatomic location nor technique.

BACKGROUND: Current clinical practices are shifting towards utilizing less invasive biopsy techniques, including fine needle aspiration (FNA) and needle core biopsies. If a patient has a suspected hematologic malignancy, a portion of the FNA sample is typically submitted for flow cytometry (FC) analysis, providing valuable immunophenotypic data. METHODS: FNA specimens were identified via a pathology database search. All cases were morphologic evaluated and a subset of cases were analyzed by FC. RESULTS: 245 hematologic FNA specimens were identified; 84% of these cases had an adequate number of cells for FC analysis, and an unequivocal morphologic diagnosis (benign or malignant) was rendered in 85%. The percentage of cases with an unequivocal diagnosis was statistically significantly higher in those with associated FC than with those without FC (90% vs 58%). Neither FNA technique nor anatomic site affected the likelihood of obtaining an adequate sample for FC analysis and/or rendering a definitive morphologic or unequivocal FC diagnosis. Likewise, tumor subtype did not affect the likelihood of acquiring enough cells for FC analysis, but occasionally resulted in equivocal FC diagnoses or discordant FNA and FC diagnoses. Aggressive B-cell lymphomas and Hodgkin lymphomas were significantly less likely to be detected by FC as compared to low-grade B-cell lymphomas. Discrepancies between FNA and FC diagnoses occurred in 13% of cases. The majority of discrepancies (78%) included FC false negatives, while only 22% of cases had atypical or positive FC with negative FNA. CONCLUSIONS: FNA with associated FC is a powerful diagnostic technique; however, lymphoma subtype may affect diagnostic sensitivity by FC, and therefore, discordant FNA and FC results should be interpreted with caution.

Combining immune check-point blockade and cryoablation in an immunocompetent hormone sensitive murine model of prostate cancer.

BACKGROUND: Prostate cancer remains the second leading cause of cancer related death in men. Immune check point blocking antibodies have revolutionized treatment of multiple solid tumors, but results in prostate cancer remain marginal. Previous reports have suggested that local therapies, in particular cryoablation might increase tumor immunogenicity. In this work, we examine potential synergism between tumor cryoabalation and check point blocking antibodies. METHODS: FVB/NJ mice were injected subcutaneously into each flank with either 1 × 106 or 0.2 × 106 isogenic hormone sensitive Myc-Cap cells to establish synchronous grafts. Mice were treated with four intraperitoneal injections of anti-PD-1 (10 mg/kg), anti-CTLA-4 (1 mg/kg), or isotype control antibody with or without adjuvant cryoablation of the larger tumor graft and with or without neo-adjuvant androgen deprivation with degarelix (ADT). Mouse survival and growth rates of tumor grafts were measured. The immune dependency of observed oncological effects was evaluated by T cell depletion experiments. RESULTS: Treatment with anti-CTLA-4 antibody and cryoablation delayed the growth of the distant tumor by 14.8 days (p = 0.0006) and decreased the mortality rate by factor of 4 (p = 0.0003) when compared to cryoablation alone. This synergy was found to be dependent on CD3+ and CD8+ cells. Combining PD-1 blockade with cryoablation did not show a benefit over use of either treatment alone. Addition of ADT to anti-PD1 therapy and cryoablation doubled the time to accelerated growth in the untreated tumors (p = 0.0021) and extended survival when compared to cryoablation combined with ADT in 25% of the mice. Effects of combining anti-PD1 with ADT and cryoablation on mouse survival were obviated by T cell depletion. CONCLUSION: Trimodal therapy consisting of androgen deprivation, cryoablation and PD-1 blockade, as well as the combination of cryoablation and low dose anti-CTLA-4 blockade showed that local therapies with cryoablation could be considered to augment the effects of checkpoint blockade in prostate cancer.

Glycophenotype of breast and prostate cancer stem cells treated with thieno[2,3-b]pyridine anticancer compound.

Tumor progression may be driven by a small subpopulation of cancer stem cells (CSCs characterized by CD44+/CD24- phenotype). We investigated the influence of a newly developed thienopyridine anticancer compound (3-amino-5-oxo-N-naphthyl-5,6,7, 8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide, 1) on the growth, survival and glycophenotype (CD15s and GM3 containing neuraminic acid substituted with acetyl residue, NeuAc) of breast and prostate cancer stem/progenitor-like cell population. MDA-MB-231 and Du-145 cells were incubated with compound 1 alone or in combination with paclitaxel. The cellular metabolic activity was determined by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. The type of cell death induced by 48-h treatment was assessed using a combination of Annexin-V-FITC and propidium iodide staining. Flow cytometric analysis was performed to detect the percentage of CD44+/CD24- cells, and GM3 and CD15s positive CSCs, as well as the expression of GM3 and CD15s per one CSC, in both cell lines. Compound 1 produces a dose- and time-dependent cytotoxicity, mediated mainly by apoptosis in breast cancer cells, and slightly (2.3%) but statistically significant lowering breast CSC subpopulation. GM3 expression per one breast CSC was increased, and the percentage of prostate GM3+ CSC subpopulation was decreased in cells treated with compound 1 compared with non-treated cells. The percentage of CD15s+ CSCs was lower in both cell lines after treatment with compound 1. Considering that triple-negative breast cancers are characterized by an increased percentage of breast CSCs and knowing their association with an increased risk of metastasis and mortality, compound 1 is a potentially effective drug for triple-negative breast cancer treatment.

Metastatic Prostate Cancer Diagnosed by Bone Marrow Aspiration in an Elderly Man Not Undergoing PSA Screening.

Prostate cancer screening by PSA testing remains controversial, particularly in the elderly. Practice guidelines from most clinical societies suggest discontinuing PSA screening at age 70 while the USPSTF recommends against screening at any age. Recent reports have demonstrated an increased incidence of metastatic prostate cancer, with men aged 75 or older accounting for roughly half of those newly diagnosed at an incurable stage. We herein describe the case of an elderly gentleman with no history of prostate cancer screening who presented with anorexia and back pain of unclear etiology. Evaluation with bone marrow aspiration revealed a diagnosis of metastatic prostate cancer.

Tumor-Infiltrating Macrophages in Post-Transplant, Relapsed Classical Hodgkin Lymphoma Are Donor-Derived.

Tumor-associated inflammatory cells in classical Hodgkin lymphoma (CHL) typically outnumber the neoplastic Hodgkin/Reed-Sternberg (H/RS) cells. The composition of the inflammatory infiltrate, particularly the fraction of macrophages, has been associated with clinical behavior. Emerging work from animal models demonstrates that most tissue macrophages are maintained by a process of self-renewal under physiologic circumstances and certain inflammatory states, but the contribution from circulating monocytes may be increased in some disease states. This raises the question of the source of macrophages involved in human disease, particularly that of CHL. Patients with relapsed CHL following allogeneic bone marrow transplant (BMT) provide a unique opportunity to begin to address this issue. We identified 4 such patients in our archives. Through molecular chimerism and/or XY FISH studies, we demonstrated the DNA content in the post-BMT recurrent CHL was predominantly donor-derived, while the H/RS cells were derived from the patient. Where possible to evaluate, the cellular composition of the inflammatory infiltrate, including the percentage of macrophages, was similar to that of the original tumor. Our findings suggest that the H/RS cells themselves define the inflammatory environment. In addition, our results demonstrate that tumor-associated macrophages in CHL are predominantly derived from circulating monocytes rather than resident tissue macrophages. Given the association between tumor microenvironment and disease progression, a better understanding of macrophage recruitment to CHL may open new strategies for therapeutic intervention.

Development of paroxysmal nocturnal hemoglobinuria in CALR-positive myeloproliferative neoplasm.

Paroxysmal nocturnal hemoglobinuria (PNH), a disease characterized by intravascular hemolysis, thrombosis, and bone marrow failure, is associated with mutations in the PIG-A gene, resulting in a deficiency of glycosylphosphatidylinositol-anchored proteins. Many hypotheses have been posed as to whether PNH and PIG-A mutations result in an intrinsic survival benefit of CD55(-)/CD59(-) cells or an extrinsic permissive environment that allows for their clonal expansion within the bone marrow compartment. Recent data have identified the concurrence of PIG-A mutations with additional genetic mutations associated with myeloproliferative disorders, suggesting that some presentations of PNH are the result of a stepwise progression of genetic mutations similar to other myelodysplastic or myeloproliferative syndromes. We report for the first time in the literature the development of clinically significant PNH in a patient with JAK2V617F-negative, CALR-positive essential thrombocythemia, providing further support to the hypothesis that the development of PNH is associated with the accumulation of multiple genetic mutations that create an intrinsic survival benefit for clonal expansion. This case study additionally highlights the utility of genomic testing in diagnosis and the understanding of disease progression in the clinical setting.

Software-automated counting of Ki-67 proliferation index correlates with pathologic grade and disease progression of follicular lymphomas.

OBJECTIVES: To examine the accuracy of software-assisted measurement of the Ki-67 proliferation index (PI) and its correlation with the grade and clinical progression of follicular lymphoma (FL). METHODS: High-power field equivalents were extracted from H&E- and Ki-67-immunostained slides of FL, and a nuclear quantitation algorithm was used to calculate a PI. Representative fields were manually counted for validation with close agreement. RESULTS: The PI was significantly higher in World Health Organization grade 3 FL than grade 1 to 2 FL. Disease progression, as defined by subsequent treatment with radiation or cytotoxic chemotherapy, was also significantly associated with elevated PI but not pathologic grade. CONCLUSIONS: These data show that software-automated quantitation of Ki-67 can provide both a useful adjunct to pathologic grade in FL and improved prognostic information for patients.

Targeted pathologic evaluation of bone marrow donors identifies previously undiagnosed marrow abnormalities.

Potential bone marrow donors are screened to ensure the safety of both the donor and recipient. At our institution, potential donors with abnormal peripheral blood cell counts, a personal history of malignancy, or age >60 years are evaluated to ensure that they are viable candidates for donation. Evaluation of the marrow includes morphologic, flow cytometric, and cytogenetic studies. A total of 122 potential donors were screened between the years of 2001 and 2011, encompassing approximately 10% of all donors. Of the screened potential donors, the mean age was 59 years and there were 59 men and 63 women. The donors were screened because of age >60 years (n = 33), anemia (n = 22), cytopenias other than anemia (n = 27), elevated peripheral blood counts without a concurrent cytopenia (n = 20), elevated peripheral blood counts with a concurrent cytopenia (n = 10), history of malignancy (n = 4), abnormal peripheral blood differential (n = 3), prior graft failure (n = 1), history of treatment with chemotherapy (n = 1), and body habitus (n = 1). Marrow abnormalities were detected in 9% (11 of 122) of donors. These donors were screened because of anemia (5 of 22, 23%), age >60 years (2 of 33, 6%), history of malignancy (2 of 4, 50%), elevated peripheral blood counts (1 of 20, 5%), and body habitus (1 of 1, 100%). Abnormalities included plasma cell dyscrasia (n = 3), abnormal marrow cellularity (n = 3), clonal cytogenetic abnormalities (n = 2), low-grade myelodysplastic syndrome (1), a mutated JAK2 V617F allele (n = 1), and monoclonal B cell lymphocytosis (n = 1). Our experience indicates that extended screening of potential donors identifies a significant number of donors with previously undiagnosed marrow abnormalities.

Flow cytometric findings in hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is an often fatal hyperinflammatory syndrome. HLH may be inherited, but it more commonly arises secondary to Epstein-Barr virus (EBV) or other infections, hematologic malignancies, or rheumatologic diseases. We identified 17 patients diagnosed with HLH who had flow cytometric analysis of peripheral blood or bone marrow performed at the time of diagnosis. Two patients had primary HLH, and the others had HLH secondary to EBV infection, hematologic malignancies, rheumatologic conditions, or tuberculosis. The marrow typically showed a reactive lymphocytosis and a marked left shift in myelopoiesis regardless of the etiology. Qualitative abnormalities were also found in several cases, including T-cell abnormalities in the majority of the EBV-associated HLH cases. While not specific, flow cytometric findings in HLH are different from the findings in uninvolved marrow samples, and care should be taken not to overinterpret immunophenotypic findings in these cases as indicative of a primary marrow disorder or lymphoma.

Clinical and pathologic features of secondary acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a relatively common form of acute myeloid leukemia (AML) that has an excellent prognosis. In contrast, secondary acute myeloid leukemias, including therapy-related AML and AML with myelodysplasia-related changes, have a relatively poor prognosis. We identified 9 cases of APL at our institution in which there was a history of chemotherapy, radiotherapy, chronic immunosuppression, or antecedent myelodysplastic syndrome. The clinical and pathologic findings in these cases of secondary APL were compared with the clinical and pathologic findings in cases of de novo APL. We found that secondary and de novo APL had abnormal promyelocytes with similar morphologic and immunophenotypic features, comparable cytogenetic findings, comparable rates of FMS-like tyrosine kinase mutations, and similar rates of recurrent disease and death. These data suggest that secondary APL is similar to de novo APL and, thus, should be considered distinct from other secondary acute myeloid neoplasms.

RNase H and multiple RNA biogenesis factors cooperate to prevent RNA:DNA hybrids from generating genome instability.

Genome instability, a hallmark of cancer progression, is thought to arise through DNA double strand breaks (DSBs). Studies in yeast and mammalian cells have shown that DSBs and instability can occur through RNA:DNA hybrids generated by defects in RNA elongation and splicing. We report that in yeast hybrids naturally form at many loci in wild-type cells, likely due to transcriptional errors, but are removed by two evolutionarily conserved RNase H enzymes. Mutants defective in transcriptional repression, RNA export and RNA degradation show increased hybrid formation and associated genome instability. One mutant, sin3Δ, changes the genome profile of hybrids, enhancing formation at ribosomal DNA. Hybrids likely induce damage in G1, S and G2/M as assayed by Rad52 foci. In summary, RNA:DNA hybrids are a potent source for changing genome structure. By preventing their formation and accumulation, multiple RNA biogenesis factors and RNase H act as guardians of the genome.

Metabolic and electrochemical mechanisms of dimeric naphthoquinones cytotoxicity in breast cancer cells.

Cancer cells reprogram their metabolism due to genetic alteration to compensate for increased energy demand and enhanced anabolism, cell proliferation, and protection from oxidative damage. Here, we assessed the cytotoxicity of three dimeric naphthoquinones against the glycolytic MCF-7 versus the oxidative MDA-453 breast carcinoma cell lines. Dimeric naphthoquinones 1 and 2 impaired MDA-453, but not MCF-7, cell growth at IC(50)=15 µM. Significant increase in reactive oxygen species, decrease in oxygen consumption and ATP production were observed in MDA-453 cells but not in MCF-7 cell. These findings suggest that oxidative stress and mitochondrial dysfunction are mechanisms by which these agents exert their cytotoxic effects. Cyclic voltammetry and semi-empirical molecular orbital calculations further characterized the electrochemical behavior of these compounds. These results also suggest that dimeric naphthoquinones may be used to selectively target cancer cells that depend on oxidative phosphorylation for energy production and macromolecular synthesis.

Gene expression pathways of high grade localized prostate cancer.

BACKGROUND: Despite advances in screening and local therapy, prostate cancer remains the second most common cause of cancer related death among American men, with those having high grade disease being at highest risk for prostate cancer mortality. Here we identify the genes and cellular pathways that distinguish high grade from low grade pathologically localized prostate cancer. METHODS: Cancer cells from low grade (Gleason 3 + 3 = 6) or high grade (4 + 4 = 8) tumors of men with localized disease were isolated by laser capture micro-dissection. Expression profiling was conducted across 18,344 unique annotated genes and data were analyzed using packages from the R/Bioconductor project to determine differential gene expression and perform gene set enrichment analysis. Publically available expression data was retrieved and analyzed individually in the same manner and in cross platform meta-analyses. RESULTS: Six hundred seventy genes were differentially expressed between Gleason sum 6 and 8 tumors with a false discovery rate of <5% (P < 0.0014) including genes previously shown to mediate prostate cancer survival, proliferation, and metastasis. Functional themes associated with Gleason grade included developmental processes, signal transduction, chemokine and embryonic stem cell pathways with specific enrichment of the androgen receptor, EGFR, TNF-alpha, and Notch signaling cascades. CONCLUSIONS: In addition to androgen receptor signaling, growth factor, and cytokine mediated pathways are active in clinically localized high grade prostate cancer. The availability of therapeutics that selectively target these pathways encourages the development of clinical trials for their selective use in the neoadjuvant or adjuvant setting in men at high risk for disease progression.

Dimeric naphthoquinones, a novel class of compounds with prostate cancer cytotoxicity.

OBJECTIVES: • To evaluate the cytotoxicity of dimeric naphthoquinones (BiQs) in prostate cancer cells. • To assess the interaction of dimeric naphthoquinones with common therapies including radiation and docetaxel. MATERIALS AND METHODS: • The cytotoxicity of 12 different dimeric naphthoquinones was assessed in androgen-independent (PC-3, DU-145) and androgen-responsive (LNCaP, 22RV1) prostate cancer cell lines and in prostate epithelial cells (PrECs). • BiQ2 and BiQ11 were selected for determination of dose response, effects on colony formation and initial exploration into mechanism of action. • Synergistic effects with radiation and docetaxel were explored using colony-forming and MTT assays. RESULTS: • At concentrations of 15µM, BiQ2, BiQ3, BiQ11, BiQ12, and BiQ15 demonstrated cytotoxicity in all prostate cancer cell lines. • Treatment with BiQs limited the ability of prostate cancer cells to form colonies in clonogenic assays. • Exposure of prostate cancer to BiQs increased cellular reactive oxygen species (ROS), decreased ATP production, and promoted apoptosis. • BiQ cytotoxicity was independent of NADP(H):quinone oxidoreductase 1 (NQO1) activity in PrECs, PC-3 and 22RV1, but not DU-145 cells. • Exposure of prostate cancer cells to radiation before treatment with BiQs increased their activity allowing for inhibitory effects well below the IC(50) s of these compounds in PrECs. • Co-administration of BiQs with docetaxel had minimal additive effects. CONCLUSIONS: • Dimeric naphthoquinones represent a new class of compounds with prostate cancer cytotoxicity and synergistic effects with radiation. The cytotoxic effect of these agents is probably contributed to by the accumulation of ROS and mitochondrial dysfunction. • Further studies are warranted to better characterize this class of potential chemo-therapeutics.

A chemical genetic screen for modulators of asymmetrical 2,2'-dimeric naphthoquinones cytotoxicity in yeast.

BACKGROUND: Dimeric naphthoquinones (BiQ) were originally synthesized as a new class of HIV integrase inhibitors but have shown integrase-independent cytotoxicity in acute lymphoblastic leukemia cell lines suggesting their use as potential anti-neoplastic agents. The mechanism of this cytotoxicity is unknown. In order to gain insight into the mode of action of binaphthoquinones we performed a systematic high-throughput screen in a yeast isogenic deletion mutant array for enhanced or suppressed growth in the presence of binaphthoquinones. METHODOLOGY/PRINCIPAL FINDINGS: Exposure of wild type yeast strains to various BiQs demonstrated inhibition of yeast growth with IC(50)s in the microM range. Drug sensitivity and resistance screens were performed by exposing arrays of a haploid yeast deletion mutant library to BiQs at concentrations near their IC(50). Sensitivity screens identified yeast with deletions affecting mitochondrial function and cellular respiration as having increased sensitivity to BiQs. Corresponding to this, wild type yeast grown in the absence of a fermentable carbon source were particularly sensitive to BiQs, and treatment with BiQs was shown to disrupt the mitochondrial membrane potential and lead to the generation of reactive oxygen species (ROS). Furthermore, baseline ROS production in BiQ sensitive mutant strains was increased compared to wild type and could be further augmented by the presence of BiQ. Screens for resistance to BiQ action identified the mitochondrial external NAD(P)H dehydrogenase, NDE1, as critical to BiQ toxicity and over-expression of this gene resulted in increased ROS production and increased sensitivity of wild type yeast to BiQ. CONCLUSIONS/SIGNIFICANCE: In yeast, binaphthoquinone cytotoxicity is likely mediated through NAD(P)H:quonine oxidoreductases leading to ROS production and dysfunctional mitochondria. Further studies are required to validate this mechanism in mammalian cells.

Induction of ectopic Myc target gene JAG2 augments hypoxic growth and tumorigenesis in a human B-cell model.

Ectopic Myc expression plays a key role in human tumorigenesis, and Myc dose-dependent tumorigenesis has been well established in transgenic mice, but the Myc target genes that are dependent on Myc levels have not been well characterized. In this regard, we used the human P493-6 B cells, which have a preneoplastic state dependent on the Epstein-Barr viral EBNA2 protein and a neoplastic state with ectopic inducible Myc, to identify putative ectopic Myc target genes. Among the ectopic targets, JAG2 that encodes a Notch receptor ligand Jagged2, was directly induced by Myc. Inhibition of Notch signaling through RNAi targeting JAG2 or the gamma-secretase Notch inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester (DAPT) preferentially inhibited the neoplastic state in vitro. Furthermore, P493-6 tumorigenesis was inhibited by DAPT in vivo. Ectopic expression of JAG2 did not enhance aerobic cell proliferation, but increased proliferation of hypoxic cells in vitro and significantly increased in vivo tumorigenesis. Furthermore, the expression of Jagged2 in P493-6 tumors often overlapped with regions of hypoxia. These observations suggest that Notch signaling downstream of Myc enables cells to adapt in the tumor hypoxic microenvironment.