National survey of anatomical pathology centres in Italy: the questionnaire.

OBJECTIVES: To obtain a picture of the work done in Italian anatomical pathology centres in 2014, and evaluate differences between the various centres in terms of the workloads of medical and non-medical staff. METHODS: A self-administered questionnaire designed by a SIAPEC working group was e-mailed to 256 centres and subsequently collected by the Anatomical Pathology Service of Bolzano. QlikView software was used to prepare the final database and check the quality of the data, which were processed using version 18.0 of SPSS for Windows statistical software. RESULTS: The questionnaire was completed by 120 of the centres (46.9%), which were staffed by a mean number of 6.6 physicians (range 1-24), 1.6 biologists (range 0-7), 10.8 laboratory technicians (range 2-47) and 2.2 administrative personnel (range 0-9). During 2014, the centres carried out a mean of 15,000 histology examinations (range 3,215-50,680), almost 11,700 immunohistochemistry examinations (range 0-54,359), and a mean of 1,471 molecular biology examinations (range 0-31,322) relating to a mean of 704 patients (range 0-9,434), and a mean of 16,509 cytology examinations (range 0-150,000) relating to 13,383 patients (range 0-120,000). Each centre physician issued a mean of 2,444 histology examinations reports (range 613-11,000); the ratio between the number of immunohistochemistry examinations and the number of histology examinations was 0.8 (range 0-2.7); and each laboratory technician had a mean overall annual workload of 3,072 histology, molecular biology and cytology examinations (range 793-9,882/year). These values varied widely among the participating centres. The mean ratio between the number of histology examinations carried out and the number of physicians was 1,982.77:1 a year in the small centres (< 10,000 histology cases/year), 2,627:1 a year in the medium-sized centres (10-24,999 histology cases/year), and 2,881.34:1 in the large centres (> 25,000 histology cases/year). There were significant differences between the small and medium-sized centres (p = 0.004) and between the small and large centres (p = 0.001), but not between the medium-sized and large centres.The ratio between the total number of histology, molecular biology and cytology examinations and the number of laboratory technicians was 1,963.34 in the small centres (< 10,000 examinations/year), 2,717.11 in the medium-sized centres (10,000-24,999 examinations/year), and 3,531.56 in the large centres (≥ 25,000 examinations/year). There were significant differences between the small and large centres (p = 0.001) and between the medium-sized and large centres (p = 0.004), but not between the small and medium-sized centres. CONCLUSIONS: The data collected by means of this survey provide an important, albeit partial, point of reference concerning the status of Italian anatomical pathology centres and their recent, everyday working situation.

Effects of miRNA-15 and miRNA-16 expression replacement in chronic lymphocytic leukemia: implication for therapy.

Chronic lymphocytic leukemia (CLL) clones are characterized by loss of a critical region in 13q14.3, (del(13)(q14)) involving the microRNA (miRNA) cluster miR-15a and miR-16-1. We have investigated the effects of replacement of miR-15a and miR-16-1. CLL cells transfected with these miRNA mimics exhibited a decrease in cell viability in vitro and impaired capacity for engraftment and growth in NOD/Shi-scid,γcnull (NSG) mice. No synergistic effects were observed when the two miRNA mimics were combined. The phenomena were not restricted to CLL with the del(13)(q14) lesion. Similar effects induced by miRNA mimics were seen in cells with additional chromosomal abnormalities with the exception of certain CLL clones harboring TP53 alterations. Administration of miRNA mimics to NSG mice previously engrafted with CLL clones resulted in substantial tumor regression. CLL cell transfection with miR-15a and miR-16-1-specific inhibitors resulted in increased cell viability in vitro and in an enhanced capacity of the engrafted cells to grow in NSG mice generating larger splenic nodules. These data demonstrate that the strong control by miR-15a and miR-16-1 on CLL clonal expansion is exerted also at the level of full-blown leukemia and provide indications for a miRNA-based therapeutic strategy.

Tumor MGMT promoter hypermethylation changes over time limit temozolomide efficacy in a phase II trial for metastatic colorectal cancer.

BACKGROUND: Objective response to dacarbazine, the intravenous form of temozolomide (TMZ), in metastatic colorectal cancer (mCRC) is confined to tumors harboring O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter hypermethylation. We conducted a phase II study of TMZ enriched by MGMT hypermethylation in archival tumor (AT), exploring dynamic of this biomarker in baseline tumor (BT) biopsy and plasma (liquid biopsy). PATIENTS AND METHODS: We screened 150 mCRC patients for MGMT hypermethylation with methylation-specific PCR on AT from FFPE specimens. Eligible patients (n = 29) underwent BT biopsy and then received TMZ 200 mg/m(2) days 1-5 q28 until progression. A Fleming single-stage design was used to determine whether progression-free survival (PFS) rate at 12 weeks would be ≥35% [H0 ≤ 15%, type I error = 0.059 (one-sided), power = 0.849]. Exploratory analyses included comparison between MGMT hypermethylation in AT and BT, and MGMT methylation testing by MethylBEAMing in solid (AT, BT) and LB with regard to tumor response. RESULTS: The PFS rate at 12 weeks was 10.3% [90% confidence interval (CI) 2.9-24.6]. Objective response rate was 3.4% (90% CI 0.2-15.3), disease control rate 48.3% (90% CI 32.0-64.8), median OS 6.2 months (95% CI 3.8-7.6), and median PFS 2.6 months (95% CI 1.4-2.7). We observed the absence of MGMT hypermethylation in BT in 62.7% of tumors. CONCLUSION: Treatment of mCRC with TMZ driven by MGMT promoter hypermethylation in AT samples did not provide meaningful PFS rate at 12 weeks. This biomarker changed from AT to BT, indicating that testing BT biopsy or plasma is needed for refined target selection.

Mutation frequencies of GNAQ, GNA11, BAP1, SF3B1, EIF1AX and TERT in uveal melanoma: detection of an activating mutation in the TERT gene promoter in a single case of uveal melanoma.

BACKGROUND: Uveal melanoma is the most frequent primary tumour of the eye. It is molecularly clearly distinct from cutaneous melanoma and shows a different pattern of driver mutations. The influence of sunlight ultraviolet (UV) exposure on the aetiology of uveal melanoma is a matter of debate. The recent identification of driver mutations in the promoter of the telomerase reverse transcriptase (TERT) gene with UV-induced cytidine-to-thymidine transitions in cutaneous melanoma prompted us to investigate whether these mutations also occur in uveal melanoma. METHODS: We analysed 50 cases of uveal melanoma obtained from enucleation surgery for mutations in the genes GNAQ, GNA11, BAP1, SF3B1, EIFAX1 and TERT, measured gene expression using microarrays and analysed gene copy numbers by SNP arrays. RESULTS: We detected a TERT mutation in only one case of a 57-year-old white male patient with clinical and histopathological features typical for uveal melanoma. The tumour showed mutations in GNA11 and EIF1AX that are typical for uveal melanoma and absent from cutaneous melanoma. No mutations were detected in GNAQ, BAP1 and SF3B1 that are frequently mutated in uveal melanoma. Both copies of chromosome 3 were retained. Several tumours among which the one carrying the TERT promoter mutation showed elevated TERT expression. Consistent with previous reports, GNAQ is inversely associated with chromosome 3 monosomy and metastasis. BAP1 mutations are significantly associated with chromosome 3 monosomy but not with relapse. CONCLUSION: These data indicate that TERT mutations are rare in uveal melanoma. No conclusion can be drawn on their potential influence on tumour progression.

Report from the OECI Oncology Days 2014.

The 2014 OECI Oncology Days was held at the 'Prof. Dr. Ion Chiricuta' Oncology Institute in Cluj, Romania, from 12 to 13 June. The focus of this year's gathering was on developments in personalised medicine and other treatment advances which have made the cost of cancer care too high for many regions throughout Europe.

Patients with systemic lupus erythematosus (SLE) having developed malignant lymphomas. Complete remission of lymphoma following high-dose chemotherapy, but not of SLE.

The development of malignant lymphomas, generally of the non-Hodgkin type (NHL), and with a preference to diffuse large cell B lymphomas (DLCBL), in systemic lupus erythematosus (SLE), has been analysed in an exhaustive recent literature. The combination of germline and somatic mutations, persistent immune overstimulation and the impairment of immune surveillance facilitated by immunosuppressive drugs, is thought to be at the origin of the increased lymphoma genesis. However the treatment and course of such affected patients is less known, and prognosis is generally estimated as poor. Out of 258 patients with complete/incomplete lupus and secondary antiphospholipid syndrome (APS) seen and treated at the institutional Day Hospital between 1982 and 2009, 6 developed lymphomas (4 DLCBL, 1 Hodgkin's and 1 indolent lymphocytic lymphoma). The first 5 patients were treated with high dose chemotherapy (HDCT) and achieved complete remissions (CR) with a follow-up comprised between 13 and 172 months. One patient relapsed of lymphoma and died 15 months following CR, with persistent lupus serology. One patient achieved complete remission (CR) of both diseases. In the other 3 lupus serology, Antinuclear and antiphospholipid antibodies (ANA, aPL) persisted, with occasional lupus flares and vascular complications. While eradication of the last cancer stem cell is tantamount to cure in neoplastic disease, persistent autoantigenic overstimulation may contribute to the refractoriness of autoimmunity. The implications of these results for the increasing utilisation of haematopoietic stem cell transplantation for severe autoimmune diseases (SADS), with lupus as a paradigm, are discussed.

Incidental finding of peripheral B-cell non-Hodgkin lymphoma, lymphocytic/CLL type, of the gallbladder in a patient with chronic cholecystitis.

Although lymphoma involvement of the gallbladder, especially by MALT and large-cell types, is rare, this possibility should be considered in patients with symptoms of acute cholecystitis. A cholecystectomy was performed in a 79-year-old male patient with a clinical diagnosis of chronic cholecystitis. Histologically, the specimen showed an incidental finding of a small lymphocytic lymphoma (CLL) by morphologic and immunophenotyping studies, subsequently confirmed with flow cytometric analysis of blood. During follow-up, multiple lymph node enlargement was detected. An axillary node, excised and submitted to our department, was positive for lymphoma involvement. The bone marrow was negative.

Intra-operative evaluation of the sentinel lymph node for T1-N0 breast-cancer patients: always or never? A risk/benefit and cost/benefit analysis.

AIM: To investigate whether omitting intra-operative staging of the sentinel lymph node (SLN) in T1-N0 breast-cancer patients is feasible and convenient because it could allow a more efficient management of human and logistic resources without leading to an unacceptable increase in the rate of delayed axillary lymph node dissection (ALND). METHODS: According to the experimental procedure, T1a-T1b-patients were to not receive any intra-operative SLN evaluation on frozen sections (FS). In all T1c-patients, the SLN was macroscopically examined; if the node appeared clearly free of disease, no further intra-operative assessment was performed; if the node was clearly metastatic or presented a dubious aspect, the pathologist proceeded with analysis on FS. T2-patients, enrolled in the study as reference group, were treated according to the institutional standard procedure; they all received SLN staging on FS. RESULTS: The study included 395 T1-N0-patients. Among the 118 T1a-T1b-patients whose SLN was not analyzed at surgery, 12 (10.2%) were recalled for ALND. In the group of 258 T1c-patients, 112 received SLN analysis on FS and 146 did not. An SLN falsely negative either at macroscopic or FS examination was found in 33 (12.8%) cases. Overall, the rate of recall for ALND was 11.6% as compared to 8.4% in T2-patients. Using the experimental protocol, the institution reached a 9.6% cost saving, as compared to the standard procedure. CONCLUSIONS: Omission of SLN intra-operative staging in T1-N0-patients is rather safe. It provides the institution with both management and economical advantages.

Heterogeneous nuclear ribonucleoprotein K: altered pattern of expression associated with diagnosis and prognosis of prostate cancer.

Using proteomic analysis of the nuclear matrix (NM), we found that heterogeneous nuclear ribonucleoprotein K (hnRNP K), a member of the hnRNP family with pleiotropic functions, was differentially expressed in prostate cancer (PCa) tissues. This study aimed to characterise the expression of hnRNP K and its subcellular localisation in PCa, utilising immunohistochemical and quantitative western blot techniques. Furthermore, the hnRNP K expression was studied in human PCa cell lines in order to determine its modulation by bicalutamide, the anti-androgen widely used in PCa therapy. Immunohistochemical staining of paraffin-embedded tissues showed that hnRNP K was overexpressed in PCa, where it was localised both in the cytoplasm and in the nucleus. Staining of non-tumour tissues showed exclusively nuclear localisation and a less intense or absent signal. Immunoblot analysis demonstrated that the hnRNP K level within the NM was higher in PCa compared with non-tumour tissues and closely correlated with Gleason score (P=0.008). Higher expression within the NM was significantly (P=0.032) associated with poor prognosis. In two-dimensional western blot analysis hnRNP K presented several isoforms; the one with pI 5.1 was the most differently expressed between non-tumour and PCa tissues. Preliminary results indicate that hnRNP K can be modulated in vitro by a non-steroidal anti-androgen. Taken together, our findings suggest that hnRNP K has potential implications at the diagnostic, prognostic and therapeutic levels in PCa.

PHOX2A and PHOX2B genes are highly co-expressed in human neuroblastoma.

The detection of PHOX2B mutations in a small proportion of patients affected with either familial or sporadic neuroblastoma (NB), has arisen interest on the possible pathogenic role of this gene in the disease determination. In this light, we have carried out a quantitative expression analysis of PHOX2B and its paralogue PHOX2A on a panel of NB cell lines and NB tumour samples to identify a possible differential expression between NB cells and their normal counterpart (adrenal medulla cells). Our results revealed that both PHOX2A and PHOX2B are over-expressed in tumour samples and NB cell lines. Particularly, the expression levels of the two genes in NB cell lines show a highly significant correlation, suggesting their possible synergistic role or a coordinated expression regulation. Furthermore, PHOX2 gene over-expression in NB tumours and cell lines suggests these genes may be widely involved in NB development through either a direct mechanism of up-regulation or a failure in maintaining proper transcript levels after embryonic development.

Patients with advanced stage breast carcinoma immunoreactive to biotinylated Herceptin are most likely to benefit from trastuzumab-based therapy: an hypothesis-generating study.

BACKGROUND: Biotin-labeled trastuzumab (BiotHER) can be used to test for HER2 by immunohistochemistry. We previously showed that BiotHER immunoreactivity is highly correlated with HER2 amplification and indicated that it could be associated with better clinical outcome in advanced breast cancer patients receiving trastuzumab. PATIENTS AND METHODS: Tumor specimens and clinical information from 234 patients who received trastuzumab-based treatments were collected from 10 institutions. HER2 amplification and BiotHER immunoreactivity were assessed centrally. The effect of BiotHER positivity on response rate (RR), time to progression and survival were studied by univariate and multivariate analysis in patients presenting HER2-amplified breast cancer. The pathologic reviews of the assays were blinded to patient outcomes. RESULTS: BiotHER was positive in 109/194 (56%) HER2-amplified breast cancers and in one not amplified tumor. RRs were 74% [95% (confidence interval) CI 64%-81%] and 47% (95% CI 36%-58%) in BiotHER-positive and -negative tumors, respectively (P < 0.001). BiotHER immunoreactivity was independently associated with increased probability of tumor response (odds ratio 3.848; 95% CI 1.952-7.582), with reduced risk of disease progression [hazard ratio (HR) 0.438; 95% CI 0.303-0.633] and with reduced risk of death (HR 0.566; 95% CI 0.368-0.870) by multivariate analysis. CONCLUSION: The results support a role for BiotHER testing in better tailoring trastuzumab-based treatments in patients with advanced HER2-amplified breast cancers.

Detection of tissue abnormalities in normal mucosa surrounding colorectal cancer using an endocytoscopy system.

The endocytoscopy system is a novel diagnostic technique that provides extremely high-magnification imaging of the gastrointestinal mucosa. We are currently using a prototype Olympus endocytoscope for clinical research in gastrointestinal tumors. In one surgical specimen obtained after resection of a cancer of the transverse colon, focal abnormalities of colonic glands were detected 7 cm away from the primary tumor, within macroscopically normal mucosa. Our finding, which was confirmed by histopathological examination, suggests the need for further clinical investigation to assess whether endocytoscopy is able to identify premalignant changes in apparently normal mucosa. This could potentially be useful for accurate evaluation before planning minimally invasive therapy.

[The Haemolymphopathology Italian Group (H.I.G.): an essential resource for the new technical and organization problems troubling modern haemolymphopathology diagnostics]. / II Gruppo Italiano di Emolinfopatologia (G.I.E.): una esigenza per le nuove e complesse problematiche tecnico-organizzative della moderna diagnostica emolinfopatologica.

Recently, many progresses have been recorded in the molecular and histogenetic characterization of the haematopoietic and lymphoid tumours, resulting in important classifying changes. As a consequence, the exact definition of lymphoma subtype requires an integration between traditional morphologic "expertise" and several bio-functional data obtained from advanced and complex ancillary techniques (immunohistochemistry, molecular biology and cytogenetics). At the same time, the data provided by gene expression profiling studies are going to deeply modify the therapies in haematological cancers. These studies are expected to allow the achievement of single-patient-tailored genic therapy; for this reason it is necessary to get biological samples of good quality. Indeed, while these progresses contribute to highlight the pathologist's diagnostic role, they should make us reflect on the state of the art of the Italian haemolymphopathology diagnostics and on its ability to cope up with the new challanges. The aim of this article is to outline a realistic picture of the present condition, and to explain the reasons for setting up, inside SIAPEC-IAP, the Haemolymphopathology Italian Group (H.I.G.). The purpose of H.I.G. will be twofold: first of all, scheduling of a series of projects so as to the haemolymphopathological diagnostic standardization; secondly, building a national network among all the pathologists involved in this exciting and complex field of the anatomic pathology.

A third generation regimen VACOP-B with or without adjuvant radiotherapy for aggressive localized non-Hodgkin's lymphoma: report from the Italian Non-Hodgkin's Lymphoma Co-operative Study Group

The objective of this multicenter prospective study was to determine the clinical efficacy and toxicity of a polychemotherapeutic third generation regimen, VACOP-B, with or without radiotherapy as front-line therapy in aggressive localized non-Hodgkin's lymphoma. Ninety-three adult patients (47 males and 46 females, median age 45 years) with aggressive localized non-Hodgkin's lymphoma, 43 in stage I and 50 in stage II (non-bulky), were included in the study. Stage I patients received VACOP-B for 6 weeks plus involved field radiotherapy and stage II patients received 12 weeks VACOP-B plus involved field radiotherapy on residual masses. Eighty-six (92.5 percent) achieved complete remission and 4 (4.3 percent) partial remission. Three patients (3.2 percent) were primarily resistant. Ten-year probability of survival, progression-free survival and disease-free survival were 87.3, 79.9 and 83.9 percent, respectively. Eighty-four patients are surviving at a median observation time of 57 months (range: 6-126). Statistical analysis showed no difference between stages I and II in terms of response, ten-year probability of survival, progression-free survival or disease-free survival. Side effects and toxicity were negligible and were similar in the two patient groups. The results of this prospective study suggest that 6 weeks of VACOP-B treatment plus radiotherapy may be the therapy of choice in stage I aggressive non-Hodgkin's lymphoma. Twelve weeks of VACOP-B treatment with or without radiotherapy was shown to be effective and feasible for stage II. These observations need to be confirmed by a phase III study comparing first and third generation protocols in stage I-II aggressive non-Hodgkin's lymphoma.

A third generation regimen VACOP-B with or without adjuvant radiotherapy for aggressive localized non-Hodgkin's lymphoma: report from the Italian Non-Hodgkin's Lymphoma Co-operative Study Group.

The objective of this multicenter prospective study was to determine the clinical efficacy and toxicity of a polychemotherapeutic third generation regimen, VACOP-B, with or without radiotherapy as front-line therapy in aggressive localized non-Hodgkin's lymphoma. Ninety-three adult patients (47 males and 46 females, median age 45 years) with aggressive localized non-Hodgkin's lymphoma, 43 in stage I and 50 in stage II (non-bulky), were included in the study. Stage I patients received VACOP-B for 6 weeks plus involved field radiotherapy and stage II patients received 12 weeks VACOP-B plus involved field radiotherapy on residual masses. Eighty-six (92.5%) achieved complete remission and 4 (4.3%) partial remission. Three patients (3.2%) were primarily resistant. Ten-year probability of survival, progression-free survival and disease-free survival were 87.3, 79.9 and 83.9%, respectively. Eighty-four patients are surviving at a median observation time of 57 months (range: 6-126). Statistical analysis showed no difference between stages I and II in terms of response, ten-year probability of survival, progression-free survival or disease-free survival. Side effects and toxicity were negligible and were similar in the two patient groups. The results of this prospective study suggest that 6 weeks of VACOP-B treatment plus radiotherapy may be the therapy of choice in stage I aggressive non-Hodgkin's lymphoma. Twelve weeks of VACOP-B treatment with or without radiotherapy was shown to be effective and feasible for stage II. These observations need to be confirmed by a phase III study comparing first and third generation protocols in stage I-II aggressive non-Hodgkin's lymphoma.

[Use of cellulose acetate millipore filters for the correct orientation of endoscopic biopsies in digestive diseases]. / Utilizzo dei filtri millipore di acetato di cellulosa per un corretto orientamento delle biopsie endoscopiche in patologia digestiva.

INTRODUCTION: Properly oriented endoscopic biopsies allow an effective assessment of some diagnostic features in non neoplastic gastrointestinal diseases. MATERIALS AND METHODS: We used cellulose acetate millipore filters (Endofilter, Bio-Optica, Milan, Italy) in order to improve the orientation of biopsies during processing. Forty biopsies were placed on filter after endoscopic sampling whereas no orientation attempt was done for other 40 filter-free biopsies (20 from esophagus and 20 from gastric antrum in each group). Both groups were compared in terms of orientation and assessability of the following morphological features: thickness of basal layer in the esophageal squamous epithelium, length of esophageal papillae, interstitial space dilatations in the esophageal squamous epithelium and gland atrophy in the gastric antrum. Both orientation and assessability of individual morphological features were graded with a score ranging from 1 (good) to 3 (poor). The impact of this procedure on costs was analysed, both in terms of material and technical workload. RESULTS: All 20 esophageal and 20 antral biopsies on filter showed acceptable (score 1 or 2) orientation. In contrast, 14/20 filter-free esophageal and 13/20 antral biopsies showed poor (score 3) orientation (p = 0.0001 for both groups). Basal layer thickness was assessable (score 1 or 2) in 20/20 esophageal biopsies on filter vs 14/20 filter-free ones (p = 0.0001) and length of papillae in 15/20 biopsies on filter vs 4/20 filter-free ones (p = 0.0002). Interstitial space dilatation assessability was not affected by orientation procedures. Gland atrophy in the antrum was assessable (score 1 or 2) in 20/20 gastric biopsies on filter vs 8/20 filter-free ones (p = 0.0001). The use of endofilters permitted the process of numerous (up to 8 for each block) samples from different biopsy sites together and produced a significant reduction in costs (18.35 in the case of 8 biopsies from 4 different biopsy sites). CONCLUSIONS: The use of millipore filters allows orientation of biopsy samples, improves the assessment of several diagnostic features in esophageal and gastric pathology and yields a significant reduction in costs when biopsies from different sites are processed together.