M2 tumor-associated macrophages play important role in predicting response to neoadjuvant chemotherapy in triple-negative breast carcinoma.

PURPOSE: Two types of macrophages are present in tumor microenvironment. M1 macrophages exhibit potent anti-tumor properties, while M2 macrophages play the pro-tumoral roles. The presence of M2 macrophages is associated with worsened overall survival in triple-negative breast carcinoma (TNBC) patients. However, the relationship between M2 macrophages and response to neoadjuvant chemotherapy (NAC) is unknown. METHODS: M2 macrophages were investigated on biopsy whole sections from 66 TNBCs treated with NAC by CD163 together with other immune checkpoint markers (PD1, PD-L1 and CD8) using a multi-color immunohistochemical multiplex assay. RESULTS: Incomplete response was significantly associated with older age, lower PD-L1 expression (tumor and stroma), lower levels of CD8-positive TILs in stroma, but higher level of CD163-positive macrophages, with the level of CD163-positive M2 macrophages in peritumoral area as the strongest factor. CONCLUSIONS: Our data have demonstrated that the level of CD163-positive M2 macrophages was significantly higher in TNBC patients with incomplete response than patients with complete response, suggesting M2 macrophages' important role in predicting TNBC patients' response to NAC.

HER2 intratumoral heterogeneity is independently associated with distal metastasis and overall survival in HER2-positive breast carcinomas.

PURPOSE: Human epidermal growth factor receptor 2 (HER2) intratumoral heterogeneity (ITH) occurs in a subset of breast cancers. Our recent study revealed it as an independent predictive factor for the response to anti-HER2 neoadjuvant therapy. In this study, we aimed to investigate its association with distal metastasis. METHODS: HER2 ITH was assessed using HER2 gene protein assay (GPA) on whole tissue sections of pretreatment biopsies from a cohort of 158 HER2-positive invasive breast carcinomas and correlated with patients' clinical follow-up outcomes along with other clinicopathologic characteristics. RESULTS: Fifty-seven cases (36%) showed HER2 ITH including 19 with genetic, 8 with both genetic and non-genetic, and 30 with non-genetic ITH. Multivariate analysis demonstrated larger tumor size, positive resected lymph node(s), negative PR, and the presence of HER2 ITH were independently associated with distal metastasis. Additionally, multivariate analysis demonstrated larger tumor size and the presence of HER2 ITH were the only independent factors associated with decreased overall survival (death). CONCLUSION: The presence of HER2 ITH is an independent factor associated with poor overall survival and increased distal metastasis in HER2-positive breast cancer patients.

Performance Analysis of Leica Biosystems Monoclonal Antibody Programmed Cell Death Ligand 1 Clone 73-10 on Breast, Colorectal, and Hepatocellular Carcinomas.

Programmed cell death receptor 1/Programmed cell death ligand 1 (PD-L1) checkpoint pathway is responsible for the control of immune cell responses. Immunotherapy using checkpoint inhibitors, such as anti-PD-L1 therapy, aids disease management and potentiates clinical outcomes. This study aimed to analyze the performance of the Leica Biosystems (LBS) USA FDA class I in vitro diagnostic monoclonal antibody (clone 73-10) to detect PD-L1 expression in breast, colorectal, and hepatocellular carcinomas compared with the class III FDA-approved PD-L1 detecting antibodies [SP263 (Ventana), 22C3 (Dako), and 28-8 (Dako)] using 208 unique tissue microarray-based cases for each tumor type. The interassay concordances between LBS 73-10 clone and other PD-L1 antibodies ranged from 0.59 to 0.95 Cohen kappa coefficient (K) and from 0.66 to 0.90 (K) for cutoff values of 1% and 50% tumor proportion score (TPS), respectively. The 73-10 clones showed inter-pathologist agreements ranging from 0.53 to 1.0 (K) and 0.34 to 0.94 (K) for cutoff values of 1% and 50% TPS, respectively. For the immune cell proportion score (IPS) using a cutoff of 1%, the Kappa coefficient of interassay concordances and inter-pathologist agreements ranged from 0.34 to 0.94. The 73-10 clone assay's sensitivity ranged from 78.3% to 100% (TPS ≥1%), 100% (TPS ≥50%), and 77.4% to 93.5% (IPS ≥1%), while its specificity was 97.9% to 100% (TPS ≥1%), 99.5% to 99.8% (TPS ≥50%), and 97.9% to 100% (IPS ≥1%). This exploratory evaluation of LBS 73-10 monoclonal antibody on a large set of breast, colorectal, and hepatocellular carcinomas showed the assay's technical performance is comparable to the FDA-approved companion/complementary diagnostics PD-L1 detection assays.

Kinetics of nuclear-cytoplasmic translocation of Foxo1 and Foxo3A in adult skeletal muscle fibers.

In skeletal muscle, the transcription factors Foxo1 and Foxo3A control expression of proteins that mediate muscle atrophy, making the nuclear concentration and nuclear-cytoplasmic movements of Foxo1 and Foxo3A of therapeutic interest in conditions of muscle wasting. Here, we use Foxo-GFP fusion proteins adenovirally expressed in cultured adult mouse skeletal muscle fibers to characterize the time course of nuclear efflux of Foxo1-GFP in response to activation of the insulin-like growth factor-1 (IGF-1)/phosphatidylinositol-3-kinase (PI3K)/Akt pathway to determine the time course of nuclear influx of Foxo1-GFP during inhibition of this pathway and to show that Akt mediates the efflux of nuclear Foxo1-GFP induced by IGF-1. Localization of endogenous Foxo1 in muscle fibers, as determined via immunocytochemistry, is consistent with that of Foxo1-GFP. Inhibition of the nuclear export carrier chromosome region maintenance 1 by leptomycin B (LMB) traps Foxo1 in the nucleus and results in a relatively rapid rate of Foxo1 nuclear accumulation, consistent with a high rate of nuclear-cytoplasmic shuttling of Foxo1 under control conditions before LMB application, with near balance of unidirectional influx and efflux. Expressed Foxo3A-GFP shuttles ∼20-fold more slowly than Foxo1-GFP. Our approach allows quantitative kinetic characterization of Foxo1 and Foxo3A nuclear-cytoplasmic movements in living muscle fibers under various experimental conditions.

Localization and regulation of the N terminal splice variant of PGC-1α in adult skeletal muscle fibers.

The transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) regulates expression of genes for metabolism and muscle fiber type. Recently, a novel splice variant of PGC-1α (NT-PGC-1α, amino acids 1-270) was cloned and found to be expressed in muscle. Here we use Flag-tagged NT-PGC-1α to examine the subcellular localization and regulation of NT-PGC-1α in skeletal muscle fibers. Flag-NT-PGC-1α is located predominantly in the myoplasm. Nuclear NT-PGC-1α can be increased by activation of protein kinase A. Activation of p38 MAPK by muscle activity or of AMPK had no effect on the subcellular distribution of NT-PGC-1α. Inhibition of CRM1-mediated export only caused relatively slow nuclear accumulation of NT-PGC-1α, indicating that nuclear export of NT-PGC-1α may be mediated by both CRM1-dependent and -independent pathways. Together these results suggest that the regulation of NT-PGC-1α in muscle fibers may be very different from that of the full-length PGC-1α, which is exclusively nuclear.

TRPS1 Expression in Breast Carcinomas: Focusing on Metaplastic Breast Carcinomas.

TRPS1 has been recently demonstrated as a highly sensitive and specific marker for breast carcinomas. To further explore TRPS1's utility in breast carcinoma, we systematically evaluated TRPS1 expression on tissue microarrays from 160 estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-positive, 94 ER-/HER2+, 117 triple-negative breast carcinomas, and 618 other primary carcinomas (cholangiocarcinoma, endometrial, colorectal, and hepatocellular carcinomas), and whole tissue sections from 64 HER2+, 76 triple-negative, and 67 metaplastic breast carcinomas. The results showed TRPS1 was highly expressed in breast carcinomas (100% of HER2+ and 97.4% of triple negative on whole tissue sections), but almost completely negative in other tested tumor types. TRPS1 was also highly expressed in metaplastic carcinoma (91%), significantly higher than GATA3 (55.2%). The different expression between TRPS1 and GATA3 was most prominent in chondroid/mesenchymal subtypes (100% vs. 36.4%), followed by spindle cell carcinoma (66.7% vs. 44.4%). In addition, TRPS1 was expressed in normal breast ductal epithelial cells with less staining than in carcinoma cells, and TRPS1 showed aberrant membranous staining in HER2+ breast carcinomas that suggests a potential cross-reactivity with HER2 protein.

Unusual staining of immunohistochemical markers PAX8 and CDX2 in breast carcinoma: a potential diagnostic pitfall.

Knowing the sensitivity and specificity of tissue-specific immunohistochemical markers is crucial for accurate determination of the primary tumor site. PAX8 has been used as a diagnostic marker for carcinomas of the gynecologic tract, kidney, and thyroid gland, and CDX2 has been used as a marker of gastrointestinal carcinoma. Neither is considered a marker for breast carcinoma (BC). However, we have encountered BCs that express PAX8 or CDX2, some of which caused diagnostic confusion. We investigated the immunohistochemical staining frequency of PAX8 and CDX2 in BC. We identified 237 BCs for which PAX8 staining results were reported (102 primary and 135 metastatic BCs); seven primary and four metastatic BCs (4.6%) were positive for PAX8, with various intensities and staining patterns. CDX2 staining results were reported for 271 BCs (78 primary and 193 metastatic); four primary BCs and one metastatic BC (1.8%) were positive for CDX2, ranging from focal and weak to diffuse and strong. We also stained primary invasive BCs with PAX8 and CDX2 using tissue microarrays. None of the 332 PAX8-stained cases was positive, while one of 143 CDX2-stained cases was positive. Four PAX8-positive and three CDX2-positive cases were stained with TRPS1, and all were positive for TRPS1. In addition, we reviewed the literature for PAX8 and CDX2 expression in BCs and found 5.5% PAX8-positive BCs (90/1625) in 17 studies and 0.8% CDX-2 positive BCs (7/909) in 20 studies. PAX8 and CDX2 are infrequently expressed in BC by immunohistochemistry, and in rare cases, the staining can be strong and diffuse. Additional diagnostic markers are necessary and helpful in distinguishing breast from other primary origins.

TRPS1, GATA3, and SOX10 expression in triple-negative breast carcinoma.

A diagnostic dilemma can be encountered when primary triple-negative breast carcinoma (TNBC) without an in situ component or metastatic TNBCs lose the currently used organ-specific marker such as GATA3, raising concerns about metastatic carcinoma from other sites. In the current study, we compared the newly identified breast marker TRPS1 with currently used breast markers GATA3 and SOX10 in whole-tissue sections from 315 cases of various subtypes of TNBC. TRPS1 was highly expressed in 100% of triple-negative primary and metastatic invasive lobular carcinomas, 99% of triple-negative primary and metastatic invasive breast carcinoma of no special type (IBC-NST), and 95% of metaplastic breast carcinomas. In contrast, GATA3 and SOX10 were expressed in 94% and 0% of invasive lobular carcinomas, 63% and 74% of IBC-NST, and 50% and 49% of metaplastic breast carcinomas, respectively. For special-type TNBCs, both TRPS1 and GATA3 were negative in acinic cell carcinomas, most cribriform adenoid cystic carcinomas, and neuroendocrine carcinomas, but positive in secretory carcinomas. Triple-negative apocrine carcinoma was the only subtype of TNBC with positive GATA3 but negative TRPS1. These data indicate that TRPS1 is a highly sensitive marker for TNBCs with positivity not only in GATA3/SOX10-positive TNBCs but also in almost all GATA3/SOX10-negative TNBCs.

Prognostic value of androgen receptor expression and molecular alterations in metastatic triple-negative or low hormone receptor breast carcinomas.

Metastatic breast carcinomas (BCs) with phenotype of triple-negative (TNBC) or low hormonal receptor levels [estrogen receptor (ER)/progesterone receptor (PR) < 10% and HER2-] are mainly treated with cytotoxic chemotherapy. Targeting androgen receptor (AR) pathway may represent a potential new therapeutic strategy in such group of BCs. We evaluated AR expression by immunohistochemistry and genetic alterations by next-generation sequencing. Among 114 metastatic BCs, 37 (32.5%) cases showed AR expression and 77 (67.5%) lacked AR expression. Statistical analysis revealed that AR expression is associated with older age, lobular carcinoma, positive ER and positive PR in primary tumors, and lymph node metastasis. Patients with AR-positive tumors had significantly longer metastatic intervals and overall survivals. In addition, AR-positive tumors had significantly higher rate of PI3CA mutation. Our results demonstrated that AR expression has prognostic value in this subgroup of metastatic BCs and tumors with AR expression had different molecular alterations compared with those without AR expression.

DNA binding sites target nuclear NFATc1 to heterochromatin regions in adult skeletal muscle fibers.

We have previously demonstrated that Ca²+/calcineurin-dependent dephosphorylation of the transcription factor nuclear factor of activated T cells subtype 1 (NFATc1) during repetitive skeletal muscle activity causes NFAT nuclear translocation and concentration in subnuclear NFAT foci. We now show that NFAT nuclear foci colocalize with heterochromatin regions of intense staining by DAPI or TO-PRO-3 that are present in the nucleus prior to NFATc1 nuclear entry. Nuclear NFATc1 also colocalizes with the heterochromatin markers trimethyl-histone H3 (Lys9) and heterochromatin protein 1α. Mutation of the NFATc1 DNA binding sites prevents entry and localization of NFATc1 in heterochromatin regions. However, fluorescence in situ hybridization shows that the NFAT-regulated genes for slow and fast myosin heavy chains are not localized within the heterochromatin regions. Fluorescence recovery after photobleaching shows that within a given nucleus, NFATc1 redistributes relatively rapidly (t(¹/2) < 1 min) between NFAT foci. Nuclear export of an NFATc1 mutant not concentrated in NFAT foci is accelerated following nuclear entry during fiber activity, indicating buffering of free nuclear NFATc1 by NFATc1 within the NFAT foci. Taken together, our results suggest that NFAT foci serve as nuclear storage sites for NFATc1, allowing it to rapidly mobilize to other nuclear regions as required.

Alpha-adrenergic signalling activates protein kinase D and causes nuclear efflux of the transcriptional repressor HDAC5 in cultured adult mouse soleus skeletal muscle fibres.

The protein kinase PKD1 has recently been linked to slow fibre-type gene expression in fast skeletal muscle through phosphorylation of class II histone deacetylase (HDAC) molecules, resulting in nuclear efflux of HDAC and consequent activation of the transcription factor MEF2. However, possible upstream activators of PKD, and the time course and signalling pathway of downstream effectors have not been determined in skeletal muscle. Using fluorescent fusion proteins HDAC5-green fluorescent protein (GFP) and PKD1-mPlum expressed in fibres isolated from predominantly slow soleus muscle and maintained for 4 days in culture, we now show that alpha-adrenergic receptor activation by phenylephrine causes a transient, PKD-dependent HDAC5-GFP nuclear efflux. Concurrent to this response, PKD1-mPlum transiently redistributes from cytoplasm to plasma membrane and nuclei, and back, during 2 h exposure to phenylephrine. The recovery may reflect alpha-receptor desensitization. In contrast, the phorbol ester PMA (phorbol-12-myristate-13-acetate, a pharmacological mimic of the downstream mediator diacylglycerol in alpha-adrenergic signalling), caused continuous PKD-dependent HDAC5-GFP nuclear efflux and maintained PKD1-mPlum redistribution. In the absence of expressed HDAC, PMA increased histone H3 acetylation and increased MEF2 reporter activity in a PKD-dependent manner, consistent with PKD phosphorylation of endogenous HDAC(s) and reduced nuclear HDAC activity due to HDAC nuclear efflux. HDAC5-GFP did not respond to PMA in fibres from predominantly fast flexor digitorum brevis (FDB) muscle, but did in FDB fibres expressing exogenous PKD1. Our results demonstrate that a PKD-mediated signalling pathway for HDAC nuclear efflux is activated in slow skeletal muscle through adrenergic input, which is typically active in parallel with motor neurone input during muscular activity.

Activity- and calcineurin-independent nuclear shuttling of NFATc1, but not NFATc3, in adult skeletal muscle fibers.

The transcription factor NFATc1 may be involved in slow skeletal muscle gene expression. NFATc1 translocates from cytoplasm to nuclei during slow fiber type electrical stimulation of skeletal muscle fibers because of activation of the Ca(2+)-dependent phosphatase calcineurin, resulting in nuclear factor of activated T-cells (NFAT) dephosphorylation and consequent exposure of its nuclear localization signal. Here, we find that unstimulated adult skeletal muscle fibers exhibit a previously unanticipated nucleocytoplasmic shuttling of NFATc1 without appreciable nuclear accumulation. In resting fibers, the nuclear export inhibitor leptomycin B caused nuclear accumulation of NFATc1 (but not of isoform NFATc3) and formation of NFATc1 intranuclear bodies independent of calcineurin. The rate of nuclear uptake of NFATc1 was 4.6 times lower in resting fibers exposed to leptomycin B than during electrical stimulation. Inhibitors of glycogen synthase kinase and protein kinase A or of casein kinase 1 slowed the decay of nuclear NFATc1 after electrical stimulation, but they did not cause NFATc1 nuclear uptake in unstimulated fibers. We propose that two nuclear translocation pathways, one pathway mediated by calcineurin activation and NFAT dephosphorylation and the other pathway independent of calcineurin and possibly independent of NFAT dephosphorylation, determine the distribution of NFATc1 between cytoplasm and nuclei in adult skeletal muscle.

The PD-1/PD-L1 Axis in HER2+ Ductal Carcinoma In Situ (DCIS) of the Breast.

OBJECTIVES: The aims were to evaluate the programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) axis in ductal carcinoma in situ (DCIS) of the breast. METHODS: We reviewed 85 pure DCIS cases treated with surgical excision at our institution, including 51 luminal A (estrogen receptor [ER] positive/human epidermal growth factor 2 [HER2] negative), 15 luminal B (ER+/HER2+), 13 HER2 (ER-/HER2+), and six basal-like (ER-/HER2-/CK5/6+). The extent and intensity of PD-1 and PD-L1 immunohistochemical staining in the tumor-infiltrating lymphocytes (TILs) and in the tumor cells were recorded. RESULTS: Our study found that moderate/severe inflammation around DCIS correlated with HER2 expression (20/28 HER2+ cases [71%] vs 21/57 HER2- cases [37%], P = .005). Of interest, over half of the TILs around the HER2 subtype expressed PD-L1 (7/13, 54%). In addition, about one-third of TILs around the HER2 subtype expressed PD-1 (4/13, 31%). CONCLUSIONS: These findings suggest that immune-based therapeutic strategies may be used as a potential therapy in DCIS cases with PD-L1 overexpression, especially those of the HER2 molecular subtype.

Interplay of RUNX1/MTG8 and DNA methyltransferase 1 in acute myeloid leukemia.

The translocation t(8;21)(q22;q22) in acute myeloid leukemia (AML) results in the expression of the fusion protein RUNX1/MTG8, which in turn recruits histone deacetylases (HDAC) to silence RUNX1 target genes [e.g., interleukin-3 (IL-3)]. We previously reported that expression of the RUNX1/MTG8 target gene IL-3 is synergistically restored by the combination of inhibitors of HDACs (i.e., depsipeptide) and DNA methyltransferases (DNMT; i.e., decitabine) in RUNX1/MTG8-positive Kasumi-1 cells. Thus, we hypothesized that DNMT1 is also part of the transcriptional repressor complex recruited by RUNX1/MTG8. By a chromatin immunoprecipitation assay, we identified a RUNX1/MTG8-DNMT1 complex on the IL-3 promoter in Kasumi-1 cells and in primary RUNX1/MTG8-positive AML blasts. The physical association of RUNX1/MTG8 with DNMT1 was shown by coimmunoprecipitation experiments. Furthermore, RUNX1/MTG8 and DNMT1 were concurrently released from the IL-3 promoter by exposure to depsipeptide or stabilized on the promoter by decitabine treatment. Finally, we proved that RUNX1/MTG8 and DNMT1 were functionally interrelated by showing an enhanced repression of IL-3 after coexpression in 293T cells. These results suggest a novel mechanism for gene silencing mediated by RUNX1/MTG8 and support the combination of HDAC and DNMT inhibitors as a novel therapeutic approach for t(8;21) AML.

The CD4/CD8 ratio of tumor-infiltrating lymphocytes at the tumor-host interface has prognostic value in triple-negative breast cancer.

Compelling evidence has demonstrated the prognostic value of tumor-infiltrating lymphocytes (TILs), especially in triple-negative breast cancer (TNBC). However, only a limited number of studies to investigate the importance of the subsets of T cells in TILs have been carried out, less so the significance of the location of these TILs. In this study, we explored in a cohort of 42 consecutive TNBC cases the prognostic significance of TIL subsets at the tumor-host interface (within 1 high-power field [0.5 mm] of the invasive front) and compared them with TILs within the intratumoral stroma. Given the reported importance of TILs in HER2-overexpressing breast cancer, a subset of such tumors was also included for comparison. The range was wide in both locations; nevertheless, the mean CD4+ and CD8+ T cell count was significantly higher at the tumor-host interface than that found within the intratumoral stroma (both P<.0001). The number of CD4+ or CD8+ T cells at either location was not significantly associated with distant relapse-free or overall survival. However, the CD4/CD8 ratio at the tumor-host interface was significantly associated with both relapse-free survival (hazard ratio 0.2, P=.002) and overall survival (hazard ratio 0.13, P=.002), whereas this association was not seen for the CD4/CD8 ratio within the intratumoral stroma. As expected, both tumor size and nodal status were significantly associated with survival outcomes. The findings further support the contention that TILs, as markers of regional immune escape, are of prognostic importance in TNBC progression and that the CD4/CD8 ratio of TILs at the tumor-host interface plays a distinctive role, thus appearing to be of clinical relevance.

Prognostic outcomes in advanced breast cancer: the metastasis-free interval is important.

Metastatic breast cancer is a heterogeneous disease with a diverse clinical course. There have been limited studies regarding prognostic outcomes in patients with de novo metastatic breast cancer versus those with metastatic recurrence, with controversial observations. In this study, we sought to examine the difference in survival outcomes among patients with advanced breast cancer stratified based on metastasis-free interval (MFI) and to further explore the role of systemic therapy in these patient groups. Of 569 consecutive patients with stage IV breast cancer between 1998 and 2013, 201 had de novo metastatic disease (metastasis at diagnosis) and 368 developed metastatic recurrence, including 168 with an MFI≤24 months and 200 with an MFI>24 months. In the 492 patients who received systemic therapy, de novo metastasis was an independent favorable prognostic factor for overall survival after metastasis when compared with metastatic recurrence irrespective of MFI. Compared with the patients with metastatic recurrence with an MFI≤24 months, those with an MFI>24 months had a superior survival outcome, although it did not reach statistical significance by multivariate analysis. In contrast, de novo metastatic breast cancer was associated with a worse prognosis when compared with recurring metastasis in the patients who did not receive systemic treatment. These findings provide more insight into the natural history of advanced breast cancer, thus necessitating further investigation into the molecular mechanism of drug resistance.

Diagnostic utility of IDH1/2 mutations to distinguish dedifferentiated chondrosarcoma from undifferentiated pleomorphic sarcoma of bone.

Histologically, it is nearly impossible to distinguish the dedifferentiated component of dedifferentiated chondrosarcoma from undifferentiated pleomorphic sarcoma (UPS) of bone when the low-grade cartilaginous component is absent. Previous studies have revealed that isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations are present in a significant number of cartilaginous tumors including most conventional chondrosarcomas and dedifferentiated chondrosarcomas. These mutations have not been studied in UPSs of bone. We sought to investigate whether an IDH1 or IDH2 mutation signature could be used as a clinically diagnostic marker for the distinction of dedifferentiated component of chondrosarcoma from UPS of bone. Sixty-eight bone tumor cases, including 31 conventional chondrosarcomas, 23 dedifferentiated chondrosarcomas, and 14 UPSs of bone, were collected for IDH1/2 mutation analysis either using the Qiagen IDH1/2 RGQ PCR Kit or using whole-exome sequencing. IDH1/2 mutations were detected in 87% (20/23) of dedifferentiated chondrosarcomas and 30% (6/20) of conventional chondrosarcomas. No mutations were detected in the IDH1/2 codon 132 or codon 172 among 14 UPSs of bone. Identification of IDH1 or IDH2 mutations supports the diagnosis of dedifferentiated chondrosarcoma rather than UPS of bone while also providing some insight into the pathogenesis of these 2 lesions.

Cellular uptake and intracellular levels of the bcl-2 antisense g3139 in cultured cells and treated patients with acute myeloid leukemia.

PURPOSE: Down-regulation of Bcl-2 by the antisense G3139, currently under clinical evaluations, could restore chemosensitivity in otherwise resistant malignant cells. To date, the mechanism of intracellular accumulation of G3139 following in vivo administration remains to be elucidated. This study aimed to assess whether detectable intracellular concentrations of G3139 are achievable in vivo and how these relate to Bcl-2 down-regulation. EXPERIMENTAL DESIGN: Cellular uptake of G3139 was studied in leukemia myeloid cell lines and blasts collected from treated patients using a newly developed, novel, and highly sensitive ELISA-based assay. Real-time reverse transcription-PCR was used to quantify Bcl-2 mRNA changes in treated cells. RESULTS: The assay was fully validated and showed a limit of quantification of 50 pmol/L. When exposed to 0.33 to 10 mumol/L G3139, K562 cells exhibited intracellular concentrations in the range of 2.1 to 11.4 pmol/mg protein. When G3139 was delivered with cationic lipids, a 10- to 25-fold increase of the intracellular concentrations was observed. There was an accumulation of G3139 in the nuclei, and the ratio of nucleus to cytoplasm was increased 7-fold by cationic lipids. Intracellular concentrations of G3139 were correlated with Bcl-2 mRNA down-regulation. Robust intracellular concentrations of G3139 were achieved in vivo in bone marrow (range, 3.4-40.6 pmol/mg protein) and peripheral blood mononuclear cells (range, 0.47-19.4 pmol/mg protein) from acute myeloid leukemia patients treated with G3139. CONCLUSIONS: This is the first evidence that measurable intracellular levels of G3139 are achievable in vivo in acute myeloid leukemia patients and that Bcl-2 down-regulation is likely to depend on the achievable intracellular concentrations rather than on plasma concentrations.

Clinicopathologic factors associated with de novo metastatic breast cancer.

While breast cancers with distant metastasis at presentation (de novo metastasis) harbor significantly inferior clinical outcomes, there have been limited studies analyzing the clinicopathologic characteristics in this subset of patients. In this study, we analyzed 6126 breast cancers diagnosed between 1998 and 2013 to identify factors associated with de novo metastatic breast cancer. When compared to patients without metastasis at presentation, race, histologic grade, estrogen/progesterone receptor (ER/PR) and HER2 statuses were significantly associated with de novo metastasis in the entire cohort, whereas age, histologic grade, PR and HER2 status were the significant parameters in the subset of patients with locally advanced breast cancer (Stage IIB/III). The patients with de novo metastatic breast cancer had a significant older mean age and a lower proportion of HER2-positive tumors when compared to those with metastatic recurrence. Further, the HER2-rich subtype demonstrated a drastically higher incidence of de novo metastasis when compared to the luminal and triple-negative breast cancers in the entire cohort [odds ratio (OR)=5.68 and 2.27, respectively] and in the patients with locally advanced disease (OR=4.02 and 2.12, respectively), whereas no significant difference was seen between de novo metastatic cancers and those with metastatic recurrence. Moreover, the luminal and HER2-rich subtypes showed bone-seeking (OR=1.92) and liver-homing (OR=2.99) characteristics, respectively, for the sites of de novo metastasis, while the latter was not observed in those with metastatic recurrence. Our data suggest that an algorithm incorporating clinicopathologic factors, especially histologic grade and receptor profile, remains of significant benefit during decision making in newly diagnosed breast cancer in the pursuit of precision medicine.

Prognostic Value of E-Cadherin and ß-Catenin in Triple-Negative Breast Cancer.

OBJECTIVES: To analyze the expression of E-cadherin and ß-catenin in triple-negative breast cancer (TNBC) to assess their prognostic significance. METHODS: The expression of E-cadherin and ß-catenin was examined semiquantitatively and correlated with other pathologic factors and survival outcomes. RESULTS: Of 72 consecutive TNBCs, 56% showed reduced membranous expression of E-cadherin or ß-catenin, with a strong correlation to each other. Of the clinicopathologic factors analyzed, tumor size and nodal status were significantly associated with overall survival and disease-specific survival, while the latter remained an independent factor by multivariate analysis. Reduced E-cadherin and ß-catenin were both significantly associated with a poor overall survival and disease-specific survival by univariate and multivariate analyses. CONCLUSIONS: E-cadherin and ß-catenin expression provides discriminative prognostic power independent of conventional pathologic factors, thus further reinforcing the important role of cell adhesion molecules in the process of tumor metastasis, especially in TNBC.