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Patients with aggressive B-cell lymphoma and MYC rearrangement at fluorescence in situ hybridization exhibit poor outcome after R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). In the last decade, 68 patients with Burkitt lymphoma ([BL] n = 46) or high-grade B-cell lymphoma ([HGBCL] single, double, or triple hit; n = 22) were treated with a dose-dense, short-term therapy termed “CARMEN regimen” at 5 Italian centers. Forty-six (68%) patients were HIV+. CARMEN included a 36-day induction with sequential, single weekly doses of cyclophosphamide, vincristine, rituximab, methotrexate, etoposide, and doxorubicin plus intrathecal chemotherapy, followed by high-dose-cytarabine–based consolidation. Patients who did not achieve complete remission (CR) after induction received BEAM (carmustina, etoposide, cytarabine, melfalan)-conditioned autologous stem cell transplantation (ASCT) after consolidation. Sixty-one (90%) patients completed induction, and 59 (87%) completed consolidation. Seventeen patients received ASCT. Grade 4 hematological toxicity was common but did not cause treatment discontinuation; grade 4 nonhematological toxicity was recorded in 11 (16%) patients, with grade 4 infections in 6 (9%). Six (9%) patients died of toxicity (sepsis in 4, COVID-19, acute respiratory distress syndrome). CR rate after the whole treatment was 73% (95% confidence interval [CI], 55% to 91%) for patients with HGBCL and 78% (95% CI, 66% to 90%) for patients with BL. At a median follow-up of 65 (interquartile range, 40-109) months, 48 patients remain event free, with a 5-year progression-free survival of 63% (95% CI, 58% to 68%) for HGBCL and 72% (95% CI, 71% to 73%) for BL, with a 5-year overall survival (OS) of 63% (95% CI, 58% to 68%) and 76% (95% CI, 75% to 77%), respectively. HIV seropositivity did not have a detrimental effect on outcome. This retrospective study shows that CARMEN is a safe and active regimen both in HIV-negative and -positive patients with MYC-rearranged lymphomas. Encouraging survival figures, attained with a single dose of doxorubicin and cyclophosphamide, deserve further investigation in HGBCL and other aggressive lymphomas.

Background The deeper knowledge of non–small-cell lung cancer (NSCLC) biology and the discovery of driver molecular alterations have opened the era of precision medicine in lung oncology, thus significantly revolutionizing the diagnostic and therapeutic approach to NSCLC. In Italy, however, molecular assessment remains heterogeneous across the country, and numbers of patients accessing personalized treatments remain relatively low. Nationwide programs have demonstrated that the creation of consortia represent a successful strategy to increase the number of patients with a molecular classification. Patients and Methods The Alliance Against Cancer (ACC), a network of 25 Italian Research Institutes, has developed a targeted sequencing panel for the detection of genomic alterations in 182 genes in patients with a diagnosis of NSCLC (ACC lung panel). One thousand metastatic NSCLC patients will be enrolled onto a prospective trial designed to measure the sensitivity and specificity of the ACC lung panel as a tool for molecular screening compared to standard methods. Results and Conclusion The ongoing trial is part of a nationwide strategy of ACC to develop infrastructures and improve competences to make the Italian research institutes independent for genomic profiling of cancer patients.

A few prospective trials in HIV-positive patients with Burkitt lymphoma (BL) or high-grade B-cell lymphoma (HGBL) have been reported. Investigated therapies have shown good efficacy but relevant safety problems, with high rates of interruptions, severe mucositis, septic complications, and fungal infections. Here, we report the results of a multicentre phase II trial addressing a new dose-dense, short-term therapy aimed at maintaining efficacy and improving tolerability. The experimental programme included a 36-day polychemotherapy induction followed by high-dose cytarabine-based consolidation and response-tailored BEAM (carmustine, etoposide, cyatarabine, and melphalan)- conditioned autologous stem cell transplantation (ASCT). This therapy would be considered active if ≥11 complete remissions (CR) after induction (primary endpoint) were recorded among 20 assessable patients. HIV-positive adults (median age 42, range 26-58; 16 males) with untreated BL (n = 16), HGBL (n = 3) or double-hit lymphoma (n = 1) were enrolled. All patients had high-risk features, with meningeal and bone marrow infiltration in five and nine patients respectively. The experimental programme was safe and active in a multicentre setting, with only two episodes of grade 4 non-haematological toxicity (hepatotoxicity and mucositis), and no cases of systemic fungal infections; two patients died of toxicity (bacterial infections). Response after induction (median duration: 47 days; interquartile range 41-54), was complete in 13 patients and partial in five [overall response rate = 90%; 95% confidence interval (CI) = 77-100]. All responders received consolidation, and five required autologous stem cell transplant. At a median follow-up of 55 (41-89) months, 14 patients are relapse-free and 15 are alive, with a five-year progression-free survival and an overall survival of 70% (95% CI = 60-80%) and 75% (95% CI = 66-84) respectively. No patient with cerebrospinal fluid (CSF)/meningeal lymphoma experienced central nervous system recurrence. With respect to previously reported regimens, this programme was delivered in a shorter period, and achieved the main goal of maintaining efficacy and improving tolerability.

Recently, the term mixed neuroendocrine non-neuroendocrine neoplasms (MiNEN) has been proposed as an umbrella definition covering different possible combinations of mixed neuroendocrine-exocrine neoplasms. Among these, the adenoma plus neuroendocrine tumor (NET) combination is among the rarest and not formally recognized by the 2019 WHO Classification. In this setting, the debate between either collision tumors or true mixed neoplasms is still unsolved. In this report, a pancreatic intraductal papillary mucinous neoplasm (IPMN) plus a NET is described, and the molecular investigations showed the presence in both populations of the same KRAS, GNAS, and CDKN2A mutations and the amplification of the CCND1 gene. These data prove clonality and support a common origin of both components, therefore confirming the true mixed nature. For this reason, mixed neuroendocrine-exocrine neoplasms, in which the exocrine component is represented by a glandular precursor lesion (adenoma/IPMN) only, should be included into the MiNEN family.

Objectives Circulating cell-free tumor DNA (ctDNA) isolated from the peripheral blood of non-small-cell lung cancer (NSCLC) patients provides biomarkers for both therapeutic target selection, particularly when direct tumor biopsy is unfeasible, and also for drug resistance monitoring. This study evaluates the reliability and feasibility of ctDNA analysis in an in-house clinical molecular diagnostic workflow. Materials and methods Mutation profiling by both standard methods and Next-Generation sequencing (NGS) was carried out and compared on 2 independent lung cancer patient cohorts. Cohort 1 consisted of 50 EGFR-mutated NSCLC patients, established on tumour biopsy, for whom ctDNA was collected at disease progression after TKI-inhibitor treatment and could be used to monitor drug resistance. Cohort 2 consisted of 50 newly diagnosed lung cancer patients for whom tumour biopsy was not possible and only ctDNA was available, providing the possibility of biomarker identification. Results ctDNA analysis of Cohort 1 verified the persistence of the tumour-detected EGFR activating mutation at disease progression by both standard and NGS methods, in 84% and 92% of the cases respectively. The T790M EGFR resistance mutation was identified in 71% of the ctDNA EGFR mutated samples providing vital information for their disease management. In newly diagnosed Cohort 2 patients, EGFR activating mutations were detected in 16% of the patients by both standard and NGS analysis of ctDNA in peripheral blood, providing indication to targeted-therapy otherwise unavailable for this group of patients. Conclusion The presented study investigated lung cancer ctDNA analysis, comparing conventional methods versus NGS sequencing, and demonstrated the successful use of plasma ctDNA as a template for targeted NGS tumor gene panel in an in-house routine clinical practice. More importantly, these data underline the advantages of the clinical application of ctDNA NGS analysis for identification of therapeutic targets, real-time monitoring of therapy, and resistance mechanisms in lung cancer patients.

Purpose To detect the presence of MYD88 L265P mutation in the aqueous humor of patients with cytologically proven vitreoretinal lymphoma. Methods Eight consecutive patients with bilateral vitreoretinal lymphoma (16 eyes) were prospectively evaluated. Genomic DNA was extracted from aqueous samples after paracentesis and vitreous humor samples after diagnostic vitrectomy. MYD88 codon 265 mutation was investigated by both amplification-refractory mutation system polymerase chain reaction approach and pyrosequencing assay in the aqueous humor of all patients and in the vitreous of 6 patients. A control group of 8 age-matched patients with established diagnosis of noninfectious uveitis was also tested for the presence of MYD88 L265P mutation in the aqueous humor. Results Eight patients (three men, five women) with mean age of 69.5 years (range 50-85 years) were considered. All the patients tested for MYD88 L265P in the vitreous (six) were positive, and this result was consistent with cytological examination in all samples but one. The MYD88 L265P mutation was found in the aqueous of 6 patients (75%), and in 3 of them, the mutation was present in both eyes. Results of MYD88 L265P mutation in aqueous and vitreous sample were consistent in 7 of the 8 eyes with available samples. The aqueous humor of the noninfectious uveitis control group was negative for the detection of MYD88 L265P mutation. Conclusion MYD88 mutation was detected in the aqueous humor of 75% of patients with cytologically proven vitreoretinal lymphoma. This technique may be considered as an additional diagnostic tool in the detection of the disease.

// Gilda Magliacane 1, * , Greta Grassini 1, * , Paola Bartocci 2, * , Ilaria Francaviglia 1 , Elena Dal Cin 1 , Gianluca Barbieri 2 , Gianluigi Arrigoni 1 , Lorenza Pecciarini 1 , Claudio Doglioni 1 , Maria Giulia Cangi 1 1 Unit of Pathology, IRCCS San Raffaele Scientific Institute, Milano, Italy 2 Diatech Pharmacogenetics Company, Jesi, Italy * These authors have contributed equally to this work Correspondence to: Maria Giulia Cangi, e-mail: cangi.mariagiulia@hsr.it Claudio Doglioni, e-mail: doglioni.claudio@hsr.it Keywords: Pathology, molecular diagnostics, solid tumors, high throughput mass-spectrometry, target therapy, mutations Received: July 01, 2015 Accepted: August 14, 2015 Published: September 02, 2015 ABSTRACT Tumor genotyping is an essential step in routine clinical practice and pathology laboratories face a major challenge in being able to provide rapid, sensitive and updated molecular tests. We developed a novel mass spectrometry multiplexed genotyping platform named PentaPanel to concurrently assess single nucleotide polymorphisms in 56 hotspots of the 5 most clinically relevant cancer genes, KRAS, NRAS, BRAF , EGFR and PIK3CA for a total of 221 detectable mutations. To both evaluate and validate the PentaPanel performance, we investigated 1025 tumor specimens of 6 different cancer types (carcinomas of colon, lung, breast, pancreas, and biliary tract, and melanomas), systematically addressing sensitivity, specificity, and reproducibility of our platform. Sanger sequencing was also performed for all the study samples. Our data showed that PentaPanel is a high throughput and robust tool, allowing genotyping for targeted therapy selection of 10 patients in the same run, with a practical turnaround time of 2 working days. Importantly, it was successfully used to interrogate different DNAs isolated from routinely processed specimens (formalin-fixed paraffin embedded, frozen, and cytological samples), covering all the requirements of clinical tests. In conclusion, the PentaPanel platform can provide an immediate, accurate and cost effective multiplex approach for clinically relevant gene mutation analysis in many solid tumors and its utility across many diseases can be particularly relevant in multiple clinical trials, including the new basket trial approach, aiming to identify appropriate targeted drug combination strategies.

Multiple myeloma develops primarily inside the bone marrow microenvironment, that confers pro-survival signals and drug resistance. 3D cultures that reproduce multiple myeloma-bone marrow interactions are needed to fully investigate multiple myeloma pathogenesis and response to drugs. To this purpose, we exploited the 3D Rotary Cell Culture System bioreactor technology for myeloma-bone marrow co-cultures in gelatin scaffolds. The model was validated with myeloma cell lines that, as assessed by histochemical and electron-microscopic analyses, engaged contacts with stromal cells and endothelial cells. Consistently, pro-survival signaling and also cell adhesion-mediated drug resistance were significantly higher in 3D than in 2D parallel co-cultures. The contribution of the VLA-4/VCAM1 pathway to resistance to bortezomib was modeled by the use of VCAM1 transfectants. Soluble factor-mediated drug resistance could be also demonstrated in both 2D and 3D co-cultures. The system was then successfully applied to co-cultures of primary myeloma cells-primary myeloma bone marrow stromal cells from patients and endothelial cells, allowing the development of functional myeloma-stroma interactions and MM cell long-term survival. Significantly, genomic analysis performed in a high-risk myeloma patient demonstrated that culture in bioreactor paralleled the expansion of the clone that ultimately dominated in vivo. Finally, the impact of bortezomib on myeloma cells and on specialized functions of the microenvironment could be evaluated. Our findings indicate that 3D dynamic culture of reconstructed human multiple myeloma microenvironments in bioreactor may represent a useful platform for drug testing and for studying tumor-stroma molecular interactions.

"The CDC25A-CDK2 pathway has been proposed as critical for the oncogenic action of. human epidermal growth factor receptor 2 (HER2) in mammary epithelial cells. In. particular, transgenic expression of CDC25A cooperates with HER2 in promoting. mammary tumors, whereas CDC25A hemizygous loss attenuates the HER2-induced. tumorigenesis penetrance. On the basis of this evidence of a synergism between. HER2 and the cell cycle regulator CDC25A in a mouse model of mammary. tumorigenesis, we investigated the role of CDC25A in human HER2-positive breast. cancer and its possible implications in therapeutic response. HER2 status and. CDC25A expression were assessed in 313 breast cancer patients and we found. statistically significant correlation between HER2 and CDC25A (P = .007).. Moreover, an HER2-positive breast cancer subgroup with high levels of CDC25A and . very aggressive phenotype was identified (P = .005). Importantly, our in vitro. studies on breast cancer cell lines showed that the HER2 inhibitor efficacy on. cell growth and viability relied also on CDC25A expression and that such. inhibition induces CDC25A down-regulation through phosphatidylinositol. 3-kinase\/protein kinase B pathway and DNA damage response activation. In line. with this observation, we found a statistical significant association between. CDC25A overexpression and trastuzumab-combined therapy response rate in two. different HER2-positive cohorts of trastuzumab-treated patients in either. metastatic or neoadjuvant setting (P = .018 for the metastatic cohort and P =. .021 for the neoadjuvant cohort). Our findings highlight a link between HER2 and . CDC25A that positively modulates HER2-targeted therapy response, suggesting that,. in HER2-positive breast cancer patients, CDC25A overexpression affects. trastuzumab sensitivity."

Background In vitro primary cultures of microvascular endothelial cells from endocrine pancreas are difficult to obtain, but can be a very helpful tool for studies of islet biology, transplantation and regenerative medicine. Methods We applied a protocol recently described for the isolation and culture of brain microvascular endothelial cells (EC) on human pancreatic islets. EC obtained were characterized in terms of morphological (light and transmission electron microscopy), phenotypical (by immunofluorescence and flow cytometry) and functional (cord formation assay and protein secretion by multiplex bead-based assay) characteristics. Results EC were obtained from 25% of islet preparations processed. Two primary endothelial cell lines showed high proliferative potential and were deeply characterized: they presented endothelial cell morphology and expressed CD31, CD49a, CD49e, CD34, von Willebrand Factor (vWF), Vascular Endothelial CAdherin (VE-CAD), Tyrosine Kinase with Ig and EGF Homology Domains-2 (TIE2), Vascular Endothelial Growth Factor Receptor 1 (VEGFR1), Ulex lectin and the endothelium endocrine-specific marker nephrin. Besides, they were able to form cordons in vitro and secreted factors involved in the process of angiogenesis such as Vascular Endothelial Growth Factor (VEGF), Monocyte Chemotactic Protein 1 (MCP-1), interleukin (IL)-8 and Melanoma Growth Stimulatory Activity Alpha (GROα). These cell lines were termed Human Islet Microvascular Endothelial Cells (HIMEC). Discussion This study establishes a simple and effective strategy for isolation and long-term culture of EC derived from human pancreatic islet. HIMEC in culture preserve phenotype and functional properties and are, therefore, a useful tool for future experiments of in vitro pancreas modelling, co-transplantation with pancreatic islets, re-vascularization of scaffold or matrix for regenerative medicine purposes. © 2017 International Society for Cellular Therapy

Monoclonal B-cell lymphocytosis (MBL) is classified as chronic lymphocytic leukemia (CLL)–like, atypical CLL, and CD5− MBL. The number of B cells per microliter divides CLL-like MBL into MBL associated with lymphocytosis (usually detected in a clinical setting) and low-count MBL detected in the general population (usually identified during population screening). After a median follow-up of 34 months we reevaluated 76 low-count MBLs with 5-color flow cytometry: 90% of CLL-like MBL but only 44.4% atypical CLL and 66.7% CD5− MBL persisted over time. Population-screening CLL-like MBL had no relevant cell count change, and none developed an overt leukemia. In 50% of the cases FISH showed CLL-related chromosomal abnormalities, including monoallelic or biallelic 13q deletions (43.8%), trisomy 12 (1 case), and 17p deletions (2 cases). The analysis of the T-cell receptor β (TRBV) chains repertoire showed the presence of monoclonal T-cell clones, especially among CD4highCD8low, CD8highCD4low T cells. TRBV2 and TRBV8 were the most frequently expressed genes. This study indicates that (1) the risk of progression into CLL for low-count population-screening CLL-like MBL is exceedingly rare and definitely lower than that of clinical MBL and (2) chromosomal abnormalities occur early in the natural history and are possibly associated with the appearance of the typical phenotype.

Ocular adnexal MALT lymphoma (OAML) is linked to Chlamydophila psittaci (Cp) infection. Viability and infectivity of Cp, demonstrated by growth in culture, has not been yet investigated in these patients. We conducted a single-center prospective case-control study to assess the prevalence, viability and infectivity of Cp in 20 OAML patients and 42 blood donors registered in a 6-month period. The presence of Cp in conjunctival swabs and peripheral blood mononuclear cells (PBMC) of patients and donors was assessed by TETR-PCR and in vitro cultures. From an epidemiological point of view, OAML patients often resided in rural areas, and reported a history of chronic conjunctivitis and prolonged contact with household animals (85% vs. 38% of donors; p = 0.00001). Cp was detected in lymphoma tissue in 15 (75%) patients. Cp DNA was detected in conjunctival swabs and/or PBMC from 10 (50%) patients and in PBMC from 1 (2%) donor (p = 0.01). Viability and infectivity of Cp, demonstrated by growth in culture, were confirmed in conjunctival swabs and/or PBMC from 5 (25%) patients, but not in donors (p = 0.002). This prospective study demonstrates, for the first time, that Cp present in the conjunctiva and PBMC of OAML patients is capable to grow and be isolated in cell cultures. Cp infection is common in OAML patients and exceptional in blood donors. Epidemiological data of OAML patients (prolonged contact with household animals and chronic conjunctivitis) are consistent with Cp exposure risk. (C) 2008 Wiley-Liss, Inc. Ocular adnexal MALT lymphoma (OAML) is linked to Chlamydophila psittaci (Cp) infection. Viability and infectivity of Cp, demonstrated by growth in culture, has not been yet investigated in these patients. We conducted a single-center prospective case-control study to assess the prevalence, viability and infectivity of Cp in 20 OAML patients and 42 blood donors registered in a 6-month period. The presence of Cp in conjunctival swabs and peripheral blood mononuclear cells (PBMC) of patients and donors was assessed by TETR-PCR and in vitro cultures. From an epidemiological point of view, OAML patients often resided in rural areas, and reported a history of chronic conjunctivitis and prolonged contact with household animals (85% vs. 38% of donors; p = 0.00001). Cp was detected in lymphoma tissue in 15 (75%) patients. Cp DNA was detected in conjunctival swabs and/or PBMC from 10 (50%) patients and in PBMC from 1 (2%) donor (p = 0.01). Viability and infectivity of Cp, demonstrated by growth in culture, were confirmed in conjunctival swabs and/or PBMC from 5 (25%) patients, but not in donors (p = 0.002). This prospective study demonstrates, for the first time, that Cp present in the conjunctiva and PBMC of OAML patients is capable to grow and be isolated in cell cultures. Cp infection is common in OAML patients and exceptional in blood donors. Epidemiological data of OAML patients (prolonged contact with household animals and chronic conjunctivitis) are consistent with Cp exposure risk. (C) 2008 Wiley-Liss, Inc.

Malignant Mesothelioma is a highly aggressive cancer, which is difficult to diagnose and treat. Here we describe the molecular, cellular and morphological characterization of a syngeneic system consisting of murine AB1, AB12 and AB22 mesothelioma cells injected in immunocompetent BALB/c mice, which allows the study of the interplay of tumor cells with the immune system. Murine mesothelioma cells, like human ones, respond to exogenous High Mobility Group Box 1 protein, a Damage-Associated Molecular Pattern that acts as a chemoattractant for leukocytes and as a proinflammatory mediator. The tumors derived from AB cells are morphologically and histologically similar to human MM tumors, and respond to treatments used for MM patients. Our system largely recapitulates human mesothelioma, and we advocate its use for the study of MM development and treatment. Malignant Mesothelioma is a highly aggressive cancer, which is difficult to diagnose and treat. Here we describe the molecular, cellular and morphological characterization of a syngeneic system consisting of murine AB1, AB12 and AB22 mesothelioma cells injected in immunocompetent BALB/c mice, which allows the study of the interplay of tumor cells with the immune system. Murine mesothelioma cells, like human ones, respond to exogenous High Mobility Group Box 1 protein, a Damage-Associated Molecular Pattern that acts as a chemoattractant for leukocytes and as a proinflammatory mediator. The tumors derived from AB cells are morphologically and histologically similar to human MM tumors, and respond to treatments used for MM patients. Our system largely recapitulates human mesothelioma, and we advocate its use for the study of MM development and treatment

Renal-cell carcinoma (RCC) constitutes a heterogeneous group of tumors with specific chromosome aberrations. Recently, a new small group of RCC, occurring in children and young adults, has been described as characterized by t(6;11)(p21;q12). It has been shown that this translocation results in the fusion of the 5′ portion of the ALPHA gene (11q12) with the transcription factor gene TFEB (6p21). Herewith, we report the first complete cytogenetic and molecular characterization of a t(6;11)-positive RCC of an adult patient, a 54-year-old woman. The tumor was histologically defined as RCC with peculiar features and it was negative for epithelial markers and positive for melanocytic markers. Chromosome QFQ banding analysis of short-term cultured cells from the RCC showed t(6;11)(p21;q12) as the sole cytogenetic abnormality. The translocation was confirmed by FISH analysis. RT-PCR analysis, performed on total RNA isolated from both neoplastic and normal tissue samples, revealed an ALPHA–TFEB chimeric transcript in the tumor sample; sequencing of the RT-PCR product defined a novel TFEB gene breakpoint cluster region, broader than the one reported thus far. Western blot analysis showed a band at the expected size of wild-type TFEB in the neoplastic tissue compared to the normal sample, supporting that the fusion gene does not encode for a chimeric protein but it causes an upregulation of the wild-type TFEB. Our data contribute to define better this rare RCC type, which is typical not only of childhood but can also be found in adulthood. © 2007 Wiley-Liss, Inc.

Immortalized cell lines representative of chronic lymphocytic leukemia (CLL) can assist in understanding disease pathogenesis and testing new therapeutic agents. At present, very few representative cell lines are available. We here describe the characterization of a new cell line (PCL12) that grew spontaneously from the peripheral blood (PB) of a CLL patient with progressive disease and EBV infection. The CLL cell origin of PCL12 was confirmed after the alignment of its IGH sequence against the "original" clonotypic sequence. The IGH gene rearrangement was truly unmutated and no CLL-related cytogenetic or genetic lesions were detected. PCL12 cells express CD19, CD20, CD5, CD23, low levels of IgM and IgD and the poor-outcome-associated prognostic markers CD38, ZAP70 and TCL1. In accordance with its aggressive phenotype the cell line is inactive in terms of LYN and HS1 phosphorylation. BcR signalling pathway is constitutively active and anergic in terms of p-ERK and Calcium flux response to α-IgM stimulation. PCL12 cells strongly migrate in vitro in response to SDF-1 and form clusters. Finally, they grow rapidly and localize in all lymphoid organs when xenotrasplanted in Rag2-/-γc-/- mice. PCL12 represents a suitable preclinical model for testing pharmacological agents.

Twist proteins have been shown to contribute to cancer development and progression by impinging on different regulatory pathways, but their mechanism of action is poorly defined. By investigating the role of Twist in sarcomas, we found that Twist1 acts as a mechanism alternative to TP53 mutation and MDM2 overexpression to inactivate p53 in mesenchymal tumors. We provide evidence that Twist1 binds p53 C terminus through the Twist box. This interaction hinders key posttranslational modifications of p53 and facilitates its MDM2-mediated degradation. Our study suggests the existence of a Twist box code of p53 inactivation and provides the proof of principle that targeting the Twist box:p53 interaction might offer additional avenues for cancer treatment. Twist proteins have been shown to contribute to cancer development and progression by impinging on different regulatory pathways, but their mechanism of action is poorly defined. By investigating the role of Twist in sarcomas, we found that Twist1 acts as a mechanism alternative to TP53 mutation and MDM2 overexpression to inactivate p53 in mesenchymal tumors. We provide evidence that Twist1 binds p53 C terminus through the Twist box. This interaction hinders key posttranslational modifications of p53 and facilitates its MDM2-mediated degradation. Our study suggests the existence of a Twist box code of p53 inactivation and provides the proof of principle that targeting the Twist box:p53 interaction might offer additional avenues for cancer treatment.

Molecular therapies targeting HER2 are part of the established drug armamentarium in breast carcinoma. Now the ToGA trial, an international multicenter phase III clinical study, involving 24 countries globally, has shown that the anti-HER2 humanized monoclonal antibody Trastuzumab is effective in prolonging survival in HER2-positive carcinoma of the stomach and the gastroesophageal junction (GEJ). Similarly to breast carcinoma, > 20% of gastric cancers show HER2 overexpression and/or amplification, and this percentage increases to 33% in GEJ tumors. Thus, as in breast carcinoma, pathologists are now asked to evaluate HER2 status in gastric carcinoma samples. As validated in the ToGA trial, the HER2 testing criteria that must be used in evaluating both gastric carcinoma biopsies and surgical specimens significantly differ from those routinely applied in breast carcinoma. The main variations with regard to the pattern of reactivity in HER2-expressing cells are as follows: the completeness of membrane staining is not a "conditio sine qua non" and the number of stained cells necessary to consider a case as positive is different. We must also take note of the much more frequent heterogeneity of HER2 positivity in gastric cancer compared with breast carcinoma and the less stringent correlation between HER2 amplification and protein overexpression that is observed in gastric carcinoma, where more than 20% of cases may carry HER2 amplification, although of low level, without HER2 expression. In these patients, in the ToGA trial, there was no apparent benefit from adding Trastuzumab to chemotherapy: for this reason the European Medicines Agency, while approving usage of Trastuzumab for metastatic adenocarcinoma treatment, indicated HER2 testing by immunohistochemistry as first evaluation assay, followed by fluorescence in situ hybridization in 2+ equivocal cases. HER2 testing in gastric carcinoma is a new field, opening several opportunities: for patients with gastric cancer, this is a new promising therapeutic option; for pathologists, strengthening our role in therapy selection and emphasizing our duty of providing accurate and reproducible HER2 testing results; for all interested in understanding the biology of gastric and GEJ cancer and in discovering new possible molecular therapy targets. ZR 0 ZS 0 ZB 51 Z8 7

Purpose: Chlamydia psittaci (Cp) has been associated to ocular adnexal lymphomas (OAL) with variable geographic distribution. Herein, we used multiple Chlamdia detection tools to identify Cp elementary bodies-containing cell and to assess Cp prevalence in both nodal and extranodal lymphomas. Experimental Design: TETR-PCR, immunohistochemistry, immunofluorescence, electron microscopy, and laser-capture microdissection were done in 35 OALs to define their effect in Chlamydia detection and, moreover, to identify the Cp cellular carrier. Cp prevalence was screened by TETR-PCR in 205 extraorbital lymphomas and 135 nonneoplastic controls. Results: Twenty-six (74%) OALs were associated with Cp infection: immunohistochemistry, immunofluorescence, and laser-capture microdissection-assisted PCR showed that monocytes/macrophages were the Cp carriers; electron microscopy showed the presence of intact CP elementary bodies into these cells. Immunohistochemistry and TETR-PCR showed a 70% concordance rate (P = 0.001). Cp DNA was equally prevalent in non-OAL, nodal, and extranodal lymphomas: among the latter, it was more common in diffuse large B-cell lymphomas of the skin (P = 0.03) and Waldeyer's ring. Conclusions: This multiparametric approach shows, for the first time, that monocytes/macrophages are the carriers of Cp, Cp seems preferentially associated with lymphomas arising in organs primarily exposed to antigens. The clinical implications of these findings deserve to be prospectively investigated. Purpose: Chlamydia psittaci (Cp) has been associated to ocular adnexal lymphomas (OAL) with variable geographic distribution. Herein, we used multiple Chlamdia detection tools to identify Cp elementary bodies-containing cell and to assess Cp prevalence in both nodal and extranodal lymphomas. Experimental Design: TETR-PCR, immunohistochemistry, immunofluorescence, electron microscopy, and laser-capture microdissection were done in 35 OALs to define their effect in Chlamydia detection and, moreover, to identify the Cp cellular carrier. Cp prevalence was screened by TETR-PCR in 205 extraorbital lymphomas and 135 nonneoplastic controls. Results: Twenty-six (74%) OALs were associated with Cp infection: immunohistochemistry, immunofluorescence, and laser-capture microdissection-assisted PCR showed that monocytes/macrophages were the Cp carriers; electron microscopy showed the presence of intact CP elementary bodies into these cells. Immunohistochemistry and TETR-PCR showed a 70% concordance rate (P = 0.001). Cp DNA was equally prevalent in non-OAL, nodal, and extranodal lymphomas: among the latter, it was more common in diffuse large B-cell lymphomas of the skin (P = 0.03) and Waldeyer's ring. Conclusions: This multiparametric approach shows, for the first time, that monocytes/macrophages are the carriers of Cp, Cp seems preferentially associated with lymphomas arising in organs primarily exposed to antigens. The clinical implications of these findings deserve to be prospectively investigated. PURPOSE: Chlamydia psittaci (Cp) has been associated to ocular adnexal lymphomas (OAL) with variable geographic distribution. Herein, we used multiple Chlamydia detection tools to identify Cp elementary bodies-containing cell and to assess Cp prevalence in both nodal and extranodal lymphomas. EXPERIMENTAL DESIGN: TETR-PCR, immunohistochemistry, immunofluorescence, electron microscopy, and laser-capture microdissection were done in 35 OALs to define their effect in Chlamydia detection and, moreover, to identify the Cp cellular carrier. Cp prevalence was screened by TETR-PCR in 205 extraorbital lymphomas and 135 nonneoplastic controls. RESULTS: Twenty-six (74%) OALs were associated with Cp infection: immunohistochemistry, immunofluorescence, and laser-capture microdissection-assisted PCR showed that monocytes/macrophages were the Cp carriers; electron microscopy showed the presence of intact Cp elementary bodies into these cells. Immunohistochemistry and TETR-PCR showed a 70% concordance rate (P = 0.001). Cp DNA was equally prevalent in non-OAL, nodal, and extranodal lymphomas: among the latter, it was more common in diffuse large B-cell lymphomas of the skin (P = 0.03) and Waldeyer's ring. CONCLUSIONS: This multiparametric approach shows, for the first time, that monocytes/macrophages are the carriers of Cp, Cp seems preferentially associated with lymphomas arising in organs primarily exposed to antigens. The clinical implications of these findings deserve to be prospectively investigated.

CDC25A phosphatase, an essential component of the cell cycle machinery, is also a key player in integrating the specific signals of checkpoint control in response to DNA damage. There are several lines of evidence that indicate a role for CDC25A in cancer (level opment, consistent with the fact that its overexpression is detected in human cancers. In particular we previously reported that CDC25A is overexpressed also in early breast carcinoma. Recent data suggest that oncogene activation during early stages of tumor development causes DNA replication stress resulting in the induction of DNA damage response (DDR) and that the selection of cells defecting in their DDR could lead to malignant progression. To address how CDC25A overexpression contributes to breast cancer development we established it cell model in which CDC25A was constitutively overexpressed in hTERT-immortalized primary human mammary epithelial cells. At the earliest passages following CDC25A transduction we observed DDR signs associated with unscheduled DNA replication origins. In the latest passages DDR was significantly impaired and, even after ionizing radiation exposition, cells failed to induce G1 and G2 checkpoints; moreover DNA replication stress conditions, such as aphidicolin treatment, highlighted increased fragile site breakages and destabilized chromosomes Just in these latest passages cells. Our data suggest that CDC25A overexpression, pushing the cell through the cell cycle transitions, induces DDR alterations that might enhance genomic instability. (C) 2008 Wiley-Liss, Inc. OI Maestro, Roberta/0000-0002-6642-5592 ZR 0 ZS 0 Z8 1 CDC25A phosphatase, an essential component of the cell cycle machinery, is also a key player in integrating the specific signals of checkpoint control in response to DNA damage. There are several lines of evidence that indicate a role for CDC25A in cancer development, consistent with the fact that its overexpression is detected in human cancers. In particular we previously reported that CDC25A is overexpressed also in early breast carcinoma. Recent data suggest that oncogene activation during early stages of tumor development causes DNA replication stress resulting in the induction of DNA damage response (DDR) and that the selection of cells defecting in their DDR could lead to malignant progression. To address how CDC25A overexpression contributes to breast cancer development we established a cell model in which CDC25A was constitutively overexpressed in hTERT-immortalized primary human mammary epithelial cells. At the earliest passages following CDC25A transduction we observed DDR signs associated with unscheduled DNA replication origins. In the latest passages DDR was significantly impaired and, even after ionizing radiation exposition, cells failed to induce G1 and G2 checkpoints; moreover DNA replication stress conditions, such as aphidicolin treatment, highlighted increased fragile site breakages and destabilized chromosomes just in these latest passages cells. Our data suggest that CDC25A overexpression, pushing the cell through the cell cycle transitions, induces DDR alterations that might enhance genomic instability.

Immortalized cell lines representative of chronic lymphocytic leukemia (CLL) can assist in understanding disease pathogenesis and testing new therapeutic agents. At present, very few representative cell lines are available. We here describe the characterization of a new cell line (PCL12) that grew spontaneously from the peripheral blood (PB) of a CLL patient with progressive disease and EBV infection. The CLL cell origin of PCL12 was confirmed after the alignment of its IGH sequence against the “original” clonotypic sequence. The IGH gene rearrangement was truly unmutated and no CLL-related cytogenetic or genetic lesions were detected. PCL12 cells express CD19, CD20, CD5, CD23, low levels of IgM and IgD and the poor-outcome-associated prognostic markers CD38, ZAP70 and TCL1. In accordance with its aggressive phenotype the cell line is inactive in terms of LYN and HS1 phosphorylation. BcR signalling pathway is constitutively active and anergic in terms of p-ERK and Calcium flux response to α-IgM stimulation. PCL12 cells strongly migrate in vitro in response to SDF-1 and form clusters. Finally, they grow rapidly and localize in all lymphoid organs when xenotrasplanted in Rag2-/-γc-/- mice. PCL12 represents a suitable preclinical model for testing pharmacological agents.

To determine the role of beta-catenin pathway in human skin carcinogenesis, 135 non-melanoma skin tumors were analyzed for beta-catenin expression and gene mutations. Intense nucleo-cytoplasmic immunoreactivity for C terminus beta-catenin antibodies was observed in all pilomatricomas and in single cases of trichoepithelioma and squamous cell carcinoma showing peculiar signs of matrical differentiation. Moderate increase of beta-catenin nuclear staining was detected in a significant proportion of basal cell carcinomas, Bowen disease, spiroadenomas, and occasionally also in squamous cell carcinomas, but in these neoplasms only a limited fraction of tumor cells accumulated beta-catenin. Molecular analysis revealed that beta-catenin gene mutations are a peculiar feature of skin tumors with matrical differentiation and correlate with a pattern of intense and diffuse beta-catenin nuclear expression. In contrast, adenomatous polyposis coli (APC) and AXIN2 mutations were not involved in skin tumorigenesis. Analysis of Wnt pathway revealed that TCF-1 and MITF-M were selectively induced in the tumor types harboring beta-catenin mutations, indicating that a Wnt/beta-catenin pathway involving TCF-1 and MITF-M is activated in these tumors. Interestingly, high expression levels of TCF-3 were found in basal cell carcinomas and spiroadenomas. TCF-3 is reported to act as a negative modulator of beta-catenin degradation pathway. Thus, the moderate increase of beta-catenin nuclear staining detected in these tumor types might, at least in part, be due to a TCF-3-dependent mechanism. Finally, we found that the presence of beta-catenin mutations significantly correlated with loss of nuclear immunoreactivity for an antibody raised against the N terminus of beta-catenin (alphaABC). Thus, a combined analysis with C terminus-beta-catenin antibodies and alphaABC Ab may represent a powerful investigative approach for the detection of beta-catenin structural alterations.

BACKGROUND Syndecan-1 is a transmembrane heparan sulphate proteoglycan that is involved in cell–cell adhesion, organization of cell–matrix adhesion, and regulation of growth factor signaling. METHODS Specimens from 254 consecutive breast carcinoma (BC) cases (110 N0, 144 N1/2) with long-term follow-up (median, 95 months) were immunostained for syndecan-1, estrogen receptor (ER), progesterone receptor (PgR), and p53; in 154 cases, c-erbB-2 status was known. Syndecan-1 mRNA and protein expression also were evaluated in 20 breast tissue samples (10 normal and tumor pairs). RESULTS Syndecan-1 was expressed at high levels in 106 (42%) BCs; syndecan-1 up-regulation was confirmed by reverse transcriptase–polymerase chain reaction (RT-PCR) studies. High syndecan-1 expression was associated with high histologic grade, large tumor size, high mitotic count, c-erbB-2 overexpression, and ER and PgR negative status. At univariate survival analysis syndecan overexpression was related to poor prognosis (P < 0.01 for both overall survival (OS) and disease-free survival). Bivariate survival analysis showed an additive adverse effect for syndecan-1 and c-erbB-2 overexpression. At multivariate analysis, syndecan-1 overexpression was independently associated with poor OS (hazard ratio [HR], 1.71; 95% confidence interval [CI], 1.08–2.69). High syndecan-1 expression also was of independent prognostic value for OS in the group of 102 ER-negative patients (HR, 2.42; 95% CI, 1.21–4.82). Stratifying patients on the basis of the type of adjuvant therapy given, high syndecan-1 expression was associated with a higher risk of death only in patients treated with the cyclophosphamide-methotrexate-fluorouracil regimen (HR, 1.9; P = 0.09); at multivariate analysis for OS, this association proved to be of independent statistical significance (P = 0.03; HR, 2.15). CONCLUSIONS Syndecan-1 is expressed at high levels in a significant percentage of breast carcinomas and is related to an aggressive phenotype and poor clinical behavior. Cancer 2003;98:474–83. © 2003 American Cancer Society. DOI 10.1002/cncr.11515

Myoepithelial cells (MCs) constitute the basal cell layer of normal mammary epithelia, and their identification is of particular diagnostic value because they are retained in most benign lesions while being lost in malignancy. Several MC immunocytochemical markers are currently available for diagnostic purposes, with special reference to smooth muscle-related antigens. p63 is a member of the p53 gene family, and its germline mutations are associated with severe mammary developmental defects in both rodents and humans. Different p63 isoforms have been identified, some of which (Delta Np63) are preferentially expressed in the epithelial basal cells of different or-ans and have been considered as possible markers of stem cells/reserve cells. We investigated immunohistochemically 384 samples of normal and diseased human breast, including 300 invasive carcinomas, using four antibodies recognizing all p63 isoforms, or the Delta Np63 isoforms. Twenty cytologic specimens were also investigated. Furthermore, snap-frozen tissue samples from three fibroadenomas and 10 invasive ductal carcinomas with their paired non-neoplastic tissues and three corresponding lymph node metastases were evaluated for the expression of p63 mRNA by RT-PCR. In normal breast tissue p63 immunoreactivity was confined to the nuclei of MCs. In all benign lesions p63-immunoreactive cells formed a continuous basal rim along the epithelial structures. Stromal cells, and in particular myofibroblasts, were consistently unreactive. Adenomyoepitheliomas showed nuclear staining in most neoplastic cells. A peripheral rim of p63-immunoreactive cells was retained surrounding lobular and ductal carcinoma in situ, although it was discontinuous as opposed to the normal structures. Invasive breast carcinomas were consistently devoid of nuclear p63 staining, with the exception of the two adenoid-cystic carcinomas, of the two ductal carcinomas with squamous metaplasia, and of 11 (4.6%) ductal carcinomas not otherwise specified, showing p63 immunoreactivity in a minor fraction (5-15%) of the neoplastic cells. In comparison with other MC markers, p63 was the most specific, being restricted exclusively to MCs, whereas antibodies to smooth muscle actin and, to a lesser extent, calponin also decorated stromal myofibroblasts. In the cytologic preparations p63 immunoreactivity was a consistent feature of "naked nuclei" and of a subset of cells surrounding benign epithelial clusters. RT-PCR experiments with primers specific for different p63 isoforms documented that normal tissues and fibroadenomas preferentially expressed the Delta Np63 isoforms. Our study demonstrates that in normal and pathologic breast tissues MCs consistently express the Delta Np63 isoforms. We suggest p63 as a reliable, highly specific, and sensitive MC marker in both histologic and cytologic preparations. Furthermore, because p63 immunoreactivity in adult epithelia is normally restricted to progenitor cells, it can be speculated that it mi-ht be a clue for the identification of the still elusive breast progenitor cells. ZR 0 Z8 16 ZB 189 ZS 2

Chorioamnionitis, a major cause of preterm birth with significant neonatal morbidity and mortality, frequently occurs in mothers who are free of symptoms. A combined clinical, radiologic, and pathologic study of 129 very low birth weight infants indicated a significant association between a markedly decreased thymic size at birth and subclinical chorioamnionitis.