Oncogenic kinase NPM/ALK induces through STAT3 expression of immunosuppressive protein CD274 (PD-L1, B7-H1).

The mechanisms of malignant cell transformation caused by the oncogenic, chimeric nucleophosmin (NPM)/anaplastic lymphoma kinase (ALK) remain only partially understood, with most of the previous studies focusing mainly on the impact of NPM/ALK on cell survival and proliferation. Here we report that the NPM/ALK-carrying T cell lymphoma (ALK+TCL) cells strongly express the immunosuppressive cell-surface protein CD274 (PD-L1, B7-H1), as determined on the mRNA and protein level. The CD274 expression is strictly dependent on the expression and enzymatic activity of NPM/ALK, as demonstrated by inhibition of the NPM/ALK function in ALK+TCL cells by the small molecule ALK inhibitor CEP-14083 and by documenting CD274 expression in IL-3-depleted BaF3 cells transfected with the wild-type NPM/ALK, but not the kinase-inactive NPM/ALK K210R mutant or empty vector alone. NPM/ALK induces CD274 expression by activating its key signal transmitter, transcription factor STAT3. STAT3 binds to the CD274 gene promoter in vitro and in vivo, as shown in the gel electromobility shift and chromatin immunoprecipitation assays, and is required for the PD-L1 gene expression, as demonstrated by siRNA-mediated STAT3 depletion. These findings identify an additional cell-transforming property of NPM/ALK and describe a direct link between an oncoprotein and an immunosuppressive cell-surface protein. These results also provide an additional rationale to therapeutically target NPM/ALK and STAT3 in ALK+TCL. Finally, they suggest that future immunotherapeutic protocols for this type of lymphoma may need to include the inhibition of NPM/ALK and STAT3 to achieve optimal clinical efficacy.

Activation of mTORC1 signaling pathway in AIDS-related lymphomas.

Using immunohistochemistry with antibodies against the phosphoserine residues in both S6rp and 4E binding protein 1, we identified the activation of the mammalian target of rapamycin (mTORC)1 pathway in 29 cases of AIDS-related lymphoma. These cases represented a diverse spectrum of histological types of non-Hodgkin lymphoma (24 cases) and classic Hodgkin lymphoma (five cases). mTORC1 was also activated in the hyperplastic but not involuted follicles of HIV-associated lymphadenopathy in eight cases, supporting the notion that mTORC1 activation is a common feature of transformed lymphocytes irrespective of either their reactive or malignant phenotype. We also found that in B-cell lines that represent diffuse large B-cell lymphoma, Burkitt lymphoma, Epstein-Barr virus-infected lymphocytes, and human herpesvirus 8-positive primary effusion lymphoma, inhibitors of Syk, MEK, and, seemingly, phosphoinositide 3 kinases suppressed mTORC1 activation, in particular when these inhibitors were used in combination. These findings indicate that AIDS-related lymphoma and other histologically similar types of lymphomas that are derived from transformed B lymphocytes may display clinical responses to inhibitors that directly target mTORC1 or, possibly, upstream activators of the mTORC1 pathway.

Role of different immunostaining patterns in HercepTest interpretation and criteria for gene amplification as determined by fluorescence in situ hybridization.

Immunohistochemical (IHC) detection of HER-Z overexpression by HercepTest on paraffin sections has been proposed for selecting patients with metastatic breast carcinoma for Herceptin therapy. Interpretation of intermediate (2+) reactivity could be subjective and has been shown to have low concordance with fluorescence in situ hybridization (FISH). FISH assay for gene amplification (GA) has been used as a confirmatory test in equivocal cases with different criteria judging gene amplification. A wide range of discordance between IHC and FISH has been reported in the literature, with ongoing debate about which assay is more accurate for HER-Z test. Little attention has been paid to the role of different criteria in judging protein overexpression on IHC and GA on FISH. We evaluated HER-Z overexpression and GA by IHC (HercepTest) and FISH (PathVysion), respectively, on paraffin sections of 142 primary breast carcinomas. For the HercepTest, in addition to scoring guidelines proposed by the manufacturer, scoring systems taking into consideration the pattern of membranous staining and staining in benign epithelium were also evaluated. For the FISH assay, the ratios of HER-Z:CEP17, HER-Z:cell, and CEP17:cell were used to evaluate the impact of the different criteria for determining GA. HER-Z overexpression was detected in 53 (37.3%), 48 (33.8%), and 36 (25.4%) cases according to the HercepTest, normal epithelium-modified, and pattern-modified scoring systems, respectively. The HER-Z gene was considered amplified in 41 (28.9%), 37 (26.1%), and 34 (23.9%) cases according to the HER-Z:cell criteria, HER-Z:CEP17 criteria, and the combined criteria, respectively. Our results suggest that a high concordance (95.8%) between HER-Z overexpression and GA can be achieved by using more stringent pattern-modified criteria for judging protein overexpression and combined criteria for GA.

IL-2- and IL-15-induced activation of the rapamycin-sensitive mTORC1 pathway in malignant CD4+ T lymphocytes.

We examined functional status, activation mechanisms, and biologic role of the mTORC1 signaling pathway in malignant CD4(+) T cells derived from the cutaneous T-cell lymphoma (CTCL). Whereas the spontaneously growing CTCL-derived cell lines displayed persistent activation of the TORC1 as well as the PI3K/Akt and MEK/ERK pathways, the IL-2-dependent cell lines activated the pathways in response to IL-2 and IL-15 but not IL-21. Activation of mTORC1 and MEK/ERK was nutrient dependent. The mTORC1, PI3K/Akt, and MEK/ERK pathways could also be activated by IL-2 in the primary leukemic, mitogen-preactivated CTCL cells. mTORC1 activation was also detected in the CTCL tissues in the lymphoma stage-dependent manner with the highest percentage of positive cells present in the cases with a large cell transformation. Rapamycin inhibited mTORC1 signaling and suppressed CTCL cell proliferation but showed little effect on their apoptotic rate when used as a single agent. Activation of the mTORC1, PI3K/Akt, and MEK/ERK pathways was strictly dependent on the Jak3 and Jak1 kinases. Finally, mTORC1 activation was transduced preferentially through the PI3K/Akt pathway. These findings document the selective gammac-signaling cytokine-mediated activation of the mTORC1 pathway in the CTCL cells and suggest that the pathway represents a therapeutic target in CTCL and, possibly, other T-cell lymphomas.

Constitutive activation of mTOR signaling pathway in post-transplant lymphoproliferative disorders.

We examined activation of the mTOR signaling pathway in situ in the primary, normal reactive and patient-derived post-transplant lymphoproliferative disorder (PTLD) tissue samples. We accomplished this analysis by immunohistochemistry on formalin-fixed, paraffin-embedded specimens using a set of highly specific antibodies that permitted us to determine phosphorylation status of the key serines in the mTOR target proteins. Our results demonstrate that the mTOR signaling pathway is activated in reactive tissue in a highly distinct fashion with positive, typically enlarged cells being present primarily in the germinal center and, to a lesser degree, in interfollicular areas with mantle zone being conspicuously negative. We could demonstrate mTOR activation in the lesional cells in the entire spectrum of PTLD subtypes, regardless of their Epstein-Barr virus genome expression status. These data demonstrate the ubiquitous activation of the mTOR signaling pathway in PTLD and indicate that mTOR inhibitors may be effective in treatment and, notably, prevention of PTLDs given their immunosuppressive properties. Furthermore, our results define potential biomarkers of the therapeutic response. Because the constitutive mTOR activation has also been identified in cells isolated from other hematologic malignancies, the ability to examine the in vivo mTOR signaling may have implications reaching beyond the PTLD field.

STAT3 induces transcription of the DNA methyltransferase 1 gene (DNMT1) in malignant T lymphocytes.

In this study, we demonstrated that STAT3, a well-characterized transcription factor expressed in continuously activated oncogenic form in the large spectrum of cancer types, induces in malignant T lymphocytes the expression of DNMT1, the key effector of epigenetic gene silencing. STAT3 binds in vitro to 2 STAT3 SIE/GAS-binding sites identified in promoter 1 and enhancer 1 of the DNMT1 gene. STAT3 also binds to the promoter 1 region and induces its activity in vivo. Treatment of the malignant T lymphocytes with STAT3 siRNA abrogates expression of DNMT1, inhibits cell growth, and induces programmed cell death. In turn, inhibition of DNMT1 by a small molecule inhibitor, 5-aza-2-deoxy-cytidine, and 2 DNMT1 antisense DNA oligonucleotides inhibits the phosphorylation of STAT3. These data indicate that STAT3 may in part transform cells by fostering epigenetic silencing of tumor-suppressor genes. They also indicate that by inducing DNMT1, STAT3 facilitates its own persistent activation in malignant T cells. Finally, these data provide further rationale for therapeutically targeting STAT3 in T-cell lymphomas and, possibly, other malignancies.

STAT3- and DNA methyltransferase 1-mediated epigenetic silencing of SHP-1 tyrosine phosphatase tumor suppressor gene in malignant T lymphocytes.

Expression of SHP-1 phosphatase, a key negative regulator of cell signaling, is lost in T cell lymphomas and other malignancies due to DNA methylation of the SHP-1 promoter by a currently undefined mechanism. We demonstrate that malignant T cells express DNA methyltransferase (DNMT) 1 and that constantly activated signal transducer and activator of transcription (STAT) 3 is capable of binding in vitro to DNA oligonucleotides corresponding to four STAT3 SIE/GAS binding sites identified in the SHP-1 promoter. STAT3, DNMT1, and histone deacetylase 1 form complexes and bind to the SHP-1 promoter in vivo. Treatment with pharmacologic grade DNMT1 anti-sense oligonucleotides and STAT3 small-interfering RNA induces in the malignant T cells DNA demethylation and expression of SHP-1 gene. These data indicate that STAT3 may, in part, transform cells by inducing epigenetic silencing of SHP-1 in cooperation with DNMT1 and, apparently, histone deacetylase 1. Reversal of such gene silencing represents an attractive aim for novel anticancer therapies.

Is the myofibrillarlytic myocyte a forme fruste apoptotic myocyte?

BACKGROUND: Myofibrillarlytic (MFL) cells are commonly observed in subendocardial myocardium in myocardial infarction. Because ischemic damage to myocytes is also known to induce apoptosis, we evaluated the prevalence of apoptosis in MFL cells in nine ischemic cardiomyopathic hearts explanted during transplantation. METHODS: Myocytes with partial or complete clearing of cytoplasm, observed commonly in the subendocardium, were recognized as MFL cells. Prevalence of apoptosis was defined by TUNEL and ISOL staining and further characterized by immunohistochemical staining for caspase-3, Bcl2, BCL-X(L), Bax, proliferating cell nuclear antigen (PCNA), and Ki67. RESULTS: Of 4131 MFL cells examined, 1305 (32%) possessed nuclei in a given histologic section; 1140 (88%) of the nucleated myocardial cells were TUNEL positive. Of 842 cells with normal appearance, 257 (31%) cells demonstrated nuclei in the given histologic section. TUNEL staining was observed in 5 (1.9%) in these control areas. All MFL cells stained positive for caspase 3. The antiapoptotic proteins, Bcl2 and BCL-X(L), demonstrated intense upregulation within and surrounding MFL cells, whereas pro-apoptotic protein Bax expression was only seen at control level. The MFL cells had Ki67 negative and PCNA positive nuclei. CONCLUSIONS: The present study demonstrates that the majority of MFL cells are apoptotic and are associated with upregulation of caspase 3. Simultaneous upregulation of Bcl2 represents a survival effort in these myocytes. This is consistent with the review of the literature that MFL cells are viable, persist in myocardium for long time and may be functionally reversible. Evidence for concurrent apoptosis and survival instinct represent a conceptual paradox and suggests that myocytes undergoing apoptosis should be amenable to reconstitution of function.

Impact of the 1998 World Health Organization/International Society of Urological Pathology classification system for urothelial neoplasms of the kidney.

The classification of urothelial neoplasms of the kidney traditionally has been similar to that of urinary bladder tumors. Several years ago, the classification of papillary urothelial neoplasms was revised. The current study focuses on the application of the 1998 World Health Organization (WHO)/International Society of Urological Pathology classification system to 102 renal pelvic urothelial neoplasms and compares it to the 1973 WHO classification scheme. In this study, all tumors were classified as urothelial carcinomas, and the majority (85%) were papillary. Most patients with papillary tumors presented with 'superficial' disease (< or = pT1). With the 1998 system, most papillary carcinomas were high grade, and were more often invasive as compared to low-grade tumors. Only 34% were low-grade papillary tumors and, of these, most (93%) were noninvasive. With the 1973 system, most papillary tumors were grade 2 or 3, with invasion more common in grade 3 tumors. By 1973 criteria, grade 2 tumors were a heterogeneous group; with 1998 criteria, nearly one-half were high grade and the other half low grade. The grade of papillary urothelial carcinomas with both the 1973 and 1998 grading methods was associated with stage (P=0.001). Our study reveals that papillomas and papillary urothelial neoplasms of low malignant potential are uncommon tumors in the kidney. Renal pelvic papillary urothelial neoplasms are most often carcinomas and are more commonly high grade than low grade. Although both the 1973 and 1998 systems showed a significant association with tumor stage, grade 2 papillary carcinomas are a heterogeneous group by 1973 criteria. The 1998 system provides useful information in that it more clearly defines a papillary tumor's grade and selects for a group of tumors, namely low-grade papillary urothelial carcinomas, for which a low likelihood of invasion can be predicted.