Aim To explore the radiosensitizing effect of AZD8931, a novel equipotent and reversible inhibitor of signaling by EGFR (HER1), HER2 and HER3 receptors, focusing on cell cycle progression, apoptosis and clonogenic capacity in the human LoVo colorectal cancer (CRC) cell line, also in comparison with the EGFR-blocking monoclonal antibody Cetuximab or the EGFR tyrosine kinase selective small molecular inhibitor Gefitinib. Materials and methods Cells were pretreated with EGFR inhibitors for 5 consecutive days and then exposed or not to ionizing radiation (IR) (2 Gy daily for 3 consecutive days). Cell proliferation, cell cycle progression and apoptosis were evaluated by flow cytometry and enzyme-linked immunosorbent assay (ELISA), clonogenic potential and radiosensitivity were studied by colony formation assay. Results AZD8931 induced cell cycle arrest and apoptosis more effectively than Gefitinib and Cetuximab and, more importantly, it was significantly more potent than Gefitinib and Cetuximab in radiosensitizing cells. This radiosensitizing action by AZD8931 mainly occurred by markedly reducing cell cycle progression into S phase, the most radioresistant phase of cell cycle, secondly by inducing apoptosis and reducing clonogenic survival. Conclusions Our results show that AZD8931 increases IR efficacy in LoVo cells, suggesting that it works as a potent radiosensitizer, even more efficient than Gefitinib and Cetuximab, opening new pathways of investigation for further in vitro and in vivo studies aimed at confirming its potential to improve local radiotherapy in CRC.

Dissecting the pathogenesis of classical Hodgkin lymphoma (cHL), a common cancer in young adults, remains challenging because of the rarity of tumor cells in involved tissues (usually <5%). Here, we analyzed the coding genome of cHL by microdissecting tumor and normal cells from 34 patient biopsies for a total of ∼50 000 singly isolated lymphoma cells. We uncovered several recurrently mutated genes, namely, STAT6 (32% of cases), GNA13 (24%), XPO1 (18%), and ITPKB (16%), and document the functional role of mutant STAT6 in sustaining tumor cell viability. Mutations of STAT6 genetically and functionally cooperated with disruption of SOCS1, a JAK-STAT pathway inhibitor, to promote cHL growth. Overall, 87% of cases showed dysregulation of the JAK-STAT pathway by genetic alterations in multiple genes (also including STAT3, STAT5B, JAK1, JAK2, and PTPN1), attesting to the pivotal role of this pathway in cHL pathogenesis and highlighting its potential as a new therapeutic target in this disease.

We investigated whether GSTT1 (“null” allele), GSTM1 (“null”allele), GSTP1 (A313G), RFC1 (G80A), MTHFR (C677T), TS (2R/3R) polymorphisms were associated with toxicity and survival in patients with early breast cancer (EBC) treated with adjuvant chemotherapy (CT).

Hairy cell leukaemia-variant (HCL-v) is an indolent mature B-cell neoplasm, distinct from HCL and characterized by shorter survival and poorer response to purine analogues (Tiacci et al, 2006). Both HCL and HCL-v are CD11c+/CD103+, but CD25 is usually expressed only in HCL. In addition, the BRAF-V600E mutation, as well as annexin-A1 (ANXA1) and phospho-ERK (pERK) expression, are typically present in HCL and absent in HCL-v (Tiacci et al, 2011, 2012, 2013). Interestingly, some HCL and HCL-v cases carry a specific immunoglobulin (Ig) heavy chain variable gene rearrangement (IGHV4-34) with no or little somatic hypermutation, and show a lower complete response rate and progression-free survival after cladribine than corresponding HCL and HCL-v patients carrying other rearrangements (Arons et al, 2009). The development of large B-cell lymphoma after HCL-v has been reported in only four patients, all of whom subsequently died within a year (Matutes et al, 2001; Razaq et al, 2006). These cases, occurring 4–14 years after HCL-v diagnosis, were only briefly described clinically, with little pathological and no genetic characterization, leaving uncertainty as to whether they represented HCL-v transformation or the development of a clonally independent (e.g., therapy-related) high-grade lymphoma. The present study clarifies this issue for the first time, in a case extensively analysed with several immunophenotyping and genetic studies (Data S1 and Table SI), and provide the first evidence that the poorer prognosis linked to a germline or lowly mutated IGHV4-34 rearrangement in HCL-v can also extend to disease transformation. In April 2006, a 67-year-old male presented with fatigue, weight loss, splenomegaly (20 cm) without lymphadenopathy and thrombocytopenia (30 × 109/l). Although his white blood cell (WBC) count was normal, 16% of WBCs displayed an atypical hairy morphology and abundant cytoplasm. Flow cytometry showed 40% of B cells CD11c+/CD103+/CD25-/CD5-/CD23-/CD10-. A bone marrow (BM) biopsy revealed a mostly intra-sinusoidal infiltration by lymphoid cells with wide cytoplasm and hairy projections, comprising 25% of the marrow cellularity and being CD20+/ANXA1-/CD5-/CD23-/BCL6-/CD10-/cyclinD1-/IRF4- (Fig 1A–B). A diagnosis of HCL-v was made, and chemotherapy with cladribine (5 mg/m2/day for 5 days) delivered. This resulted in splenomegaly regression and clearance of the BM leukaemic infiltrate (down to 2%), but only minor platelet count improvement (up to 69 × 109/l). This response lasted only until March 2007 when the patient relapsed with constitutional symptoms, splenomegaly (21 cm) and BM infiltration (20%) by HCL-v. The patient received 4 weekly doses of rituximab, without response. A splenectomy, performed in September 2007, confirmed the diagnosis of HCL-v, showing an atrophic white pulp and an expanded red pulp infiltrated by lymphoid cells with wide cytoplasm and small nucleolus (Fig 1C) that were CD20+/CD103+/CD5-/CD23-/CD10-/IRF4-/BCL6-/CD25-/pERK-/ANXA1- (Fig 1C/inset-1D–1E). CyclinD1 was weakly expressed in a minor fraction of tumour cells and the proliferative index was low (~5%, Fig 1F). As expected, BRAF-V600E was not detected in the spleen DNA by allele-specific polymerase chain reaction. HCL-v can harbour MAP2K1 activating mutations (Waterfall et al, 2014), which however were not detected by Sanger sequencing of the spleen DNA, in keeping with the lack of pERK (the target of MAP2K1/MEK1 kinase) on immunohistochemistry. Conversely, Sanger sequencing identified a clonal heterozygous hot-spot missense mutation (S34F) of U2AF1 (Fig S1). U2AF1 encodes U2 small nuclear RNA auxiliary factor 1, a component of the RNA splicing machinery, and is recurrently mutated in myelodysplastic syndromes and other myeloid neoplasms (like other splicing factor genes) (Je et al, 2013), as well as, at lower frequency, in lung adenocarcinomas (Imielinski et al, 2012). More recently, the U2AF1 S34F mutation was observed in 3/24 patients with HCL-v (Waterfall et al, 2014), the only lymphoid malignancy where this mutation has been reported so far. Finally, by Sanger sequencing, we detected in the splenic sample a clonal productive IGHV4-34 rearrangement with low mutation load (0·94%, 2/213 mutated nucleotides). After splenectomy, the patient gained weight and platelets rose to 275 × 109/l. However, in August 2013, he presented again with constitutional symptoms, thrombocytopenia (49 × 109/l) and leukaemic lymphocytosis (WBC 14·05 × 109/l, lymphocytes 70%). At this time, however, he also had fever, hepatomegaly and a generalized lymphadenopathy with a high standardized-uptake value (SUVmax 20) on positron emission tomography–computerized tomography (PET-CT). A BM biopsy documented 80% infiltration by typical HCL-v. However, a node biopsy showed a complete effacement of the normal architecture by medium-to-large lymphoma cells with prominent nucleolus (Fig 2A) that were CD20+/CD103+/CD5-/CD23-/CD10-/CD25-/IRF4-/CD10-/ANXA1- (Fig 2B–C). These cells differed from the BM and splenic HCL-v cells not only by the larger nucleolus and cell size, but also for their strong BCL6 and cyclinD1 expression (Fig 2D–E) in the absence of CCND1 gene alterations by fluorescence in situ hybridization. Furthermore, the proliferative index was much higher in lymphoma cells (up to 60%) than in HCL-v cells (~5%; Figs 1F and 2F). The lymph node DNA carried the same clonal heterozygous U2AF1 point mutation as the spleen DNA (Fig S1). Furthermore, DNA fragment length analysis and sequencing of the immunoglobulin genes documented identically sized kappa-light chain and heavy-chain rearrangements (Fig S1), and an identical sequence of the clonal IGHV4-34+ rearrangement in the spleen and lymph node. Thus, the lymphoma was genetically related to the relapsed HCL; yet, it diverged clinically with nodal involvement and fever, and phenotypically with the acquisition of strong expression of BCL6 and CCND1, two oncogenes whose deregulated expression plays key roles in de novo diffuse large B-cell and mantle cell lymphomagenesis respectively. No clonal TP53 mutations were observed in the lymphoma by massively parallel sequencing, ruling out that a TP53 mutation, potentially present as rare subclonal event in HCL-v, expanded under the selection pressure of the subsequent chemotherapy and contributed to lymphoma transformation. After one cycle of R-CVP (rituximab/cyclophosphamide/vincristine/prednisolone) and 5 cycles of R-COMP (R-CVP plus liposomal doxorubicin), the leukaemic lymphocytosis disappeared, a BM biopsy was negative and PET-CT documented the resolution of splenomegaly and lymphadenopathies. Complete remission was confirmed at two subsequent CT scans, 4 and 9 months later. The patient was alive and well in remission at the last follow-up, 16 months after transformation. This contrasts with the four high-grade lymphomas reported so far after HCL-v, all leading to death within a year and none characterized for its clonal relationship with the preceding indolent leukaemia. Supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC IG-14447 to E.T.), Hairy Cell Leukaemia Foundation (to B.F. and E.T.), Ministero dell'Istruzione, Università e Ricerca (MIUR; Futuro in Ricerca 2010-RBFR10WT2K to E.T.; PRIN 20104HBZ8E to B.F), and the Associazione Umbra contro le Leucemie e i Linfomi (AULL, to B.F.). E.T. is a Scholar in Clinical Research of the Leukaemia and Lymphoma Society (Contract no. 2030-14). MZ and MR selected the case, analysed and interpreted the data generated from the corresponding biopsy samples, and wrote the manuscript. RV contributed to writing the manuscript. SP, EF and RL performed the laboratory work to generate the data. MIADC provided the clinical data of the case. GFO contributed to generate the immunohistological data. SA analysed and interpreted the data, and contributed to writing the manuscript. ET and BF analysed and interpreted the data, wrote the manuscript and coordinated the work. All the authors have approved the final draft. The authors wish to thank Dr. Gianluca Schiavoni, Dr. Alessandra Venanzi, Barbara Bigerna, Alessandra Pucciarini, Roberta Pacini and Alessia Tabarrini at the Institute of Haematology in Perugia (Italy) for technical assistance; and Dr. Monica Pessino at University of California, Santa Barbara, (USA) and Veronica Pessino PhD candidate in Biophysic at University of California San Francisco (USA), for help in the linguistic revision of the manuscript. The authors report no potential conflict of interest. Data S1. Materials and methods. Fig S1. Clonal relatedness of the HCL-v with the subsequent aggressive lymphoma. Table SI. Immunohistochemical and genetic analyses. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

In a phase I/II trial, patients with locally advanced rectal cancer received preoperative radiotherapy (RT) and concurrent with 5-fluorouracil (5-FU) and gefitinib. Results were promising. To elucidate the molecular and biological effects, we replicated the schedule in the LoVo human colorectal adenocarcinoma cell line.

IntroductionThis prospective study examined association between circulating plasma DNA, microsatellite alterations (MA), p53 mutations with time to relapse and survival in surgically treated non-small cell lung cancer (NSCLC) patients (pts).MethodsPlasma samples, adjacent lung tissue, and lung tumor tissue specimens were collected from consecutive patients with stage I–III NSCLC. Blood samples of 66 matched healthy donors with positive smoking history were collected as controls. The plasma DNA amount was determined by real-time PCR. The analysis of MA at loci D3S1300, D3S1289, D3S1266, and D3S2338 on chromosome 3p was performed by radiolabeled PCR. p53 Mutations (exons 5, 6, 7, and 8) were detected by PCR-single-strand conformational polymorphism assay.ResultsThere were 76 patients, 65 men; median age was 68 years (range, 42–86), 20 had stage I, 40 stage II, and 16 stage III, the majority of pts (48.7%) had squamous-cell histology. Sixty-nine (91%) were smokers and most had good Eastern Cooperative Oncology Group performance status (0/1:72/4). Mean circulating DNA of all pts was 60 ng/ml versus 5 ng/ml in smoker-matched controls (p < 0.0001). In pts without recurrence, mean circulating DNA was 48.5 ng/ml at baseline, 32.8 ng/ml at 3rd month, and 20.6 ng/ml at 12th month after surgery. In pts with recurrence, mean circulating DNA at baseline was 97.1 ng/ml. At 3rd month after surgery, mean DNA concentration was significantly lower in disease-free pts than in patients with recurrent disease (32.8 versus 292.7 ng/ml; p = 0.0016). MA in at least one locus was found in 39.5% of NSCLC tumors. p53 Genomic mutations were observed in 54.0% of tumor samples. Statistically significant associations were observed between MA and squamous-cell histotype (p = 0.007) and between p53 mutations and lymph node involvement (p = 0.012). MA and p53 mutations were found to be significantly associated with recurrence of disease (p = 0.033 and 0.026, respectively).ConclusionOur results suggest that MA and p53 mutations in tumor DNA have a potential prognostic role for disease recurrence in NSCLC patients, and elevated levels of plasma circulating DNA identify patients with possible systemic disease at diagnosis. This might be proposed as an early detection test of disease recurrence.

We previously demonstrated that when human bone marrow-derived mesenchymal cells (hMSCs) were co-cultured with CD4+/CD25+ T regulatory cells (Tregs) they maintained the T regulatory phenotype and function over time. Here we studied the TGF-beta/Smad signalling and mitogen-activated protein kinase (MAPK) pathways after Tregs/hMSCs co-culture. After 7 days co-culture of highly purified, immuno-selected Tregs and hMSCs from healthy donors, TGF-beta and IL-10 concentrations were dosed and mRNA of a panel of genes (NFAT, Smad4, Smad7, AP-1, Foxp3 and CD127) was quantified on harvested lymphocytes. Western blotting was performed by incubation with antiSMAD2, anti-pSMAD2, anti-ERK1/2 and anti-pERK1/2 polyclonal antibodies. After immuno-magnetic cell separation the final Treg fraction showed a mean purity of 93.6%±1. The CD4+/CD25+bright constituted 29%±7 of Tregs, FoxP3 cells constituted 51.9%±15.1 and CD127+ cells 19%±11.5. CD4+/CD25+ cells inhibited CD4+/CD25− cells (mean inhibition percentage: 52.1±29.6% (ratio 1:1). Tregs produced no TGF-beta and only a small quantity of IL-10 (8.67±4 pg/ml). hMSCs produced high quantity of TGF-beta (229.3 ± 54.8 pg/ml) associated with little IL-10 (2.7 ± 1.2 pg/ml). After co-culture, the TGF-beta concentration was 91.5±48.5 pg/ml while the IL-10 concentration was no different to baseline. To identify TGF-beta signaling pathways, we focused on Smad2, a central element in the cascade. In Tregs the strongly expressed pSmad2 signal after selection rapidly decreased after 7 days culture. When Tregs were co-cultured in presence of hMSCs the phosphorylated form of Smad2 did not change, indicating TGF-beta signaling was up-regulated. After co-culture mRNA quantification showed hMSCs down-regulated expression of SMAD7 (−8.9± 4.4 times vs Tregs without hMSCs), a negative regulator of TGF-beta signaling. After co-culture with hMSCs non-regulatory CD4+/CD25− control cells did not show any differences in SMAD7 expression. To study the MAPK kinase pathways, harvested Tregs were assayed for ERK1/2 kinase activity by determining their phosphorylated forms. pERK1/2 was almost absent in selected Tregs; it rapidly increased after 7 days in vitro culture without hMSCs but remained low after co-culture with hMSCs, indicating impaired ERK1/2 activation. AP-1 activation was also reduced (AP-1 mRNA −32±29 times less than controls). In conclusion, co-cultures of hMSCs and Tregs have the potential to set up a positive feedback loop that heightens responsiveness to TFG-beta in Treg cells. In fact, hMSCs produce TGF-beta which is consumed in co-culture with Tregs; TGF-beta1 signaling in Tregs is maintained through pSMAD2 up-regulation and SMAD7 down-regulation; hMSCs also inhibit ERK phosphorylation which consequently down-regulates AP-1 mRNA. Thus Tregs signalling is maintained through culture on a layer of hMSCs. Manipulation of Tregs signaling through culture on a layer of hMSCs may represent a novel strategy for maintenance of Treg phenotype and function.

in patients with recurrent disease (32.8 versus 292.7 ng/ml; p 0.0016). MA in at least one locus was found in 39.5% of NSCLC tumors. p53 Genomic mutations were observed in 54.0% of tumor samples. Statistically significant associations were observed between MA and squamous-cell histotype (p 0.007) and between p53 mutations and lymph node involvement (p 0.012). MA and p53 mutations were found to be significantly associated with recurrence of disease (p 0.033 and 0.026, respectively). Conclusion: Our results suggest that MA and p53 mutations in tumor DNA have a potential prognostic role for disease recurrence in NSCLC patients, and elevated levels of plasma circulating DNA identify patients with possible systemic disease at diagnosis. This might be proposed as an early detection test of disease recurrence.

RATIONALE:We hypothesized that antigen-activated T cells could acquire CysLT-R, increasing their susceptibility to apoptosis after in vitro exposure to Montelukast (MNT).METHODS: Enriched T cells stimulated by plastic-coated anti-CD3 mAb for 24 hours were exposed to MNT concentrations ranging from 10 - 5 to 10 -8 M. Percentage of apoptotic cells was enumerated by propidium iodide staining.We then used cDNA arrays technology (SuperArrays Inc., Frederick, MD) to determine the expression profile of genes encoding ligands, receptors, intracellular modulators, and transcription factors involved in the regulation of programmed cell death.RESULTS: Dose-response and time-course studies indicate that T cells needs to be pre-activated (at least for 24 hours) in order to acquire CysLT receptors and the sensitivity to the pro-apoptotic action of MNT at lowest concentration of 10 -6 M, that is maximal after 48 hours incubation.While genes such as TNF receptors and/or ligands, TRAF or Bcl-2 family did not show any difference with respect to negative controls, a striking overexpression of p63 cDNA, together with mdm2 inhibition was observed in samples exposed to MNT.CONCLUSIONS: Taken together, results seem to indicate that MNTinduced programmed cell death requires antigen stimulation of T cells to express CysLT-R, which in turn can mediate p53 pathway activation through over-expression of p63 cDNA.

Background Montelukast (MNT), a cysteinyl‐leukotriene receptor (Cys‐LTR) antagonist, has anti‐inflammatory activity in the treatment of allergic diseases. If this effect is due only to blocking leukotrienes or also owing to inhibiting proliferation and survival of inflammatory cells, is actually unknown.

Thirty-six sex-mismatched transplants were studied using fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) methods. Molecular cytogenetics was performed using interphase FISH with a centromeric probe for chromosome Y, and PCR amplification was performed with a set of VNTR microsatellite loci. In addition, reverse transcriptase-PCR (RT-PCR) for BCR-ABL fusion was used to investigate cases of Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). Our integrated approach of post-transplant monitoring was helpful in documenting successful transplants and in controlling the size of Ph-positive clones in CML. A striking overlap was found between results from FISH analysis and PCR for polymorphic loci.