Enrico Duranti

Therapeutic targeting of Chk1 in NSCLC stem cells during chemotherapy

Cancer stem cell (SC) chemoresistance may be responsible for the poor clinical outcome of non-small-cell lung cancer (NSCLC) patients. In order to identify the molecular events that contribute to NSCLC chemoresistance, we investigated the DNA damage response in SCs derived from NSCLC patients. We found that after exposure to chemotherapeutic drugs NSCLC-SCs undergo cell cycle arrest, thus allowing DNA damage repair and subsequent cell survival. Activation of the DNA damage checkpoint protein kinase (Chk) 1 was the earliest and most significant event detected in NSCLC-SCs treated with chemotherapy, independently of their p53 status. In contrast, a weak Chk1 activation was found in differentiated NSCLC cells, corresponding to an increased sensitivity to chemotherapeutic drugs as compared with their undifferentiated counterparts. The use of Chk1 inhibitors in combination with chemotherapy dramatically reduced NSCLC-SC survival in vitro by inducing premature cell cycle progression and mitotic catastrophe. Consistently, the co-administration of the Chk1 inhibitor AZD7762 and chemotherapy abrogated tumor growth in vivo, whereas chemotherapy alone was scarcely effective. Such increased efficacy in the combined use of Chk1 inhibitors and chemotherapy was associated with a significant reduction of NSCLC-SCs in mouse xenografts. Taken together, these observations support the clinical evaluation of Chk1 inhibitors in combination with chemotherapy for a more effective treatment of NSCLC.

Differential Modulation of Uncoupling Protein 2 in Kidneys of Stroke-Prone Spontaneously Hypertensive Rats Under High-Salt/Low-Potassium Diet

The stroke-prone spontaneously hypertensive rat (SHRsp) represents an animal model of increased susceptibility to high-salt diet–induced cerebral and renal vascular injuries. High blood pressure and genetic factors are viewed as major contributing factors. In high-salt–loaded SHRsp and stroke-resistant SHR animals, we determined blood pressure levels, degree of kidney lesions, renal uncoupling protein 2 (UCP2) gene and protein expression levels along with rattus norvegicus (rno)-microRNA (miR) 24 and 34a gene expression, nuclear factor-&kgr;B protein levels, and oxidative stress. In vitro, UCP2 gene silencing was performed in renal mesangial cells. We found more severe degree of renal damage in SHRsp at the end of 4-week high-salt dietary treatment as compared with stroke-resistant SHR, despite comparable blood pressure levels, along with increased rate of inflammation and oxidative stress. Kidney UCP2 gene and protein expression levels were significantly downregulated under high-salt diet in SHRsp, but not in stroke-resistant SHR. Differential UCP2 regulation was paralleled by differential expression of kidney rno-miR 24 and 34a, known to target UCP2 gene, in the 2 strains. UCP2 gene silencing in renal mesangial cells led to increased rate of reactive oxygen species generation, increased inflammation and apoptosis, reduced cell vitality, and increased necrosis. In conclusion, high-salt diet downregulates the antioxidant UCP2-dependent mechanism in kidneys of SHRsp, but not of stroke-resistant SHR. A parallel differential kidney miR regulation under high-salt diet in the 2 strains may contribute to the differential UCP2 modulation. UCP2 is a critical protein to prevent oxidative stress damage in renal mesangial cells in vitro.

Tyr1068-phosphorylated epidermal growth factor receptor (EGFR) predicts cancer stem cell targeting by erlotinib in preclinical models of wild-type EGFR lung cancer

Tyrosine kinase inhibitors (TKIs) have shown strong activity against non-small-cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations. However, a fraction of EGFR wild-type (WT) patients may have an improvement in terms of response rate and progression-free survival when treated with erlotinib, suggesting that factors other than EGFR mutation may lead to TKI sensitivity. However, at present, no sufficiently robust clinical or biological parameters have been defined to identify WT-EGFR patients with greater chances of response. Therapeutics validation has necessarily to focus on lung cancer stem cells (LCSCs) as they are more difficult to eradicate and represent the tumor-maintaining cell population. Here, we investigated erlotinib response of lung CSCs with WT-EGFR and identified EGFR phosphorylation at tyrosine1068 (EGFRtyr1068) as a powerful biomarker associated with erlotinib sensitivity both in vitro and in preclinical CSC-generated xenografts. In contrast to the preferential cytotoxicity of chemotherapy against the more differentiated cells, in EGFRtyr1068 cells, erlotinib was even more active against the LCSCs compared with their differentiated counterpart, acquiring potential value as CSC-directed therapeutics in the context of WT-EGFR lung cancer. Although tumor growth was inhibited to a similar extent during erlotinib or chemotherapy administration to responsive tumors, erlotinib proved superior to chemotherapy in terms of higher tolerability and reduced tumor aggressiveness after treatment suspension, substantiating the possibility of preferential LCSC targeting, both in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) tumors. We conclude that EGFRtyr1068 may represent a potential candidate biomarker predicting erlotinib response at CSC-level in EGFR-WT lung cancer patients. Finally, besides its invariable association with erlotinib sensitivity in EGFR-WT lung CSCs, EGFRtyr1068 was associated with EGFR-sensitizing mutations in cell lines and patient tumors, with relevant diagnostic, clinical and therapeutic implications.

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.

COX-2 is induced by HGF stimulation in Met-positive thyroid papillary carcinoma cells and is involved in tumour invasiveness.

Thyroid papillary carcinoma (TPC) cells express high levels of cytoplasmic cyclo‐oxygenase 2 protein. Analysis of microdissected samples of the tumour and of the paired normal thyroid tissue confirmed that mRNA transcripts for cyclo‐oxygenase 2 (COX‐2) were significantly more numerous in the tumour (7.6 ± 13‐fold; p = 0.01). High levels of COX‐2 mRNA were not associated with age, sex, tumour size or lymph node metastasis. COX‐2 was not homogeneously expressed throughout the tumour, but was significantly higher at the tumour invasion front. Hepatocyte growth factor (HGF) can up‐regulate the expression of COX‐2 mRNA. A marked increase in COX‐2 mRNA levels was observed in 8/8 primary TPC cultures after HGF stimulation (6.3 ± 6‐fold) and in two papillary carcinoma cell lines (TPC‐1 and NPA). Specific involvement of the high‐affinity HGF receptor (Met protein) was suggested by the observation that PHA‐665752, an inhibitor of the catalytic activity of c‐Met kinase, caused a 54% reduction of the hepatocyte growth factor‐induced COX‐2 up‐regulation. The possibility that HGF–Met interactions also had a causative role in the up‐regulation of COX‐2 in vivo was investigated in 30 tumour samples, where it was found that there was a statistically significant correlation (p = 0.001, r = 0.85) in the levels of expression of MET and COX‐2 RNAs. The biological role of COX‐2 in TPC cells was investigated by treating the TPC cell lines with the specific COX‐2 inhibitor NS‐398. It was found that NS‐398 treatment significantly reduced the migration (50–75%) and invasiveness (47–92%) of tumour cells, but did not alter cell proliferation. Our data suggest that the increased expression of Met protein in TPC cells has a role in up‐regulating the expression of COX‐2, which in turn contributes to the invasive capacity of TPC cells. Copyright

Mek inhibition results in marked antitumor activity against metastatic melanoma patient-derived melanospheres and in melanosphere-generated xenografts

One of the key oncogenic pathways involved in melanoma aggressiveness, development and progression is the RAS/BRAF/MEK pathway, whose alterations are found in most patients. These molecular anomalies are promising targets for more effective anti-cancer therapies. Some Mek inhibitors showed promising antitumor activity, although schedules and doses associated with low systemic toxicity need to be defined. In addition, it is now accepted that cancers can arise from and be maintained by the cancer stem cells (CSC) or tumor-initiating cells (TIC), commonly expanded in vitro as tumorspheres from several solid tumors, including melanoma (melanospheres). Here, we investigated the potential targeting of MEK pathway by exploiting highly reliable in vitro and in vivo pre-clinical models of melanomas based on melanospheres, as melanoma initiating cells (MIC) surrogates. MEK inhibition, through PD0325901, provided a successful strategy to affect survival of mutated-BRAF melanospheres and growth of wild type-BRAF melanospheres. A marked citotoxicity was observed in differentated melanoma cells regardless BRAF mutational status. PD0325901 treatment, dramatically inhibited growth of melanosphere-generated xenografts and determined impaired tumor vascularization of both mutated- and wild type-BRAF tumors, in the absence of mice toxicity. These results suggest that MEK inhibition might represent a valid treatment option for patients with both mutated- or wild type-BRAF melanomas, affecting tumor growth through multiple targets.

Epstein-Barr virus (EBV) positive classical Hodgkin lymphoma of Iraqi children: an immunophenotypic and molecular characterization of Hodgkin/Reed-Sternberg cells.

Classical Hodgkin lymphoma (cHL) in children is often associated with EBV infection, more commonly in developing countries.

Papillary Carcinoma of the Thyroid: High Expression of COX-2 and Low Expression of KAI-1/CD82 Are Associated with Increased Tumor Invasiveness

BACKGROUND We have previously demonstrated that expression of COX-2 is upregulated by hepatocyte growth factor in thyroid papillary carcinoma (TPC) cells and is associated with increased invasiveness of tumor cells. COX-2 upregulation was associated with downregulation of KAI-1/CD82, a metastasis suppressor molecule that has been associated with the metastatic potential of several solid tumors. In the present study, we have investigated the possibility that downregulation of KAI-1/CD82 may contribute to the invasiveness of papillary carcinoma cells. METHODS Expression of KAI-1/CD82 and its relation to COX-2 levels were investigated in 6 primary cultures of TPC, in 2 tumor cell lines (TPC-1 and K1), and in 55 tumor samples of TPC. The biological role of KAI-1/CD82 in regulating tumor invasiveness was investigated in TPC cell lines and primary cultures transfected with a pCDNA3.1/Hygro.KAI-1; transfected cells were tested in functional studies of cell migration and invasiveness. Finally, the role of KAI-1/CD82 in influencing TPC metastasis was investigated in vivo using nu/nu mice injected with K1-transfected cells. RESULTS We provide evidence that COX-2 and KAI-1/CD82 are inversely regulated in TPC primary cultures and in TPC-1 tumor cells. In fact, inhibition of COX-2 with NS398 is associated with a 2-9-fold upregulation of KAI-1/CD82 RNA. Moreover, a possible relation between COX-2 and KAI-1/CD82 was confirmed by the presence of a statistically significant inverse correlation in the expression of the two genes in 55 tumor samples of TPC (r = -0.513; p = 0.001). In 36 of 55 cases, tumor areas contained lower levels of KAI-1/CD82 RNA as compared with the corresponding normal tissue. Low expression of KAI-1/CD82 RNA in the tumor area was associated with extrathyroid extension of the disease in 16 of 19 cases (p < 0.04) and with lymph node metastasis in 11 of 14 cases (not significant). KAI-1/CD82 re-expression in tumor cells was associated with a significant decrease in their migratory (50-76% reduction) and invasive (46-65% reduction) capacity, even after hepatocyte growth factor stimulation. Finally, nu/nu mice injected with KAI-1/CD82-transfected K1 TPC cells developed fewer and smaller metastasis as compared with mice injected with vector-transfected K1 cells (p=0.016). CONCLUSION Our findings raise the possibility that downregulation of KAI-1/CD82 in TPC cells is one of the molecular mechanisms regulating their invasive and metastatic potential.

Primary malignant tumour of the lung with neuroendocrine and melanoma differentiation.

Melanocytic differentiation has been described in rare cases of melanin-containing neuroendocrine tumours of the lung [1–5], thyroid [6] and thymus [7]. In some of the cases, neuroendocrine granules and melanosomes were lying free in the cytoplasm of tumour cells, thus indicating true coexpression of melanocytic and neuroendocrine differentiation. On the other hand, neuroendocrine differentiation, consisting in the presence of neuroendocrine granules at ultrastructural level and of intense immunostaining for chromogranin-A and synaptophysin, was described in three cases of cutaneous and mucosal malignant melanomas [8]. Divergent differentiation towards unrelated embryological tissue has been reported in malignant melanoma [9]. So far, cases of neuroendocrine carcinoma with associated melanomatous component in the lung have not been described. There is growing evidence that tumour development may derive from neoplastic transformation of adult stem cells [10]. In a recent publication, it was reported that in the human skin there is a SOX2-positive stem cell capable of differentiation along the neuroendocrine or melanocytic lines [11]. In the present report, we describe the first case of a malignant neoplasm of the lung showing unambiguous differentiation towards neuroendocrine carcinoma and malignant melanoma.