Arianna Di Napoli

Increased expression of Met protein is associated with up-regulation of hypoxia inducible factor-1 (HIF-1) in tumour cells in papillary carcinoma of the thyroid

Met protein, the high affinity receptor for hepatocyte growth factor (HGF), was highly expressed by the tumour cells of 64 well‐differentiated papillary carcinomas of the thyroid. The p145 mature form and the p170 precursor form of the protein were both isolated from the tumours. Enhanced expression of Met protein was associated with a 9.5 ± 5‐fold increase in MET RNA transcript levels, suggesting increased transcription of the gene. In the same tumours, the levels of RNA transcripts for hypoxia inducible factor‐1 (HIF‐1), a potent stimulator of met gene transcription, were 4.5 ± 3‐fold higher than those present in the surrounding normal thyroid tissues. HIF‐1 is generally induced by hypoxia. Histological features suggestive of a hypoxia were observed in 37 of 50 tumours and included coagulative necrosis, psammoma bodies, cystic changes, intratumoural haemorrhage, and hyalinization of the fibrous stroma. Immunostaining for Met protein was particularly intense in some cells located at the tumour periphery which were characterized by an invasive phenotype. Microdissection of tumour cell nests from the invading front revealed that the levels of RNA transcripts for MET/HIF were higher than in the centre of the tumour in four of nine cases. Taken together, the findings of this study suggest that HIF‐1, perhaps driven by hypoxia, may be one of the factors leading to the increased transcription of met gene in papillary carcinoma and that this event is often more pronounced at the tumour periphery. Copyright

Papillary carcinoma of the thyroid: evidence for a role for hepatocyte growth factor (HGF) in promoting tumour angiogenesis.

The pattern of vascularization of papillary carcinoma was investigated in tumour sections from 31 cases and in primary cultures from 12 cases. Tumour sections were immunostained for von Willebrand Factor (vWF) to visualize blood vessels; for endothelial‐specific nitric‐oxide‐synthase (EC‐NOS), as a marker of endothelial cell activation; and for Ki‐67 to evaluate endothelial cell proliferation. It was found that endothelial cells lining venous vessels located in peritumoural fibrous tissue were intensely EC‐NOS‐positive and occasionally Ki‐67‐positive. Capillary vessels of tumour papillae were not stained for Ki‐67 and were weakly EC‐NOS‐positive. Primary cultures of papillary carcinoma cells were used as a potential source of factors active on endothelial cells. It was found that thyroid tumour cells contain RNAs for angiopoietin, vascular endothelial growth factor (VEGF), and VEGF‐C; moreover, they release large amounts of VEGF into culture supernatants and exert chemotactic activity in vitro for the endothelial cell line SIEC. The ability of papillary carcinoma cells to release angiogenic factors could be stimulated in vitro. Hepatocyte growth factor (HGF; 25 ng/ml) induced a 1.2‐ to 5‐fold increase in the amount of VEGF released by tumour cells and a 1.2‐ to 4.2‐fold increase in the amount of chemotactic activity present in culture supernatants. Met protein, the high affinity HGF‐receptor, is overexpressed in a large proportion of cases of papillary carcinoma. These findings are consistent with the possibility that HGF–Met protein interaction is one of the molecular mechanisms promoting the vascularization of papillary carcinoma of the thyroid. Copyright

Invariant NKT cells contribute to chronic lymphocytic leukemia surveillance and prognosis

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant CD5+ B lymphocytes in blood, bone marrow, and lymphoid organs. CD1d-restricted invariant natural killer T (iNKT) cells are innate-like T lymphocytes strongly implicated in tumor surveillance. We investigated the impact of iNKT cells in the natural history of the disease in the Eμ-Tcl1 (Tcl1) CLL mouse model and 68 CLL patients. We found that Tcl1-CLL cells express CD1d and that iNKT cells critically delay disease onset but become functionally impaired upon disease progression. In patients, disease progression correlates with high CD1d expression on CLL cells and impaired iNKT cells. Conversely, disease stability correlates with negative or low CD1d expression on CLL cells and normal iNKT cells, suggesting indirect leukemia control. iNKT cells indeed hinder CLL survival in vitro by restraining CD1d-expressing nurse-like cells, a relevant proleukemia macrophage population. Multivariable analysis identified iNKT cell frequency as an independent predictor of disease progression. Together, these results support the contribution of iNKT cells to CLL immune surveillance and highlight iNKT cell frequency as a prognostic marker for disease progression.

Circulating MMP11 and specific antibody immune response in breast and prostate cancer patients

BackgroundTumor Associated Antigens are characterized by spontaneous immune response in cancer patients as a consequence of overexpression and epitope-presentation on MHC class I/II machinery. Matrix Metalloprotease 11 (MMP11) expression has been associated with poor prognosis for several cancer types, including breast and prostate cancer.MethodsMMP11 expression was determined by immunoistochemistry in breast and prostate cancer samples. Circulating MMP11 protein as well as the spontaneous immune responses against MMP11 were analyzed in a set of breast and prostate cancer patients.ResultsIn plasma samples MMP11 protein was present in 5/13 breast cancer patients and in 1/12 prostate cancer patients. An antibody response was observed in 7/13 breast cancer patients and in 3/12 prostate cancer patients.ConclusionsThese findings further suggest MMP11 as a promising biomarker for these tumor types and a suitable target for cancer immunotherapy strategies.

Peripheral T Cell Lymphoma with Cytotoxic Phenotype: An Emerging Disease in HIV-Infected Patients?

Recently, a 15-fold increased risk of T cell lymphomas has been estimated in HIV-infected populations. This increase has been observed for all T cell lymphoma subtypes. In the present report we describe clinical and pathological features of three consecutive cases of peripheral T cell lymphoma (PTCL) with cytotoxic phenotype in HIV-positive patients that came to our attention in May-September 2002. The diagnosis of PTCL was made in lymph node (two cases) and in needle biopsies from liver and bone marrow of the same patient. The patients were two females (31 and 45 years old) and one male (49 years old). The risk factor for each patient was heterosexual, injecting drug user, and homosexual, respectively. CD4 cell counts were low (79-81 cells/mm3). Two patients were naive for antiretroviral therapy. At histological examination, all the involved tissues were effaced by a neoplastic proliferation of CD3+/CD8+ medium to large pleomorphic cells containing TIA-1+ cytotoxic granules and a few granzyme B+ granules. Neoplastic cells were not infected by EBV or by HHV-8. They were negative for the B cell antigens CD20 and CD79a, for CD30 and for CD56. Clonal T cell receptor-g (TCR-g) rearrangements were demonstrated in the three cases.

Targeted next generation sequencing of breast implant-associated anaplastic large cell lymphoma reveals mutations in JAK/STAT signalling pathway genes, TP53 and DNMT3A

Breast implant-associated anaplastic large cell lymphoma (BI-ALCL) is an uncommon neoplasm occurring in women with either cosmetic or reconstructive breast implants (Clemens et al, 2016). Until now, most studies have focused on defining the clinico-pathological features of BI-ALCL, leading to its inclusion as a new provisional entity, a subtype of anaplastic lymphoma kinase (ALK)-negative ALCL, in the revised World Health Organization classification of lymphoid malignancies (Swerdlow et al, 2016). BI-ALCL is characterized by the presence of CD30 large atypical lymphocytes frequently confined to the peri-implant seroma fluid. Nevertheless, solid infiltrating masses and cases pursuing an aggressive clinical course have been reported. The surgical and pathological staging system designed by Clemens et al (2016) suggests that BI-ALCL has a pattern of progression similar to that of solid tumours rather than non-Hodgkin lymphomas, and that the effusionand solid-types might represent different stages of the same disease rather than two distinct variants. The molecular pathogenesis and mechanisms of progression of BI-ALCL, however, remain largely unknown, thus limiting the identification of biomarkers that enable disease prognostication and optimal treatment. Hence, we performed targeted next generation sequencing of seven BI-ALCL, identified in the archives of three institutions over 7 years, to investigate the presence of underlying somatic mutations. Informed consent was obtained from patients and the study was performed in accordance with the Declaration of Helsinki. DNA extracted from micro-dissected tumour cells of formalin-fixed paraffin-embedded BI-ALCL samples (QIAamp DNA Mini kit; Qiagen, Germantown, MD, USA) was used to prepare DNA libraries (Sureselect kit; Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed on a HiSeq2500 (Illumina, San Diego, CA, USA) using a panel of 465 cancerassociated genes (Table SI). The sequence data were aligned to the human reference genome (hg19) and variants were identified using NextGENe (SoftGenetics, State College, PA, USA). The average read depth of the samples was 4009 (Table SII). Somatic mutations were identified by comparison of variants detected in lymphoma with those from matched constitutional DNA. Common variants (>1% frequency) present in the 1000 genomes database, and the database of Columbia University were removed. Somatic mutations were classified using the prior literature, and two different prediction algorithms (SIFT http://sift.bii.a-star.edu.sg and Polyphen-2 [PP2] http://genetics.bwh.harvard.edu/pph2/). The exonic somatic variants were confirmed by bidirectional Sanger sequencing using Big-Dye terminators v3.1 (Applied Biosystems, Carlsbad, CA, USA). The clinical and pathological features of the patients are summarized in Table I. Informative results were obtained in five of seven cases (Table SII); analysis failed in two cases due to the poor quality of DNA. Five somatic variants affecting four genes were identified in two cases: one intronic and four within coding regions (Fig 1 and Table SIII). A STAT3 missense variant (p.S614R) affecting the SH2 domain, which mediates STAT3 dimerization, was detected in one of these two BI-ALCLs. JAK/STAT signalling is implicated in cell proliferation, differentiation and apoptosis, and aberrant activation of STAT3 has been reported in several human cancers associated with persistent immune stimulation and/or inflammation. Notably, the gain-of-function mutation (S614R) was recently described in one BI-ALCL (Blombery et al, 2016), and has been reported in angioimmunoblastic T cell lymphomas, chronic lymphoproliferative disorders of natural killer cells, and T-cell large granular lymphocyte leukaemias (Odejide et al, 2014). Moreover, gain-of-function mutations in STAT3 have been reported in 18% of systemic ALK-negative ALCLs and 5% of cutaneous ALCLs (Crescenzo et al, 2015). An in vitro study using BI-ALCL-derived cell lines also showed activation of the JAK/STAT pathway through autocrine production of interleukin 6, suggesting a possible pathogenic mechanism (Lechner et al, 2012). A frameshift deletion causing a premature stop codon in SOCS1 (p.P83Rfs*20) was detected in the BI-ALCL harbouring the STAT3 mutation. SOCS1 is a negative feedback regulator of the JAK/STAT pathway. The p.P83Rfs*20 mutation deletes the C-terminal SOCS box domain and partially deletes the SH2 domain, which downregulates the kinase activity of JAK. Loss-of-function mutations of SOCS1, leading to constitutive activation of JAK/STAT signalling, have been described in B-cell lymphomas and in classical Hodgkin lymphomas (Mottok et al, 2009). Moreover, SOCS1 was found to be silenced by miR-155 in ALK-negative ALCL (Merkel et al, 2015). Mutations in STAT3 and SOCS1 suggest that deregulated activation of the JAK/STAT pathway may contribute to the development of BI-ALCL. A missense mutation of TP53 (p.D259Y) affecting the DNA binding domain was also observed in the Correspondence

Iatrogenic EBV-positive lymphoproliferative disorder with features of EBV+ mucocutaneous ulcer: Evidence for concomitant TCRγ/IGH rearrangements in the Hodgkin-like neoplastic cells

Iatrogenic immunodeficiency-associated lymphoproliferative disorders are a heterogeneous group of rare diseases related to therapy with immunosuppressive drugs for conditions other than the transplant setting [1]. The first description concerned patients who developed Hodgkin’s disease or Hodgkin-like lymphoproliferations after longterm, low-dose methotrexate therapy [2–4]. Very recently, in a multicenter study, 26 cases of a distinct clinicopathologic entity, the EBV+ mucocutaneous ulcer, were described [5]. The disease affects immunosuppressed and/or elderly patients, and consists of isolated, sharply circumscribed ulcers involving oropharyngeal mucosa, skin or gastrointestinal tract. Lesions are characterized by the presence of a polymorphous inflammatory-like infiltrate, in which various numbers of atypical large B cells with Hodgkin/Reed-Sternberg-like (H/RS-like) morphology are embedded. The atypical B cells are EBER+/CD30+/CD15+/ PAX5+/MUM-1+. IGH clonality was detected in 7 of 18 cases (39%); TCR clonality in 6 of 16 cases (38%); moreover, a monoclonal IGH rearrangement associated with restricted/ clonal T cell responses was observed in 3 of 16 cases. In the present report, we describe a case of a malignant lymphoproliferative disorder of the large intestine with features of Epstein–Barr virus (EBV)+ mucocutaneous ulcer; the novel contribution provided by our study is the demonstration of concomitant clonal IGH and TCRγ rearrangements in the neoplastic B cells with H/RS-like morphology.

Hepatocyte growth factor (HGF) downregulates thrombospondin 1 (TSP-1) expression in thyroid papillary carcinoma cells

This study investigates the expression of thrombospondin‐1 in papillary carcinoma of the thyroid and the role of Met–HGF interaction in TSP‐1 regulation. In tissue sections, immunostaining for TSP‐1 was associated with the fibrous tumour stroma, and showed areas of marked intensity adjacent to the basal membrane of tumour cells. Investigation of TSP‐1 RNA expression showed that, in 10 of 14 cases, TSP‐1 mRNA levels were significantly lower in tumour tissue (20–100% reduction; mean = 55% ± 20; p = 0.001) than in the corresponding normal thyroid. Since it has been reported that HGF can downregulate the expression of TSP‐1 mRNA, TSP‐1 mRNA levels were measured in 7 primary cultures, established from thyroid papillary carcinomas (TPC), and in 1 TPC cell line prior to, or after, stimulation with HGF. A marked decrease in TSP‐1 mRNA levels was observed after HGF stimulation in 6/7 primary cultures (60–100% decrease (mean = 79 ± 15%; p = 0.006) and in the TPC cell line; moreover, the decrease in TSP‐1 mRNA in cell extracts was associated with a decrease in TSP‐1 protein in culture supernatants. The HGF activity was dose dependent and the downregulation lasted for at least 48 h after stimulation. The high‐level expression of Met protein, the high‐affinity receptor for HGF, in most cases of papillary carcinoma of the thyroid is consistent with the possibility that HGF–Met interaction plays a crucial role in regulating the expression of TSP‐1 in this tumour type. Copyright

Clinicopathologic Characterization of Diffuse-Large-B-Cell Lymphoma with an Associated Serum Monoclonal IgM Component

Recently, diffuse-large-B-cell lymphoma (DLBCL) associated with serum IgM monoclonal component (MC) has been shown to be a very poor prognostic subset although, detailed pathological and molecular data are still lacking. In the present study, the clinicopathological features and survival of IgM-secreting DLBCL were analyzed and compared to non-secreting cases in a series of 151 conventional DLBCL treated with R-CHOP. IgM MC was detected in 19 (12.5%) out of 151 patients at disease onset. In 17 of these cases secretion was likely due to the neoplastic clone, as suggested by the expression of heavy chain IgM protein in the cytoplasm of tumor cells. In IgM-secreting cases immunoblastic features (p<.0001), non-GCB-type (p = .002) stage III-IV(p = .003), ≥2 extra nodal sites (p<.0001), bone-marrow (p = .002), central-nervous-system (CNS) involvement at disease onset or relapse (p<.0001), IPI-score 3–5 (p = .009) and failure to achieve complete remission (p = .005), were significantly more frequent. FISH analyses for BCL2, BCL6 and MYC gene rearrangements detected only two cases harboring BCL2 gene translocation and in one case a concomitant BCL6 gene translocation was also observed. None of the IgM-secreting DLBCL was found to have L265P mutation of MYD88 gene. Thirty-six month event-free (11.8% vs 66.4% p<.0001), progression-free (23.5% vs 75.7%, p<.0001) and overall (47.1% vs 74.8%, p<.0001) survivals were significantly worse in the IgM-secreting group. In multivariate analysis IgM-secreting (p = .005, expB = 0.339, CI = 0.160-0.716) and IPI-score 3–5 (p = .010, expB = 0.274, CI = 0.102–0.737) were the only significant factors for progression-free-survival. Notably, four relapsed patients, who were treated with salvage immmunochemotherapy combined with bortezomib or lenalidomide, achieved lasting remission. Our data suggests that IgM-secreting cases are a distinct subset of DLBCL, originating from activated-B-cells with terminally differentiated features, prevalent extra nodal dissemination and at high risk of CNS involvement.

Tumor-associated and immunochemotherapy-dependent long-term alterations of the peripheral blood NK cell compartment in DLBCL patients

Natural Killer (NK) cells are a key component of tumor immunosurveillance and thus play an important role in rituximab-dependent killing of lymphoma cells via an antibody-dependent cellular cytotoxicity (ADCC) mechanism. We evaluated the phenotypic and functional assets of peripheral blood NK cell subsets in 32 newly-diagnosed diffuse large B-cell lymphoma (DLBCL) patients and in 27 healthy controls. We further monitored long-term modifications of patient NK cells for up to 12 months after rituximab-based immunochemotherapy. At diagnosis, patients showed a higher percentage of CD56dim and CD16+ NK cells, and a higher frequency of GrzB+ cells in CD56dim, CD56bright, and CD16+ NK cell subsets than healthy controls. Conversely, DLBCL NK cell killing and interferon γ (IFNγ) production capability were comparable to those derived from healthy subjects. Notably, NK cells from refractory/relapsed patients exhibited a lower “natural” cytotoxicity. A marked and prolonged therapy-induced reduction of both “natural” and CD16-dependent NK cytotoxic activities was accompanied by the down-modulation of CD16 and NKG2D activating receptors, particularly in the CD56dim subset. However, reduced NK cell killing was not associated with defective lytic granule content or IFNγ production capability. This study firstly describes tumor-associated and therapy-induced alterations of the systemic NK cell compartment in DLBCL patients. As these alterations may negatively impact rituximab-based therapy efficacy, our work may provide useful information for improving immunochemotherapeutic strategies.