Publications by authors named "Marcello Gambacorta"

39 Publications

Primary cutaneous B-cell lymphoma other than marginal zone: clinicopathologic analysis of 161 cases: Comparison with current classification and definition of prognostic markers.

Cancer Med 2016 10 26;5(10):2740-2755. Epub 2016 Sep 26.

Unit of Anatomic Pathology, Department of Molecular Medicine, University of Pavia and Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.

Categorization of primary cutaneous B-cell lymphomas (PCBCL) other than marginal zone (MZL) represents a diagnostic challenge with relevant prognostic implications. The 2008 WHO lymphoma classification recognizes only primary cutaneous follicular center cell lymphoma (PCFCCL) and primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT), whereas the previous 2005 WHO/EORTC classification also included an intermediate form, namely PCDLBCL, other. We conducted a retrospective, multicentric, consensus-based revision of the clinicopathologic characteristics of 161 cases of PCBCL other than MZL. Upon the histologic features that are listed in the WHO classification, 96 cases were classified as PCFCCL and 25 as PCDLBCL-LT; 40 further cases did not fit in the former subgroups in terms of cytology and/or architecture, thus were classified as PCDLBCL, not otherwise specified (PCDLBCL-NOS). We assigned all the cases a histogenetic profile, based on the immunohistochemical detection of CD10, BCL6, and MUM1, and a "double hit score" upon positivity for BCL2 and MYC. PCDLBCL-NOS had a clinical presentation more similar to PCFCCL, whereas the histology was more consistent with the picture of a diffuse large B-cell lymphoma, as predominantly composed of centroblasts but with intermixed a reactive infiltrate of small lymphocytes. Its behavior was intermediate between the other two forms, particularly when considering only cases with a "non-germinal B-cell" profile, whereas "germinal center" cases resembled PCFCCL. Our data confirmed the aggressive behavior of PCDLBC-LT, which often coexpressed MYC and BCL2. The impact of single factors on 5-year survival was documented, particularly histogenetic profile in PCDLBCL and BCL2 translocation in PCFCCL. Our study confirms that a further group-PCDLBCL-NOS-exists, which can be recognized through a careful combination of histopathologic criteria coupled with adequate clinical information.
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http://dx.doi.org/10.1002/cam4.865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5083727PMC
October 2016

Assessment of a HER2 scoring system for colorectal cancer: results from a validation study.

Mod Pathol 2015 Nov 9;28(11):1481-91. Epub 2015 Oct 9.

Niguarda Cancer Center, Ospedale Niguarda Ca' Granda, Milano, Italy.

We sought to develop criteria for ERBB2-positivity (HER2) in colorectal cancer to ensure accurate identification of ERBB2-amplified metastatic colorectal cancer patients suitable for enrollment in a phase II trial of ERBB2-targeted therapy (HERACLES trial). A two-step approach was used. In step 1, a consensus panel of pathologists adapted existing protocols for use in colorectal cancer to test ERBB2 expression and amplification. Collegial revision of an archival test cohort of colorectal cancer samples led to specific recommendations for adapting current breast and gastric cancer criteria for scoring ERBB2 in colorectal cancer. In step 2, from September 2012 to January 2015, colorectal-specific ERBB2 testing protocols and ERBB2 scoring criteria were used to centrally screen for ERBB2-positive KRAS wild-type colorectal cancer patients to be enrolled in the HERACLES trial (clinical validation cohort). In both archival test (N=256) and clinical validation (N=830) cohorts, a clinically sizeable 5% fraction of KRAS wild-type colorectal cancer patients was found to be ERBB2-positive according to the colorectal cancer-specific ERBB2 scoring criteria. ERBB2-positive tumors showed ERBB2 immunostaining consisting of intense membranous ERBB2 protein expression, corresponding to homogenous ERBB2 amplification, in >50% of cells. None of the immunohistochemistry 0 or 1+ cases was amplified. Concordance between SISH and FISH was 100%. In conclusion, we propose specific criteria for defining ERBB2-positivity in colorectal cancer (HERACLES Diagnostic Criteria). In a phase II trial of trastuzumab and lapatinib in a cetuximab-resistant population, HERACLES Diagnostic Criteria shaped the selection of patients and defined ERBB2 as a predictive marker for response to ERBB2-targeted therapy in metastatic colorectal cancer.
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http://dx.doi.org/10.1038/modpathol.2015.98DOI Listing
November 2015

Predictive value of computerized morphometric analysis of steatosis in donor livers.

Anal Quant Cytopathol Histpathol 2014 Jun;36(3):137-46

Objective: To describe, by computerized morphometry, the degree and the type of steatosis in liver transplants that developed primary nonfunction and to compare the results with the quantification by pathologist.

Study Design: Twelve patients who developed primary nonfunction after liver transplantation were matched with 23 transplanted patients with a regular postoperative clinical course. Morphology of the liver biopsy included many stereological parameters; all cases were evaluated by an operator blinded to the diagnosis and to the clinical history. The assessment of steatosis by morphometry was compared with the pathologist's evaluation. Moreover, to assess the reproducibility of the morphometric model, another operator applied the morphometric model in a blinded fashion to a randomly selected sample of cases.

Results: The percentage of hepatocytes with microsteatosis and the ratio of macro/microsteatosis were higher in primary nonfunction. The pathologist's evaluation of steatosis showed a marked overestimation when compared to morphometry. Lastly, the comparison between the results of 2 blinded operators of morphometric analysis showed a high reproducibility with a low interobserver variability.

Conclusion: Our quantitative estimation of the degree and the quality of steatosis avoids interobserver interpretations. Moreover, our analysis shows that the quantification of steatosis in liver transplantation by the current assessment must be reviewed in order to reevaluate the real impact of steatosis.
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June 2014

The TPM3-NTRK1 rearrangement is a recurring event in colorectal carcinoma and is associated with tumor sensitivity to TRKA kinase inhibition.

Mol Oncol 2014 Dec 12;8(8):1495-507. Epub 2014 Jun 12.

Nerviano Medical Sciences S.r.l., Nerviano (Milan), Italy.

The NTRK1 gene encodes Tropomyosin-related kinase A (TRKA), the high-affinity Nerve Growth Factor Receptor. NTRK1 was originally isolated from a colorectal carcinoma (CRC) sample as component of a somatic rearrangement (TPM3-NTRK1) resulting in expression of the oncogenic chimeric protein TPM3-TRKA, but there has been no subsequent report regarding the relevance of this oncogene in CRC. The KM12 human CRC cell line expresses the chimeric TPM3-TRKA protein and is hypersensitive to TRKA kinase inhibition. We report the detailed characterization of the TPM3-NTRK1 genomic rearrangement in KM12 cells and through a cellular screening approach, the identification of NMS-P626, a novel highly potent and selective TRKA inhibitor. NMS-P626 suppressed TPM3-TRKA phosphorylation and downstream signaling in KM12 cells and showed remarkable antitumor activity in mice bearing KM12 tumors. Finally, using quantitative reverse transcriptase PCR and immunohistochemistry (IHC) we identified the TPM3-NTRK1 rearrangement in a CRC clinical sample, therefore suggesting that this chromosomal translocation is indeed a low frequency recurring event in CRC and that such patients might benefit from therapy with TRKA kinase inhibitors.
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http://dx.doi.org/10.1016/j.molonc.2014.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5528583PMC
December 2014

MicroRNA as potential biomarker in HCV-associated diffuse large B-cell lymphoma.

J Clin Pathol 2014 Aug 9;67(8):697-701. Epub 2014 Jun 9.

Division of Pathology, Department of Pathophysiology and Transplantation, University of Milan, IRCCS Ca' Granda-Maggiore Policlinico Hospital Foundation, Milan, Italy.

Aims: To identify molecular characteristics to hepatitis C virus (HCV)-associated diffuse large B-cell lymphoma (DLBCL) through a comprehensive miRNAs expression profiling.

Methods: In this study, miRNA profiles were obtained from 37 patients with DLBCLs and 60 patients with reactive lymph nodes, equally distributed according to HCV presence. Germinal centres, from reactive lymph nodes were used as controls. Clinical features at presentation were available for all patients.

Results: A set of 52 miRNAs define a signature for HCV-associated DLBCL. Importantly, decreased expression of miR-138-5p and increased expression of miR-147a, miR-147b and miR-511-5p in HCV DLBCL was found to be a poor prognostic factor for HCV-positive DLBCL patients.

Conclusions: These data reveal molecular differences in diffuse DLBCL patients according to HCV presence, potentially useful as novel prognostic or therapeutic biomarkers.
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http://dx.doi.org/10.1136/jclinpath-2014-202352DOI Listing
August 2014

Beyond the learning curve of the Retzius-sparing approach for robot-assisted laparoscopic radical prostatectomy: oncologic and functional results of the first 200 patients with ≥ 1 year of follow-up.

Eur Urol 2013 Dec 8;64(6):974-80. Epub 2013 Jul 8.

Department of Urology, Niguarda Ca' Granda Hospital, Milan, Italy. Electronic address:

Background: Robot-assisted laparoscopic radical prostatectomy (RARP) has become the main surgical option for localized prostate cancer. We recently developed a new approach for RARP, passing through the pouch of Douglas and avoiding all the Retzius structures involved in continence and potency preservation.

Objective: To report the functional and oncologic results of our first 200 patients operated on using this new approach.

Design, Setting, And Participants: This was a prospective, noncontrolled case series including the first 200 consecutive patients undergoing this kind of surgery (January the 1st, 2010 to December the 31st, 2011).

Surgical Procedure: Retzius-sparing RARP.

Outcome Measurements And Statistical Analysis: All perioperative, oncologic, and functional data were prospectively recorded. Potency was defined as an International Index of Erectile Function-5 questionnaire score >17; continence was defined as use of no pad or of one safety liner. Oncologic results were reported as positive surgical margins (PSM) and 1-yr biochemical disease-free survival (1y-bDFS). Recurrence was defined as a repeated prostate-specific antigen >0.2 ng/ml. Complications were graded according to the Clavien-Dindo system. The first 100 patients (group 1) were compared with the second 100 (group 2) to evaluate the learning curve effects.

Results And Limitations: The median patient age was 65 yr. Comparing the two groups, transfusions were needed in 8% versus 4% of cases in groups 1 and 2, respectively (p=0.02). There was one Clavien-Dindo grade 3b in group 1 versus one grade 3a complication in group 2. In patients with pT2 disease, PSMs were recorded in 22.4% of those in group 1 versus 10.1% in group 2 (p=0.045). 1y-bDFS was 89% in group 1 versus 92% in group 2. For groups 1 and 2, respectively, immediate continence was reached in 92% versus 90% of patients, and the 1-yr continence rate was 96% versus 96%. Considering the 77 potent patients aged <65 yr who underwent bilateral intrafascial nerve-sparing surgery, 40.4% of those in group 1 versus 40% of those in group 2 reached their first intercourse within 1 mo; at 1 yr of follow-up, these figures had increased to 81% versus 71%, respectively (p=0.162). The main limitation of this study is its noncontrolled nature.

Conclusions: We demonstrated Retzius-sparing RARP to be oncologically safe and to result in high early continence and potency rates. Long-term, prospective, comparative, and possibly randomized studies are needed.
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http://dx.doi.org/10.1016/j.eururo.2013.06.046DOI Listing
December 2013

The reticulin algorithm for adrenocortical tumor diagnosis: a multicentric validation study on 245 unpublished cases.

Am J Surg Pathol 2013 Sep;37(9):1433-40

Departments of *Oncology §§Clinical and Biological Sciences, University of Turin at San Luigi Hospital, Orbassano, Torino †Department of Medicine (DIMED), Pathology and Cytopathology Unit ††Department of Medicine (DIMED), Endocrinology Unit ∥Department of Statistics, University of Padua, Padova Divisions of ‡Pathology **Endocrinology, University of Florence at Careggi Hospital, Firenze Divisions of §Endocrinology ¶Pathology, Niguarda Ca' Granda Hospital, Milano #Department of Pathology and Molecular Genetics, General Hospital, Treviso ‡‡Department of Medical Oncology, University of Brescia, Italy.

The pathologic diagnosis of adrenocortical carcinoma (ACC) still needs to be improved, because the renowned Weiss Score (WS) system has a poor reproducibility of some parameters and is difficult to apply in borderline cases and in ACC variants. The "reticulin algorithm" (RA) defines malignancy through an altered reticulin framework associated with 1 of the 3 following parameter: necrosis, high mitotic rate, and vascular invasion. This study aimed at validating the interobserver reproducibility of reticulin stain evaluation in an unpublished series of 245 adrenocortical tumors (61 adenomas and 184 carcinomas) from 5 Italian centers, classified according to the WS. Eight pathologists reviewed all reticulin-stained slides. After training, a second round of evaluation on discordant cases was performed 10 weeks later. The RA reclassified 67 cases (27%) as adenomas, including 44 with no reticulin alterations and 23 with an altered reticulin framework but lacking the subsequent parameters of the triad. The other 178 cases (73%) were carcinomas according to the above-mentioned criteria. A complete (8/8 pathologists) interobserver agreement was reached in 75% of cases (κ=0.702), irrespective of case derivation, pathologists' experience, and histologic variants, and was further improved when only those cases with high WS and clinically malignant behavior were considered. After the training, the overall agreement increased to 86%. We conclude that reticulin staining is a reliable technique and an easy-to-interpret system in adrenocortical tumors; moreover, it has a high interobserver reproducibility, which supports the notion of using such a method in the proposed 2-step RA approach for ACC diagnosis.
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http://dx.doi.org/10.1097/PAS.0b013e31828d387bDOI Listing
September 2013

Amplification of the MET receptor drives resistance to anti-EGFR therapies in colorectal cancer.

Cancer Discov 2013 Jun 2;3(6):658-73. Epub 2013 Jun 2.

Department of Oncology, University of Torino, Torino, Italy.

EGF receptor (EGFR)-targeted monoclonal antibodies are effective in a subset of metastatic colorectal cancers. Inevitably, all patients develop resistance, which occurs through emergence of KRAS mutations in approximately 50% of the cases. We show that amplification of the MET proto-oncogene is associated with acquired resistance in tumors that do not develop KRAS mutations during anti-EGFR therapy. Amplification of the MET locus was present in circulating tumor DNA before relapse was clinically evident. Functional studies show that MET activation confers resistance to anti-EGFR therapy both in vitro and in vivo. Notably, in patient-derived colorectal cancer xenografts, MET amplification correlated with resistance to EGFR blockade, which could be overcome by MET kinase inhibitors. These results highlight the role of MET in mediating primary and secondary resistance to anti-EGFR therapies in colorectal cancer and encourage the use of MET inhibitors in patients displaying resistance as a result of MET amplification.
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http://dx.doi.org/10.1158/2159-8290.CD-12-0558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4078408PMC
June 2013

Results of the First Italian External Quality Assurance Scheme for somatic EGFR mutation testing in non-small-cell lung cancer.

J Thorac Oncol 2013 Jun;8(6):773-8

Research Department, Cell Biology and Biotherapy Unit, INT Fondazione G.Pascale, Via M. Semmola, Naples, Italy.

Introduction: The Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytology organized an external quality assessment (EQA) scheme for EGFR mutation testing in non-small-cell lung cancer.

Methods: Ten specimens, including three small biopsies with known epidermal growth factor receptor (EGFR) mutation status, were validated in three referral laboratories and provided to 47 participating centers. The participants were requested to perform mutational analysis, using their usual method, and to submit results within a 4-week time frame. According to a predefined scoring system, two points were assigned to correct genotype and zero points to false-negative or false-positive results. The threshold to pass the EQA was set at higher than 18 of 20 points. Two rounds were preplanned.

Results: All participating centers submitted the results within the time frame. Polymerase chain reaction (PCR)/sequencing was the main methodology used (n = 37 laboratories), although a few centers did use pyrosequencing (n = 8) or real-time PCR (n = 2). A significant number of analytical errors were observed (n = 20), with a high frequency of false-positive results (n = 16). The lower scores were obtained for the small biopsies. Fourteen of 47 centers (30%) that did not pass the first round, having a score less than or equal to 18 points, used PCR/sequencing, whereas 10 of 10 laboratories, using pyrosequencing or real-time PCR, passed the first round. Eight laboratories passed the second round. Overall, 41of 47 centers (87%) passed the EQA.

Conclusion: The results of the EQA for EGFR testing in non-small-cell lung cancer suggest that good quality EGFR mutational analysis is performed in Italian laboratories, although differences between testing methods were observed, especially for small biopsies.
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http://dx.doi.org/10.1097/JTO.0b013e31828c2b08DOI Listing
June 2013

Postsurgical intrapericardial adhesions: mechanisms of formation and prevention.

Ann Thorac Surg 2013 May 6;95(5):1818-26. Epub 2013 Apr 6.

Department of Cardiac Surgery, Niguarda Ca' Granda Hospital, Milan, Italy.

Postsurgical intrapericardial adhesions are still considered an unavoidable consequence of cardiothoracic operations. They increase the technical difficulty and the risk of reoperations. The pathogenesis of postsurgical adhesions is a multistep process, and the main key players are (1) loss of mesothelial cells, (2) accumulation of fibrin in areas devoid of mesothelial cells, (3) loss of normal pericardial fibrinolysis, and (4) local inflammation. Today, very promising methods to reduce adhesions are available for clinical use. This report reviews the process of formation of adhesions and the methods to prevent them, classified according to the mechanism of action.
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http://dx.doi.org/10.1016/j.athoracsur.2012.11.020DOI Listing
May 2013

Histological findings following use of CoSeal in a patient with a left ventricular assist device.

Surg Innov 2013 Dec 14;20(6):NP35-7. Epub 2013 Feb 14.

1Department of Cardiac Surgery, Niguarda Ca' Granda Hospital, Milan, Italy.

Adhesions are a formidable challenge in patients undergoing reoperative cardiac surgery, particularly in those supported by an intracorporeal left ventricular assist device (LVAD) and undergoing heart transplantation. This report describes the pathological findings following the clinical use of a surgical sealant (CoSeal, Baxter Healthcare, Fremont, CA), in a patient who underwent LVAD implantation. On the treated surfaces, a minimal amount of adhesions were observed, whereas in untreated surfaces adhesions were present.
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http://dx.doi.org/10.1177/1553350612443899DOI Listing
December 2013

KRAS gene amplification in colorectal cancer and impact on response to EGFR-targeted therapy.

Int J Cancer 2013 Sep 16;133(5):1259-65. Epub 2013 Mar 16.

Division of Pathology, Ospedale Niguarda Ca' Granda, Piazza Ospedale Maggiore 3, Milano, Italy.

KRAS mutations are the most common oncogenic event in colorectal cancer (CRC) progression and their occurrence is associated with lack of response to anti epidermal growth factor receptor (EGFR) targeted therapies. Using preclinical models and patients' samples we recently reported that the emergence of KRAS mutations but also KRAS amplification is associated with acquired resistance to the EGFR inhibitors cetuximab or panitumumab. We reasoned that KRAS amplification may also be responsible for primary resistance to these agents. Furthermore, while the prevalence of KRAS mutations has been well established in CRC, little is known about the frequency of KRAS amplification in large CRC series. We performed a screening of 1,039 CRC samples to assess the prevalence of KRAS amplification in this tumor type and further evaluated the role of this genetic alteration on the sensitivity to anti EGFR therapies. We detected KRAS amplification in 7/1,039 (0.67%) and 1/102 evaluable CRC specimens and cell lines, respectively. KRAS amplification was mutually exclusive with KRAS mutations. Tumors or cell lines harboring this genetic lesion are not responsive to anti-EGFR inhibitors. Although KRAS amplification is an infrequent event in CRC, it might be responsible for precluding response to anti-EGFR treatment in a small proportion of patients.
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http://dx.doi.org/10.1002/ijc.28106DOI Listing
September 2013

Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer.

Nature 2012 Jun;486(7404):532-6

Laboratory of Molecular Genetics, Institute for Cancer Research and Treatment, 10060 Candiolo (Torino), Italy.

A main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), is effective in a subset of KRAS wild-type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here we show that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance, but resistant cells remained sensitive to combinatorial inhibition of EGFR and mitogen-activated protein-kinase kinase (MEK). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.
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http://dx.doi.org/10.1038/nature11156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3927413PMC
June 2012

IRTA1 is selectively expressed in nodal and extranodal marginal zone lymphomas.

Histopathology 2012 Nov 20;61(5):930-41. Epub 2012 Jun 20.

Institute of Haematology, University of Perugia, Ospedale S Maria della Misericordia, Perugia, Italy.

Aims: The aim of this study was to search for a molecule selectively expressed by marginal zone (MZ) lymphomas (MZLs), whose diagnosis is currently based on morphological criteria and negativity for markers detectable in other B-cell lymphomas.

Methods And Results: Two thousand one hundred and four peripheral lymphomas of various types were immunostained with a monoclonal antibody against immunoglobulin superfamily receptor translocation-associated 1 (IRTA1), which recognizes the equivalents of MZ in human lymphoid tissues other than spleen. IRTA1 expression was restricted to extranodal (93%) and nodal MZLs (73%) and to lymphomas with MZ differentiation. Extranodal MZL cells with the strongest IRTA1 expression were usually located adjacent to epithelia, mimicking the IRTA1 expression pattern of normal and acquired mucosa-associated lymphoid tissue (MALT). The cytological features, growth pattern and IRTA1 positivity in nodal MZLs suggest they may derive from IRTA1(+) perifollicular B cells or monocytoid B cells detectable in reactive lymph nodes. Double immunostaining for IRTA1/bcl-6 tracked the colonization of B-cell follicles by MZL cells, and showed modulation of their phenotype (e.g. acquisition of bcl-6) during recirculation through germinal centres. MZL cells differentiating into plasma cells usually lost IRTA1.

Conclusions: These results further expand our knowledge of the biology of MZLs, and highlight IRTA1 as the first positive marker for MZLs, enabling more accurate diagnosis of these neoplasms.
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http://dx.doi.org/10.1111/j.1365-2559.2012.04289.xDOI Listing
November 2012

A molecularly annotated platform of patient-derived xenografts ("xenopatients") identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal cancer.

Cancer Discov 2011 Nov 2;1(6):508-23. Epub 2011 Sep 2.

Laboratory of Molecular Pharmacology, Division of Surgical Oncology, Unit of Pathology, Institute for Cancer Research and Treatment, Torino, Italy.

Unlabelled: Only a fraction of patients with metastatic colorectal cancer receive clinical benefit from therapy with anti-epidermal growth factor receptor (EGFR) antibodies, which calls for the identification of novel biomarkers for better personalized medicine. We produced large xenograft cohorts from 85 patient-derived, genetically characterized metastatic colorectal cancer samples ("xenopatients") to discover novel determinants of therapeutic response and new oncoprotein targets. Serially passaged tumors retained the morphologic and genomic features of their original counterparts. A validation trial confirmed the robustness of this approach: xenopatients responded to the anti-EGFR antibody cetuximab with rates and extents analogous to those observed in the clinic and could be prospectively stratified as responders or nonresponders on the basis of several predictive biomarkers. Genotype-response correlations indicated HER2 amplification specifically in a subset of cetuximab-resistant, KRAS/NRAS/BRAF/PIK3CA wild-type cases. Importantly, HER2 amplification was also enriched in clinically nonresponsive KRAS wild-type patients. A proof-of-concept, multiarm study in HER2-amplified xenopatients revealed that the combined inhibition of HER2 and EGFR induced overt, long-lasting tumor regression. Our results suggest promising therapeutic opportunities in cetuximab-resistant patients with metastatic colorectal cancer, whose medical treatment in the chemorefractory setting remains an unmet clinical need.

Significance: Direct transfer xenografts of tumor surgical specimens conserve the interindividual diversity and the genetic heterogeneity typical of the tumors of origin, combining the flexibility of preclinical analysis with the informative value of population-based studies. Our suite of patient-derived xenografts from metastatic colorectal carcinomas reliably mimicked disease response in humans, prospectively recapitulated biomarker-based case stratification, and identified HER2 as a predictor of resistance to anti-epidermal growth factor receptor antibodies and of response to combination therapies against HER2 and epidermal growth factor receptor in this tumor setting.
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http://dx.doi.org/10.1158/2159-8290.CD-11-0109DOI Listing
November 2011

Morphometric analysis of hepatocellular nodular lesions in HCV cirrhosis.

Pathol Res Pract 2012 Apr 18;208(4):240-4. Epub 2012 Mar 18.

Dipartimento di Morfologia Umana e Scienze Biomediche Città Studi, Faculty of Pharmacy, University of Milan, Italy.

We generated a computerized morphometric model to evaluate and quantify the morphological features in large regenerative nodules (LRN), high-grade dysplastic nodules (HGDN) and hepatocellular carcinoma (HCC). Sixteen LRN, 10 HGDN and 16 HCC in HCV-cirrhotic livers were stained with H&E, smooth muscle actin, CD34, CD31 and reticulin to evaluate volume and surface fractions. On H&E stains, the most discriminatory features between LRN, HGDN and HCC were volume fraction and the number of hepatocyte nuclei in unit volume and hepatocyte nuclear/cytoplasmic ratio. On immunohistochemistry, volume fractions of capillarised sinusoids, capillary units and isolated arteries were significantly different among all groups and highest in HCC; surface fraction of reticulin was markedly decreased in HCC. Our morphometric model is an objective method for quantification of the morphological changes of the nodular lesions, and it could be applied to studies involving histological evaluation of the spectrum of nodular lesions arising in the cirrhotic liver.
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http://dx.doi.org/10.1016/j.prp.2012.02.007DOI Listing
April 2012

A quantitative-PCR protocol rapidly detects αGAL deletions/duplications in patients with Anderson-Fabry disease.

Mol Genet Metab 2012 Apr 21;105(4):687-9. Epub 2012 Jan 21.

Health Sciences Department, University of Molise, Campobasso, Italy.

The Anderson-Fabry disease (AFD) is an X-linked glycosphingolipidosis leading to a progressive systemic disease. A particular variant of the disease of AFD presents only with left ventricular hypertrophy (LVH). Molecular diagnosis with bidirectional sequencing fails to detect genomic re-arrangements in female patients because of the presence of the wt allele. We here propose a quantitative PCR-based method alternative/complementary to the MLPA.
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http://dx.doi.org/10.1016/j.ymgme.2012.01.012DOI Listing
April 2012

KRAS mutations testing in colorectal carcinoma patients in Italy: from guidelines to external quality assessment.

PLoS One 2011 21;6(12):e29146. Epub 2011 Dec 21.

Cell Biology and Biotherapy Unit, INT Fondazione G. Pascale, Naples, Italy.

Background: Monoclonal antibodies directed against the epidermal growth factor receptor (EGFR) have been approved for the treatment of patients with metastatic colorectal carcinoma (mCRC) that do not carry KRAS mutations. Therefore, KRAS testing has become mandatory to chose the most appropriate therapy for these patients.

Methodology/principal Findings: In order to guarantee the possibility for mCRC patients to receive an high quality KRAS testing in every Italian region, the Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytopathology -Italian division of the International Academy of Pathology (SIAPEC-IAP) started a program to improve KRAS testing. AIOM and SIAPEC identified a large panel of Italian medical oncologists, pathologists and molecular biologists that outlined guidelines for KRAS testing in mCRC patients. These guidelines include specific information on the target patient population, the biological material for molecular analysis, the extraction of DNA, and the methods for the mutational analysis that are summarized in this paper. Following the publication of the guidelines, the scientific societies started an external quality assessment scheme for KRAS testing. Five CRC specimens with known KRAS mutation status were sent to the 59 centers that participated to the program. The samples were validated by three referral laboratories. The participating laboratories were allowed to use their own preferred method for DNA extraction and mutational analysis and were asked to report the results within 4 weeks. The limit to pass the quality assessment was set at 100% of true responses. In the first round, only two centers did not pass (3%). The two centers were offered to participate to a second round and both centers failed again to pass.

Conclusions: The results of this first Italian quality assessment for KRAS testing suggest that KRAS mutational analysis is performed with good quality in the majority of Italian centers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0029146PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3244430PMC
May 2012

Standardisation of EGFR FISH in colorectal cancer: results of an international interlaboratory reproducibility ring study.

J Clin Pathol 2012 Mar 30;65(3):218-23. Epub 2011 Nov 30.

The FalcK Division Of Medical Oncology, Ospedale Niguarda Ca' Granda, Milan, Italy.

Aims: Epidermal growth factor receptor (EGFR) gene copy number evaluated by fluorescence in situ hybridisation (FISH) can discriminate among KRAS wild-type patients those with better outcome to EGFR-targeted therapy in metastatic colorectal cancer, further enhancing selection of patients. Nevertheless, enumeration of gene copies is challenging and the lack of analytical standardisation has limited incorporation of the test into the clinical practice. We therefore assessed EGFR FISH interlaboratory consensus among five molecular diagnostic reference centres.

Methods: A set of 12 colorectal cancer samples circulated among laboratories, and samples were scored according to commonly agreed guidelines. Reproducibility was quantified using the standard error of measurement (SEM).

Results: A SEM of 0.865 and a within-subject coefficient of variation (WSCV) of 26.8% for mean EGFR gene/nuclei and a SEM of 0.235 and a WSCV of 19.4% for the mean EGFR gene/CEP7 ratio were observed. Measurement of the fraction of cells displaying chromosome 7 polysomy showed WSCV of 46.6%, 34.0% and 51.0% for percentage of cells displaying ≤2, ≥3 and ≥4 EGFR signals, respectively. Among different slides of the same specimen, the WSCV was 6.1% for mean EGFR gene/nuclei and 3.9% for mean of EGFR gene/CEP7 ratios.

Conclusions: Molecular diagnosis of EGFR gene copy number by FISH varied largely among pathology centres, with fluctuations covering the whole range of proposed cut-offs of predictive usefulness from literature. Definition of a detailed scoring system and implementation of comprehensive training programmes for laboratories are therefore necessary before including the test into clinical practice.
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http://dx.doi.org/10.1136/jclinpath-2011-200353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4612522PMC
March 2012

Molecular characterization of post-transplant lymphoproliferative disorders of donor origin occurring in liver transplant recipients.

J Pathol 2009 Aug;218(4):478-86

Division of Hematology, Department of Clinical and Experimental Medicine & BRMA, Amedeo Avogadro University of Eastern Piedmont, Novara, Italy.

Post-transplant lymphoproliferative disorders (PTLDs) represent a frequent complication of solid organ transplantation. Although most PTLDs arise from recipient lymphoid cells, a considerable fraction of cases may arise from donor B-cells. In an attempt to clarify the histogenesis and pathogenesis of PTLDs derived from donor B-cells, monoclonal PTLDs occurring in liver transplant recipients were chosen as a model to compare donor (D-PTLDs) versus recipient PTLDs (R-PTLDs). The tumour panel included nine D-PTLDs and six R-PTLDs. D-PTLDs were early-onset, EBV-infected lymphoproliferations classified as polymorphic PTLD (P-PTLD; n = 7) or diffuse large B-cell lymphoma (DLBCL; n = 2) with tumour localization confined to the hepatic hilum. All R-PTLDs were late-onset DLBCLs and showed extrahepatic localization. A BCL-6(-)/MUM1(+)/CD138(+/-) phenotype, consistent with a post-germinal centre (GC) stage of pre-terminal B-cell differentiation, was observed in all D-PTLDs and in 2/6 R-PTLDs, whereas a BCL6(+)/MUM1(-)/CD138(-) profile, reminiscent of GC B-cells, was detected in 4/6 R-PTLDs. The presence of somatic IGHV hypermutation was observed in 6/9 D-PTLDs and in 4/6 R-PTLDs, suggesting derivation from antigen-experienced B-cells. IGHV4-39 was the IGHV gene most frequently encountered, being rearranged in 3/9 D-PTLDs. Among IGHV-mutated PTLDs, a mutational profile suggesting antigen stimulation and/or selection was observed in 4/6 D-s and in 2/4 R-PTLDs. The presence of ongoing IGHV mutations was detected in 2/4 D-PTLDs. Aberrant SHM was detected in 10/15 (66.7%) PTLDs, including 6/9 D-PTLDs and 4/6 R-PTLDs. Our findings suggest that (i) D-PTLDs show a clinical presentation distinct from R-PTLDs; (ii) immunophenotypic and genetic features of D-PTLDs are consistent with mature, GC-experienced B-cells; (iii) transformed donor-derived B-cells may experience antigen-driven stimulation and selection, and may acquire genetic lesions during neoplastic expansion in the recipient environment; and (iv) EBV infection and expression of viral oncoproteins may be relevant in the pathogenesis of D-PTLDs.
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http://dx.doi.org/10.1002/path.2555DOI Listing
August 2009

EML4-ALK rearrangement in non-small cell lung cancer and non-tumor lung tissues.

Am J Pathol 2009 Feb 15;174(2):661-70. Epub 2009 Jan 15.

Institute of Hematology, University of Perugia, Perugia, Italy.

A fusion gene, echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK), with transforming activity has recently been identified in a subset of non-small cell lung cancer (NSCLC), but its pathogenetic, diagnostic, and therapeutic roles remain unclear. Both frequency and type of EML4-ALK transcripts were investigated by reverse transcription PCR in 120 frozen NSCLC specimens from Italy and Spain; non-neoplastic lung tissues taken far from the tumor were used as controls. In cases carrying the fusion transcript, we determined EML4-ALK gene and protein levels using fluorescence in situ hybridization, Western blotting, and immunoprecipitation. We also analyzed ALK protein levels in paraffin samples from 662 NSCLC specimens, including the 120 cases investigated in the molecular studies. EML4-ALK transcripts (variants 1 and 3) were detected in 9 of 120 NSCLC samples but were not specific for NSCLC since they were also found in non-cancerous lung tissues taken far from the tumor. Notably, no transcripts were detected in matching tumor samples from these patients. Fluorescence in situ hybridization analysis of cases expressing EML4-ALK transcripts showed that only a minority of cells harbored the EML4-ALK gene. None of these cases was found to express the EML4-ALK protein as examined by immunohistochemistry, Western blotting, and immunoprecipitation. The EML4-ALK transcript cannot be regarded as a specific diagnostic tool for NSCLC. Our results show therefore that the causal role and value of EML4-ALK as a therapeutic target remain to be defined.
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http://dx.doi.org/10.2353/ajpath.2009.080755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2630573PMC
February 2009

High expression of dopamine receptor subtype 2 in a large series of neuroendocrine tumors.

Cancer Biol Ther 2008 Dec 9;7(12):1970-8. Epub 2008 Dec 9.

Endocrine Unit, Ospedale Niguarda, Milano, Italy.

Aim: To evaluate by immumohistochemistry the presence of DR subtype 2 (D2R) in well differentiated NETs of different sites and in normal islet cells.

Background: Recent data in vitro and in vivo support that dopaminergic drugs might exert an inhibitory effect on hormone secretion and, possibly, on tumor growth in neuroendocrine tumors (NET)s. Their potential therapeutic role needs the demonstration of dopamine receptors (DR) in tumor cells. Little is known on the expression of DR in NETs.

Results: 85% of samples (100% of bronchial carcinoids and 93% of islet cell tumors) showed positivity for D2R; intensity of immunoreaction in NETs was similar or higher than in pituitary (54% and respectively 31% of cases). D2R positivity in more than 70% of tumor cells was observed in 46% of samples. Same intensity of D2R-immunoreactivity was found in pituitary and normal islet cells. No differences in D2R expression were recorded on considering tumor grading, size, proliferative activity, presence of metastases, endocrine activity and gender. A significant difference (62.5% vs 96.4%, p = 0.039) was observed in the prevalence of D2R expression between patients with more aggressive tumors and patients without recurrence/progression of disease during follow-up.

Methods: 46 NET samples from 44 patients and normal endocrine pancreatic tissue were studied. D2R-staining was performed on NETs and compared with six non-secreting pituitary adenomas and related to clinical-pathological data.

Conclusion: The present data demonstrate a high expression of D2R in NETs; this finding is of clinical relevance in view of the potential role of dopaminergic drugs in inhibiting secretion and/or cell proliferation in NETs.
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http://dx.doi.org/10.4161/cbt.7.12.6957DOI Listing
December 2008

Epidermal growth factor receptor gene copy number and clinical outcome of metastatic colorectal cancer treated with panitumumab.

J Clin Oncol 2007 Aug;25(22):3238-45

Divisione Oncologia Falck, Ospedale Niguarda Ca' Granda, Piazza Ospedale Maggiore 3, 20162 Milan, Italy.

Purpose: In a previous cohort study, we proposed that responsiveness of metastatic colorectal cancer (mCRC) to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies has a genetic basis, being associated with increased EGFR gene copy number (GCN) as measured by fluorescence in situ hybridization (FISH) in individual tumors. The present study was aimed at assessing the predictive role of EGFR GCN, in terms of clinical outcome, in patients treated with panitumumab.

Patients And Methods: Patients with mCRC refractory to standard therapies were a subset of patients from a phase III trial of panitumumab plus best supportive care (BSC; n = 58) versus BSC alone (n = 34) who were selected on the basis of availability of tumor samples adequate for FISH.

Results: In patients treated with panitumumab, a mean EGFR GCN of less than 2.5/nucleus or less than 40% of tumor cells displaying chromosome 7 polysomy within the tumor predicted for shorter progression-free survival (PFS; P = .039 and P = .029, respectively) and overall survival (P = .015 and P = .014, respectively). None of the treated patients with mean EGFR GCN of less than 2.47/nucleus or less than 43% of tumor cells displaying chromosome 7 polysomy obtained objective response compared with six of 20 and six of 19 patients with values greater than these cutoff limits, respectively (P = .0009 and P = .0007, respectively). Evaluation of BSC-treated patients showed no correlation between EGFR GCN or chromosome 7 polysomy status and PFS.

Conclusion: In a larger and more homogeneous series than in previous studies, present exploratory data suggest that mCRC patients with tumors distinguishable by FISH analysis of EGFR as homogenously disomic or with low chromosome 7 polysomy have a reduced likelihood of response to panitumumab.
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http://dx.doi.org/10.1200/JCO.2007.11.5956DOI Listing
August 2007

Response of thymoma to cetuximab.

Lancet Oncol 2007 May;8(5):449-50

Department of Oncology, Fatebenefratelli and Ophthalmic Hospital, Milan, Italy.

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http://dx.doi.org/10.1016/S1470-2045(07)70141-9DOI Listing
May 2007

The role of chromogranin A in the management of patients with phaeochromocytoma.

Clin Endocrinol (Oxf) 2006 Sep;65(3):287-93

Department of Endocrinology, Ospedale Niguarda Ca Granda, Milan, Italy.

Objective: Chromogranin A (CgA) is the most accurate general marker of neuroendocrine tumours. Supranormal CgA concentrations have been recorded in patients with tumours of neuroectodermal origin such as phaeochromocytoma and paraganglioma.

Design: The present study was performed to assess the role of CgA determination in the management of patients with phaeochromocytoma, in comparison with urinary catecholamines and their metabolites.

Patients: The patients studied included 22 cases with phaeochromocytoma at initial presentation or at relapse some years after surgical cure or during follow-up of a malignant phaeochromocytoma. Seventeen patients were evaluated before and after surgical removal of phaeochromocytoma. To assess the specificity of the hormonal parameters, 20 subjects were enrolled as controls; they were from a group of patients referred to our observation for possible phaeochromocytoma and who were subsequently proven not to have the disease.

Results: Urinary epinephrine and norepinephrine were supranormal in 82% and 77% of patients, respectively. Urinary metanephrines and normetanephrines were supranormal in 84% and 89% of patients, respectively. The combination of urinary metanephrine and normetanephrine had a sensitivity of 100% in identifying a phaeochromocytoma. CgA was supranormal in 91% of patients. Combining the results of CgA and urinary catecholamines (epinephrine and norepinephrine), the sensitivity for diagnosis of phaeochromocytoma was 100%. Urinary catecholamines, metabolites (metanephrine and normetanephrine) and CgA levels in patients with malignant phaeochromocytoma did not differ significantly from those of patients with benign lesions. In most cases, CgA normalized after surgery.

Conclusions: Our results indicate that CgA is a good marker of phaeochromocytoma; measurement of CgA could have a role in the follow-up of patients operated on for phaeochromocytoma.
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http://dx.doi.org/10.1111/j.1365-2265.2006.02591.xDOI Listing
September 2006

Splenic marginal zone lymphoma: a prognostic model for clinical use.

Blood 2006 Jun 21;107(12):4643-9. Epub 2006 Feb 21.

Division of Hematology, IRCCS Policlinico San Matteo, University of Pavia, 27100 Pavia, Italy.

The Integruppo Italiano Linfomi (IIL) carried out a study to assess the outcomes of splenic marginal zone lymphoma and to identify prognostic factors in 309 patients. The 5-year cause-specific survival (CSS) rate was 76%. In univariate analysis, the parameters predictive of shorter CSS were hemoglobin levels below 12 g/dL (P < .001), albumin levels below 3.5 g/dL (P = .001), International Prognostic Index (IPI) scores of 2 to 3 (P < .001), lactate dehydrogenase (LDH) levels above normal (P < .001), age older than 60 years (P = .01), platelet counts below 100,000/microL (P = .04), HbsAg-positivity (P = .01), and no splenectomy at diagnosis (P = .006). Values that maintained a negative influence on CSS in multivariate analysis were hemoglobin level less than 12 g/dL, LDH level greater than normal, and albumin level less than 3.5 g/dL. Using these 3 variables, we grouped patients into 3 prognostic categories: low-risk group (41%) with no adverse factors, intermediate-risk group (34%) with one adverse factor, and high-risk group (25%) with 2 or 3 adverse factors. The 5-year CSS rate was 88% for the low-risk group, 73% for the intermediate-risk group, and 50% for the high-risk group. The cause-specific mortality rate (x 1000 person-years) was 20 for the low-risk group, 47 for the intermediate-risk group, and 174 for the high-risk group. This latter group accounted for 54% of all lymphoma-related deaths. In conclusion, with the use of readily available factors, this prognostic index may be an effective tool for evaluating the need for treatment and the intensity of therapy in an individual patient.
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http://dx.doi.org/10.1182/blood-2005-11-4659DOI Listing
June 2006