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Summary The World Health Organization’s (WHO) updated classification of head and neck tumors (2017) defined odontogenic fibroma as a rare neoplasm. In this report, we describe an unusual, typical and rare variant of a central odontogenic fibroma with diffuse amyloid-like protein stromal deposition, and discuss the differential diagnosis with other entities. Radiographically, this lesion presented as a well-defined radiolucency of the mandible, partially cystic. Histologically, the lesion showed a unique confluence of odontogenic epithelial rests in a moderately cellular connective tissue. Immunohistochemical staining highlighted a mixture of benign epithelial and Langerhans cells within connective tissue with diffuse amyloid-like stromal deposition. The importance of recognizing this variant of odontogenic fibroma is due to its benign prognosis and clinical course.

Background: Rapid and reliable diagnosis of tuberculosis (TB) represents a diagnostic challenge in compartmentalized extrapulmonary TB infection because of the small number of mycobacteria (MTB) and the frequent lack of fresh samples to perform culture. Here, we estimate the performances of homemade droplet digital PCR (ddPCR)-based assays against culture in 89 biopsies, for those fresh and formalin-fixed and paraffin-embedded (FFPE) subsamples were available.Methods: MTB diagnosis in fresh subsamples was performed by culture. Fresh subsamples were also analyzed for acid-fast bacilli smear-microscopy (AFB) and Xpert® MTB/RIF (Xpert). MTB examination was repeated in blind in the 89 FFPE subsamples by in-house ddPCR assays targeting the IS6110 and rpoB. Analytical sensitivity of ddPCR assays was evaluated using serial dilution of H37Rv strain. Limit of detection (LOD) was calculated by probit analysis. Results were expressed in copies/106 cells.Results: IS6110 and rpoB ddPCR assays showed a good linear correlation between expected and observed values (R2: 0.9907 and 0.9743, respectively). Probit analyses predicted a LOD of 17 and 40 copies/106 cells of MTB DNA for IS6110 and rpoB, respectively. Of the 89 biopsies, 68 were culture positive and 21 were culture negative. Considering mycobacterial culture as reference method, IS6110 assay yielded positive results in 67/68 culture-positive samples with a median interquartile range (IQR) of 1,680 (550–8,444) copies/106 cells (sensitivity: 98.5%; accuracy: 98.9). These performances were superior to those reported by the rpoB assay in FFPE subsamples (sensitivity: 66.20%; accuracy: 74.1) and even superior to those reported by Xpert and AFB in fresh subsamples (sensitivity: 79.4 and 33.8%, respectively; accuracy: 84.3 and 49.4, respectively). When Xpert and AFB results were stratified according to mycobacterial load detected by rpoB and IS6110 ddPCR, bacterial load was lower in Xpert and AFB negative with respect to Xpert and AFB-positive samples (p = 0.003 and 0.01 for rpoB and p = 0.01 and 0.11 for IS6110), confirming the poor sensitivity of these methods in paucibacillary disease.Conclusion: ddPCR provides highly sensitive, accurate, and rapid MTB diagnosis in FFPE samples, as defined by the high concordance between IS6110 assay and culture results. This approach can be safely introduced in clinical routine to accelerate MTB diagnosis mainly when culture results remain unavailable.

We report the case of a 63-year-old white man who, 3 days after stent removal of endoscopic drainage of pancreatic cysts, developed a penile necrosis due to purpura fulminans (PF) that has been misdiagnosed as Fournier's gangrene. Penile necrosis was rapidly followed by a lethal multiorgan failure due to disseminated intravascular coagulopathy (DIC), triggered by the subsequent development of a severe acute pancreatitis. PF describes a rare syndrome involving intravascular thrombosis and hemorrhagic infarction of the skin. Although reports of penile necrosis secondary to various causes are documented in the literature, penile necrosis secondary to PF in the setting of acute pancreatitis is a rare event. Histopathologic studies of the skin showing an occlusive nonvasculitic vasculopathy are the first step to achieve an accurate diagnosis.

BackgroundHER2 amplification is detected in 3% of patients with colorectal cancer (CRC), making tumors in the metastatic setting vulnerable to double pharmacological HER2 blockade. Preclinical findings show that it also might impair response to anti-epidermal growth factor receptor (EGFR) treatment.Subjects and MethodsPatients with KRAS exon 2 wild-type metastatic CRC underwent molecular screening of HER2 positivity by HERACLES criteria (immunohistochemistry 3+ or 2+ in ≥50% of cells, confirmed by fluorescence in situ hybridization). A sample of consecutive HER2-negative patients was selected as control. A regression modeling strategy was applied to identify predictors explaining the bulk of HER2 positivity and the association with response to previous anti-EGFR treatment.ResultsFrom August 2012 to April 2018, a total of 100 HER2-positive metastatic CRC tumors were detected out of 1,485 KRAS exon 2 wild-type screened patients (6.7%). HER2-positive patients show more frequently lung metastases (odds ratio [OR], 2.04; 95% confidence interval [CI], 1.15–3.61; p = .014) and higher tumor burden (OR, 1.48; 95% CI, 1.10–2.01; p = .011), and tumors were more likely to be left sided (OR, 0.50; 95% CI, 0.22–1.11; p = .088). HER2-positive patients who received treatment with anti-EGFR agents (n = 79) showed poorer outcome (objective response rate, 31.2% vs. 46.9%, p = .031; progression-free survival, 5.7 months vs. 7 months, p = .087).ConclusionTesting for HER2 should be offered to all patients with metastatic CRC because the occurrence of this biomarker is unlikely to be predicted based on main clinicopathological features. Patients with HER2-amplified metastatic CRC are less likely to respond to anti-EGFR therapy.Implications for PracticePatients with HER2-amplified/overexpressed metastatic colorectal cancer (mCRC) harbor a driver actionable molecular alteration that has been shown in preclinical models to hamper efficacy of the anti-epidermal growth factor receptor (EGFR) targeted therapies. The present study confirmed that this molecular feature was associated with worse objective tumor response and shorter progression-free survival in response to previous anti-EGFR therapies. Moreover, it was found that the occurrence of this biomarker is unlikely to be predicted based on main clinicopathological features. Therefore, HER2 status assessment should be included in the molecular diagnostic workup of all mCRC for speedy referral to clinical trials encompassing HER2-targeted double blockade independently of previous anti-EGFR treatment.

DRESS/DiHS is a complex and potentially fatal drug reaction. Little is known about risk factors and elements that can help to identify patients with a severe reaction early. The aim of the study was to investigate those factors favoring the disease and its severity by analyzing the clinical conditions and therapies preceding the reaction. We conducted a retrospective analysis on patients admitted to our center between 2010 and 2020 who were discharged with a diagnosis of DRESS. We used the RegiSCAR diagnostic criteria. We defined the severity of DRESS using the criteria of Mizukawa et al. We included 25 patients (15 females) with a median age of 66 years. Skin involvement, eosinophilia, and liver injury were the most important aspects. Allopurinol was found to be the most involved drug. Reaction severity was significantly associated with the number of daily medications (p = 0.0067) and an age of at least 68 years (p = 0.013). In addition, 75% of severe cases had at least three comorbidities in history, and most of the severe cases were female. In our study the advanced age, the high number of comorbidities and home therapies, and the inflammatory state were found to be predisposing elements to the development of the disease and its severity.

The cytogenetic hallmark of Chronic Myeloid Leukemia (CML) is the presence of Philadelphia (Ph) chromosome, which results from a reciprocal translocation t(9;22)(q34;q11). In this report, we describe a CML patient with no evidence of Ph chromosome but trisomy of chromosome 8 as single cytogenetic abnormality and a typical e14a2 (b3a2) BCR-ABL1 fusion transcript. Fluorescence In Situ Hybridization (FISH) analysis revealed an uncommon signal pattern: the fusion signals were located on both copies of chromosome 22. During the course of the disease the appearance of the p.(Tyr315Ile) mutation was recorded. To the best of our knowledge this is the first Ph chromosome-negative CML case with e14a2 (b3a2) BCR-ABL1 transcript and p.(Tyr315Ile) mutation.

The definition of oligometastatic colorectal cancer (CRC) identifies a peculiar subpopulation of patients characterized by a limited metastatic spread of disease.1 Oligometastatic disease is defined as the involvement of up to two or occasionally three sites with five or sometimes more metastases that for their anatomic localization is amenable to local ablative therapies, thus rendering the patient free of disease.1-3 Thus, this subgroup is wide and has a significant cohort of patients with CRC. Among patients with oligometastatic CRC, those with liver-limited disease represent a more refined subset and should always be discussed in multidisciplinary teams since they appear to more likely benefit from multimodal approaches with curative intent.2,4-6 A perioperative systemic treatment integrated with surgical liver metastasectomy should be regarded as the best multimodal approach.2,7 However, the best drug regimen to be adopted for this subset of patients with CRC is still debatable and should be tailored case-by-case. Considering left-sided microsatellite stable, RAS and BRAF wild-type CRC, doublet cytotoxic regimens (FOLFIRI or FOLFOX) plus an anti-epidermal growth factor receptor (EGFR) drug can represent the best option on the basis of significant response rate (RR).8-10 However, triplet cytotoxic regimens FOLFOXIRI plus bevacizumab or anti-EGFR drug demonstrate an impressive RR up to 87%, which are increasingly regarded as potential novel neoadjuvant standard strategies.5,11 However, severe treatment-related toxicities have been reported in up to 80% of patients.11,12 Hence, both doublet or triplet combinations and anti-EGFR or antivascular endothelial growth factor are feasible options in this subset of patients. The generation of preclinical models such as patient-derived organoids (PDOs), recapitulating patient tumor histology and genetics, is emerging as a tool to predict treatment efficacy in oncology.13,14 Although genomics has already improved treatment choice in patients with CRC, especially for those carrying RAS/BRAF wild type, coclinical trials are becoming more and more important to directly test different treatment options in patient-derived tumors.15 Here, we present a proof-of-concept case report about the potential role of drug sensitivity testing in PDOs in the clinical decision making of oligometastatic CRC.

open 25 no Background HER2 is a therapeutic target for metastatic colorectal cancer (mCRC), as demonstrated in the pivotal HERACLES-A (HER2 Amplification for Colo-rectaL cancer Enhanced Stratification) trial with trastuzumab and lapatinib. The aim of HERACLES-B trial is to assess the efficacy of the combination of pertuzumab and trastuzumab-emtansine (T-DM1) in this setting. Methods HERACLES-B was a single-arm, phase II trial, in patients with histologically confirmed RAS/BRAF wild-type and HER2+ mCRC refractory to standard treatments. HER2 positivity was assessed by immunohistochemistry and in situ hybridisation according to HERACLES criteria. Patients were treated with pertuzumab (840 mg intravenous load followed by 420 mg intravenous every 3 weeks) and T-DM1 (3.6 mg/kg every 3 weeks) until disease progression or toxicity. Primary and secondary end points were objective response rate (ORR) and progression-free survival (PFS). With a Fleming/Hern design (H0=ORR 10%; α=0.05; power=0.85), 7/30 responses were required to demonstrate an ORR ≥30% (H1). Results Thirty-one patients, 48% with ≥4 lines of previous therapies, were treated and evaluable. ORR was 9.7% (95% CI: 0 to 28) and stable disease (SD) 67.7% (95% CI: 50 to 85). OR/SD ≥4 months was associated with higher HER2 immunohistochemistry score (3+ vs 2+) (p = 0.03). Median PFS was 4.1 months (95% CI: 3.6 to 5.9). Drug-related grade (G) 3 adverse events were observed in two patients (thrombocytopaenia); G≤2 AE in 84% of cycles (n = 296), mainly nausea and fatigue. Conclusions HERACLES-B trial did not reach its primary end point of ORR; however, based on high disease control, PFS similar to other anti-HER2 regimens, and low toxicity, pertuzumab in combination with T-DM1 can be considered for HER2+mCRC as a potential therapeutic resource. open Sartore-Bianchi A.; Lonardi S.; Martino C.; Fenocchio E.; Tosi F.; Ghezzi S.; Leone F.; Bergamo F.; Zagonel V.; Ciardiello F.; Ardizzoni A.; Amatu A.; Bencardino K.; Valtorta E.; Grassi E.; Torri V.; Bonoldi E.; Sapino A.; Vanzulli A.; Regge D.; Cappello G.; Bardelli A.; Trusolino L.; Marsoni S.; Siena S. Sartore-Bianchi A.; Lonardi S.; Martino C.; Fenocchio E.; Tosi F.; Ghezzi S.; Leone F.; Bergamo F.; Zagonel V.; Ciardiello F.; Ardizzoni A.; Amatu A.; Bencardino K.; Valtorta E.; Grassi E.; Torri V.; Bonoldi E.; Sapino A.; Vanzulli A.; Regge D.; Cappello G.; Bardelli A.; Trusolino L.; Marsoni S.; Siena S.

Melanoma progression is generally associated with increased transcriptional activity mediated by the Yes‐associated protein (YAP). Mechanical signals from the extracellular matrix are sensed by YAP, which then activates the expression of proliferative genes, promoting melanoma progression and drug resistance. Which extracellular signals induce mechanotransduction, and how this is mediated, is not completely understood. Here, using secretome analyses, we reveal the extracellular accumulation of amyloidogenic proteins, i.e. premelanosome protein (PMEL), in metastatic melanoma, together with proteins that assist amyloid maturation into fibrils. We also confirm the accumulation of amyloid‐like aggregates, similar to those detected in Alzheimer disease, in metastatic cell lines, as well as in human melanoma biopsies. Mechanistically, beta‐secretase 2 (BACE2) regulates the maturation of these aggregates, which in turn induce YAP activity. We also demonstrate that recombinant PMEL fibrils are sufficient to induce mechanotransduction, triggering YAP signaling. Finally, we demonstrate that BACE inhibition affects cell proliferation and increases drug sensitivity, highlighting the importance of amyloids for melanoma survival, and the use of beta‐secretase inhibitors as potential therapeutic approach for metastatic melanoma., The accumulation of PMEL fibrils in the metastatic melanoma microenvironment modulates YAP activity. Preventing aggregate formation reduces metastatic cell proliferation and chemosensitivity, suggesting beta‐secretase inhibition as treatment for metastatic melanoma.

Introduction: The first-line therapy for patients with low-risk myelodysplastic syndromes (MDSs) commonly consists of erythropoietin stimulating agents (ESAs), with a response rate ranging from 34 to 62%. For nonresponder patients, outside clinical trials, blood transfusions are the most frequent therapeutic option, with detrimental effect on the quality of life and with risks of iron-overload. Since no studies have been yet conducted on this topic, we investigated the potential predictive role of bone marrow (BM) histological evaluation in patients treated with ESAs. Materials and Methods: We performed a morphological and immunohistochemical retrospective analysis of BM biopsies of 96 patients with low-risk MDSs subsequently treated with ESAs. Results: In our series, substantial morphological overlap was found between responder and nonresponder patients. On the contrary, patients with a percentage of CD34-positive blasts >3% or with p53 protein expression Conclusions: Our study reinforces the role of BM biopsy as diagnostic tool in MDSs, being also able to supply information related to response to ESAs and to its loss over time.

Granuloma annulare (GA) presenting as a painful lesion with acral distribution has been previously described.

Melanoma progression is generally associated to increased Yes-associated protein (YAP) mediated transcription. Actually, mechanical signals from the extracellular matrix are sensed by YAP, which activates proliferative genes expression, promoting melanoma progression and drug resistance. Which and how extracellular signals induce mechanotransduction is not completely understood.Herein, by secretome studies, we revealed an extracellular accumulation of amyloidogenic proteins, i.e. premelanosome protein (PMEL), together with proteins that assist amyloids maturation into fibrils. Indeed, we confirmed the presence of amyloid-like aggregates similar to those detected in Alzheimer disease. These aggregates were enriched in metastatic cell lines as well as in human melanoma biopsies, compared to their primitive counterpart. Mechanistically, we proved that beta-secretase (BACE) regulates the maturation of these aggregates and that its inhibition hampers YAP activity. Moreover, recombinant PMEL fibrils induce per se mechanotransduction promoting YAP activation. Finally, BACE inhibition affects cell proliferation and increases drug sensitivity. These results highlight the importance of amyloids for melanoma survival and the potential of beta-secretase inhibitors as new therapeutic approach to metastatic melanoma.

Key Clinical Message We describe an High‐grade B‐cell lymphoma case, in which a complex translocation t(3;8;14) with effects on the genes BCL6, MYC, and IGH, was detected. This case could be the first double‐hit lymphoma with a single chromosome rearrangement causing the double effect with three genes involved.

AimsNeurotrophic Tropomyosin Kinase Receptor 1 (NTRK1) gene encodes for the protein Tropomyosin-related kinase A (TRKA). Deregulated activity of TRKA has been shown to have oncogenic potential. We present here the results of an immunohistochemical (IHC) observational cohort study of TRKA expression together with gene copy number (GCN) assessment in various solid tumours.MethodsFormalin-fixed, paraffin-embedded consecutive samples of different tumour types were tested for TRKA expression. Samples showing TRKA IHC staining in at least 10% of cells were analysed by fluorescence in situ hybridisation to assess NTRK1 gene rearrangements and/or individual GCN gain. All patients underwent this molecular assessment within the phase I ALKA-001 clinical trial.Results1043 samples were tested and annotation for histology was available in 1023. Most of the samples were colorectal adenocarcinoma (CRC) (n=550, 52.7%) and lung adenocarcinoma (n=312, 29.9%). 24 samples (2.3%) were biliary tract carcinoma (BTC). Overall, 17 (1.6%) samples were characterised by TRKA IHC expression (four weak, eight moderate, five strong): 9/17 lung adenocarcinoma, 3/17 CRC, 3/17 BTC, 1/17 thyroid cancer and 1/17 cancer of unknown primary. Of these, 1/17 with strong TRKA IHC staining displayed NTRK1 gene rearrangement and 15/17 NTRK1 GCN gain by FISH. No correlation was found between intensity of TRKA IHC staining and number of copies of NTRK1.ConclusionsTRKA expression can be found in 1.6% of solid tumours and can be paralleled by NTRK1 gene rearrangements or mostly GCN gain. The prognostic and translational therapeutic impact of the latter remains to be established.

Background Colorectal cancer screening may reduce disease-related mortality by early-stage detection of cancers. Aims To study the effect of a single immunochemical faecal occult blood test (i-FOBt) screening round on reduction in colorectal cancer-related-mortality among average risk subjects. Methods Comparison of 5-year mortality rates in 3 cohorts from a Northern Italian province: (1) colorectal cancers detected at the 1st biennial round of a mass-screening programme targeting 50–69 years old subjects, (2) non-screening cancers symptomatically diagnosed during the same time period, and (3) cancers detected in the pre-screening biennium. Multivariate analyses were performed with the Cox regression model including tumour node metastasis (TNM) stage at diagnosis, anatomical distribution of cancers, age at diagnosis, gender and patient group. Kaplan–Meyer survival estimates and log-rank test for equality of survivor functions were calculated. Results Stage distribution significantly differed between screening and non-screening colorectal cancers: 73% of screen-detected colorectal cancers were stages I and II versus 43% and 40% of non-screening and pre-screening colorectal cancers. Cumulative 5-year mortality rate was significantly lower in screening compared to non-screening or pre-screening colorectal cancers patients (19% versus 37% and 41%, p Conclusions Colorectal cancers were detected at earlier stages in i-FOBT-positive subjects in comparison with non-screening patients; colorectal cancers found at screening had a significantly improved 5-year survival.

This study, performed on behalf of the Italian Registry of Thrombocythaemias (Registro Italiano Trombocitemie), aimed to test the inter-observer reproducibility of the histological parameters proposed by the WHO classification for the diagnosis of the Philadelphia chromosome-negative myeloproliferative neoplasms. A series of 103 bone marrow biopsy samples of Philadelphia chromosome-negative myeloproliferative neoplasms consecutively collected in 2004 were classified according to the WHO criteria as follows: essential thrombocythaemia (n=34), primary myelofibrosis (n=44) and polycythaemia vera (n=25). Two independent groups of pathologists reviewed the bone marrow biopsies. The first group was asked to reach a collegial 'consensus' diagnosis. The second group reviewed individually all the cases to recognize the main morphological parameters indicated by the WHO classification and report their results in a database. They were subsequently instructed to individually build a 'personal' diagnosis of myeloproliferative neoplasms subtype just assembling the parameters collected in the database. Our results indicate that high levels of agreement (≥70%) have been reached for about all of the morphological features. Moreover, among the 18 evaluated histological features, 11 resulted statistically more useful for the differential diagnosis among the different Philadelphia chromosome-negative myeloproliferative neoplasms. Finally, we found a high percentage of agreement (76%) between the 'personal' and 'consensus' diagnosis (Cohen's kappa statistic >0.40). In conclusion, our results support the use of the histological criteria proposed by the WHO classification for the Philadelphia chromosome-negative myeloproliferative neoplasms to ensure a more precise and early diagnosis for these patients.

Recently, the PARP4 gene has been identified as a possible susceptibility gene of primary thyroid and breast cancers. We analyzed PARP4 in 53 patients with multiple primary cancers including a thyroid cancer (TC), in 74 patients with TC alone, and in 88 healthy donors. Two PARP4 intronic variants within the IVS29 (c.3543 + 44T > C) and the IVS22 (c.2758 + 9G > A) were found only in the two patient groups. Moreover, we found a rare variant (r.522C > A) within a PARP4 pseudogene (PARP4P2) in one patient with four primary tumors, and with a familial cancer history. PARP4 mRNA was absent in all primary tumors and matched normal tissues, whereas the pseudogene variant transcript was always expressed. Consistently, immunostaining for PARP4 protein was negative at nuclear level in all tissues, thus suggesting that PARP4P2 pseudogene variant could alter its regulatory role on PARP4, inducing the down-regulation of PARP4 expression at both tumor and normal tissues level. In conclusion, germline intronic PARP4 variants could be a risk factor for the development of TC, and PARP4P2 pseudogene variations associated with PARP4 down-regulation may confer susceptibility to develop multiple metachronous cancers.

TPS2659 Background: Metastatic colorectal cancer (CRC) harbouring genetic defects in the mismatch-repair pathway (MMRd) presents with a high tumor mutational burden (TMB), and is highly sensitive to anti–programmed cell death protein 1 (PD-1) immune checkpoint inhibitors. We recently showed in preclinical models that the pharmacological treatment with temozolomide (TMZ) can induce the inactivation of MMR genes, and consequently the increase of TMB and immunogenic neoantigens, thus suggesting that TMZ could be used to prime MMR proficient (MMRp) tumors for response to checkpoint inhibitors. Accordingly, mCRC patients recruited in previous clinical trials where TMZ was administered, acquired alterations of MMR genes upon treatment and showed remarkable increase in TMB at disease progression (PD). We thus designed the ARETHUSA clinical trial to test whether a priming course with TMZ in patients can sensitize mCRC to the anti-PD1 inhibitor pembrolizumab. Methods: Arethusa (NCT03519412) is a 2-cohorts, phase II trial consisting of three different phases. In the SCREENING, 348 mCRC RAS-mutated patients will be tested for MMR status. MMRd patients will proceed directly to TRIAL for immediate pembrolizumab treatment (expected 14). MMR-proficient (MMRp) patients will be further tested for expression of O6-methylguanine-DNA methyltransferase (MGMT) by immunohistochemistry and by promoter methylation analysis. IHC-negative, promoter methylation-positive MMRp patients (expected 67) will enter in the PRIMING phase and will be treated with TMZ until PD. TMB will then be assessed on tumor biopsies at resistance. Those patients that will have > 20 mutations/megabase will proceed to TRIAL (expected 20) and will be treated with pembrolizumab. Overall response rate (primary outcome), Progression Free, and Overall Survival, and treatment related toxicities (secondary outcomes) in MMRp pembrolizumab-treated patients will be estimated., while the MMRd cohort will be used for comparison. Tissue biopsies, longitudinal blood and stool collection will be used for discovery of predictive molecular biomarkers and assessment of tumor evolution. Clinical trial information: NCT03519412.

Myelodysplastic syndromes (MDS) are haematopoietic stem-cell disorders characterised by peripheral cytopenia, dysplastic haematopoiesis, and a substantial risk of progression into acute myeloid leukaemia (AML). We report a case of low-risk MDS, according to the international prognostic scoring systems (IPSS), which evolved into AML simultaneously to metastatic bone marrow localisation of malignant melanoma. A 70-year-old man was diagnosed with myelodysplastic syndrome in 2006. He presented with complaints of weakness for a month. He referred arterial hypertension and myocardial infarction 3 years before, treated with coronary revascularisation. Blood analysis revealed mild macrocytic anaemia. Bone marrow biopsy and aspirate evaluation at diagnosis showed a slightly hypercellular marrow, with significant dysplasia in the erythroid lineage, less than 5% of CD34-positive haematopoietic precursors and absence of marrow fibrosis. Cytogenetic analysis showed normal karyotype. According to the updated WHO classification, a diagnosis of refractory anaemia was made, and he was stratified as low risk by means of IPSS scoring systems.1 He was included in a clinical trial with thalidomide for 9 months, which was interrupted for inefficacy, worsening of anaemia and appearance of moderate neutropenia. At this point, in January 2007, he was referred to our centre presenting with bicytopenia in the peripheral blood. Bone marrow re-evaluation was consistent with refractory cytopenia with multilineage …

Introduction: Erythropoietin stimulating agents (ESAs) are first-line therapy for International Prognostic Scoring System (IPSS)-low risk myelodysplastic syndrome (MDS) when symptomatic anaemia (Hb Attempts have been made to build prognostic scores (integrating transfusion need, erythropoietin level, R-IPSS and ferritin) able to predict response to ESAs (6-7). No data concerning the predictive value of morphological and immunohistochemical analysis on bone marrow biopsy in low risk MDS patients are emerged till now. Methods: We retrospectively examined 96 IPSS low/int-1 MDS patients treated with ESAs in order to evaluate the morphological and immunohistochemical features of the bone marrow biopsies performed at baseline. All the patients had hemoglobin (Hb) ≤10 g/dL and serum EPO A detailed analysis of the morphological features (including quantitative and qualitative changes in the erythroid, myeloid and megakariopoyetic lineages) was performed, together with an immunohistochemical evaluation of p53 expression and CD34-positive blasts percentage. Results: Sixty-eight percent of the patients were classified as responder while 32% as non-responder. The morphologic profiles of the responder and non-responder did not differ significantly. A significant correlation was found between the response to the therapy and a percentage of CD34-positive blasts > 3% (univariate analysis: p= .0211). Moreover p53 expression in less than 1% of the nucleated cells correlated with the treatment response (univariate analysis: p = .0086). These results were also confirmed on multivariate analysis (p= .002). Fifty-eight percent of patients maintained response to ESAs for the entire duration of the follow up while 42% lost the response (median follow up: 35 months). More than 3% CD34-positive blasts was associated with a higher probability to lose the response to ESAs (p = .00003). Kaplan-Meier method was than applied to estimate the response duration. Patients with more than 3% CD34-positive blasts showed earlier loss of response in comparison with those with a lower percentage of blasts (p value= .0003; HR=3.9, IC 95% 1.8154-8.6238). Finally, the expression of p53 in more than ≥ 1% of the nucleated cells correlated with the loss of response before 24 mounts (p value= 0.029). Conclusions: Our study identifies 2 well-known parameters that could be useful to better predict outcome of ESAs therapy in low risk MDS patients. Therefore role of bone marrow biopsy together with its diagnostic contribution in the clinical evaluation of MDS patients (cellularity, morphologic dysplasia, percentage of CD34-positive blasts, grading of bone marrow fibrosis) could be helpful as a predictive tool in ESAs candidate patients. Further studies based on larger series of patients are needed to confirm these preliminary results. Disclosures Reda: Gilead: Consultancy; ABBVIE: Consultancy; Janssen and Cilag: Consultancy; Celgene: Consultancy. Riva:Jannsen and Cilag: Consultancy; Novartis: Consultancy; Celgene: Consultancy. Molteni:Janssen and Cilag: Consultancy; Novartis: Consultancy; Italfarmaco: Consultancy; Celgene: Consultancy; AMGEN: Consultancy. Cortelezzi:abbvie: Consultancy; novartis: Consultancy; roche: Consultancy; janssen: Consultancy.

This study defined the clinical features and assessed the prognosis of 47 patients (17 males, 30 females, median age 63 years) with primary nodal marginal zone B-cell lymphoma. Forty-five per cent had stage IV disease. Hepatitis C virus serology was positive in 24%. According to the Follicular Lymphoma International Prognostic Index (FLIPI), 33% were classified as low-risk, 34% as intermediate-risk, and 33% as high-risk. The 5-year overall survival (OS) was 69%. In univariate analysis worse OS was associated with: FLIPI (P = 0.02), age > 60 years (P = 0.05) and raised lactate dehydrogenase (P = 0.05). In multivariate analysis, only FLIPI predicted a worse OS (P = 0.02).

Diffuse large B-cell lymphoma (DLBCL) has been correlated to hepatitis C virus (HCV) infection in few series, but characteristics and outcome of these patients remain undefined.We analyzed 156 previously untreated consecutive HCV-positive patients with DLBCL observed between 1994 and 2004 in three major institutions from northern Italy.Median age at presentation was 63 years and 8% of patients had DLBCL transformed from low-grade lymphomas. Spleen was the most frequently involved extranodal site, followed by liver and stomach. Treatment was delivered with cure-intent in 132 patients, while the remaining 24 patients received monochemotherapy or radiotherapy alone due to old age or seriously impaired hepatic function. Only five patients (4%) had to discontinue chemotherapy due to severe liver function impairment. The addition of rituximab did not seem to affect patients' tolerance to treatment. Five-year overall survival of the entire cohort was 72%, while 5-year progression-free survival (PFS) of the 132 patients treated with cure-intent was 51%. Hepatitis B virus co-infection, advanced Ann Arbor stage and nodal origin of the tumor resulted the strongest adverse prognostic factors.Patients with HCV-positive DLBCL share distinctive clinical features. Future studies should prospectively evaluate the association between HCV and aggressive lymphomas.

We investigated whether dendritic cells (DCs) play a role in favoring granular lymphocyte (GL) proliferation in patients with lymphoproliferative disease of granular lymphocytes (LDGL). The presence of in vivo circulating DCs was studied in 11 patients (5 CD3+ and 6 CD3- LDGL). Autologous immature (iDCs) and mature (mDCs) DCs generated in vitro were studied for stimulatory activity on cell proliferation of CD3+ and CD3- GLs. The topographic organization of GLs and DCs was also studied in bone marrow (BM) biopsies. Peripheral blood (PB) CD3- GLs from patients showed significant proliferative activity in the presence of iDCs and mDCs. Conversely, monoclonal CD3+ GLs were unresponsive to autologous and allogeneic PB DCs. Analysis of BM biopsies demonstrated a topographic distribution of DCs and GLs that indicates contact between the 2 cell types. On functional assays, DCs obtained from BM were more efficient than PB DCs in stimulating CD3- GLs, and surprisingly, a low but definite stimulatory effect was demonstrated also on CD3+ GLs. The putative contact between DCs and GLs in the BM and, more crucial, the proliferative response of discrete GL populations to DC stimulation suggest the presence of a specific antigen within BM DCs, providing evidence for a role of DCs in the pathogenesis of LDGL.

AimsTo identify molecular characteristics to hepatitis C virus (HCV)-associated diffuse large B-cell lymphoma (DLBCL) through a comprehensive miRNAs expression profiling.MethodsIn 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.ResultsA 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.ConclusionsThese data reveal molecular differences in diffuse DLBCL patients according to HCV presence, potentially useful as novel prognostic or therapeutic biomarkers.

Experimental studies performed in Folkman laboratories suggest that angiogenesis is involved in the biology of tumor dormancy. We determined the vascular index in a series of 190 women operated of node-positive invasive breast cancer treated with adjuvant chemotherapy (CMF schedule) and we studied the relationship between vascularity of primary tumors with the behaviour in time of metastasis. The study of the hazard function of recurrence (in any site) was performed resorting to a generalized linear modelling approach with a binominal error according to Efron. A total of 80 cases developed recurrences during the period of observation. We found that the hazard function of metastasis in time presented two peaks of incidence at 20 and 60 months, respectively. We also plotted the curves of the hazard function by considering three values of microvessel counts corresponding to the quartiles of their distribution. The risk of first recurrence was associated with vascular index, and the patients of the third quartile of distribution of microvessels had the highest risk. In the final full model for the risk of recurrence at 5 years vascular index provided the highest prognostic contribution followed by the number of involved axillary lymph nodes. The observation that the patients with highly angiogenic tumors are at high risk of recurrence coupled with the identification of the second peak of incidence after 5 years which was also mainly sustained by angiogenic tumors suggest that a fraction of breast cancers promote metastasis after a period of tumor dormancy. The clinical and therapeutic implications of our results are discussed.

Summary Background The prognostic implications of p53 accumulation, bcl-2 immunoreactivity and tumour proliferative fraction in ovarian carcinomas are still debated. Patients and Methods One hundred twelve ovarian carcinomas were immunostained for p53 protein,for bcl-2 and for the cell cycle-associated Ki-67 antigen. The immunostaining results were correlated with conventional clinico-pathological variables, response to induction chemotherapy, and patient survival. Results p53 accumulation and bcl-2 immunoreactivity in more than 10% of neoplastic cells were detected in 61 (54.5%) and 42 (37.5%) cases, respectively. A positive correlation between p53 accumulation and high (more than 30% neoplastic cells) MIBl labelling index (r = 0.235; P = 0.015) was ascertained, whereas no significant association was found between bcl-2 immunoreactivity and p53 accumulation or MIBI labeling index. Both p53 accumulation and MIBI immunoreactivity correlated significantly with a reduced overall survival, but the association was lost in multivariate analysis. However, patients with tumours simultaneously showing p53 accumulation and MIBI labelling index higher than 30% had significantly reduced overall survivals, in both univariate and multivariate analyses. Conclusions The simultaneous evaluation of p53 accumulation and MIBI labelling index has independent prognostic implications in common epithelial malignancies of the ovary, irrespective of the disease stage.

Background: The clinical outcome is generally positive for patients with node-negative breast carcinoma (i.e., those who do not have detectable metastases in the lymph nodes) who have been treated with surgery or surgery plus radiation therapy. In about 30% of the patients, however, the disease recurs, and they are at risk of death. Determination of valid new prognostic indicators would improve the ability to identify patients at high risk of recurrence. Breast cancer can entail substantial development of new blood vessels within the tumor tissue, and it is known that the growth and metastasis of solid tumors are dependent on such angiogenesis. The conversion of tumor cells to an angiogenic phenotype may be preceded by a change in the balance of angiogenic growth factors and angiogenesis inhibitors. Purpose: This study was conducted to determine if the levels of vascular endothelial growth factor (VEGF) protein, a potent endothelial growth factor and mediator of vascular permeability and angiogenesis, measured in the primary tumors of women with node-negative breast cancer are associated with known prognostic factors and patient survival. Methods: By use of a selective enzymatic immunoassay, levels of VEGF protein were measured in cytosolic extracts of primary tumor tissue surgically obtained from 260 women with node-negative breast carcinoma who had been treated with surgery with or without radiation therapy but not with adjuvant therapy and who had been followed for a median time of 66 months. The relationships between VEGF concentrations and other prognostic dichotomous variables or clinical outcome were tested by the use of the Kolmogorov-Smirnov test and univariate and multivariate Cox analyses, respectively. The relationship between VEGF and hormone receptors (i.e., those for estrogen and progesterone) was examined by the use of Spear-man's correlation analyses. All P values resulted from the use of two-sided statistical tests. Results: Tumors from 247 (95%) of the 260 patients had detectable VEGF, ranging in concentration from 5.0 to 6523 pg/mg protein (median, 126.25 pg/mg protein). No statistically significant associations were found between VEGF and the other prognostic factors (e.g., age, menopausal status, histologic tumor type, tumor size, and hormone receptors) examined. Levels of VEGF were found to be prognostic for both relapse-free and overall survival in univariate and multivariate analyses (likelihood ratio tests; all four P values

Primary cutaneous lymphomas (PCL) represent a heterogeneous group of skin neoplasms, which sometimes can pose difficulties in their diagnosis and in the differential diagnosis with benign lymphoid proliferations.

Experimental studies suggest that angiogenesis plays an important role in the pathogenesis of ascites and progression of ovarian cancer. To evaluate the association of intratumoral microvessel density (IMD) with the conventional clinicopathologic features and to determine the capability of these factors in predicting responsiveness to platinum-based chemotherapy and overall survival (OS) we studied 112 ovarian carcinomas. IMD was determined using the anti-CD31 antibody and immunocytochemistry. In the entire series, we correlated IMD with the other features. In the subgroup of patients with FIGO stage III-IV (60 cases), we correlated the factors studied, determined prior of treatment, with response to therapy and prognosis. The median IMD value, in the "hot spot", in the entire series was of 48 microvessels/field. IMD values were significantly higher in mucinous carcinomas than in the other histologic types. In FIGO stage III-IV patients IMD, age and performance status (PS) were significantly associated with the probability of pathologic response to chemotherapy in univariate analysis. However, only IMD and PS retained significance in multivariate analysis. The overall capability of the 2 variables to predict response was high. In FIGO stage III-IV patients IMD, age, PS, the amount of post-operative residual disease (PORD), histologic type and response to chemotherapy were significant prognostic indicators of OS in univariate analysis. In multivariate analysis only histologic type, PORD and PS retained significance. The overall capability of these 3 variables to predict OS was satisfactory.

AimsThe authors investigated vascular endothelial growth factor receptor 1 (VEGFR-1) protein expression in a series of Philadelphia chromosome-negative myeloproliferative neoplasms (Ph- MPNs) and its correlations with microvessel density (MVD) and vascular endothelial growth factor (VEGF).Methods83 bone marrow biopsies of Ph- MPNs patients, including 27 essential thrombocythaemia (ET), 21 polycythaemia vera (PV) and 35 primary myelofibrosis (PMF), and 10 normal controls (NCs) were investigated by immunohistochemistry.ResultsPatients with PV and PMF showed an increased MVD (PV: 20.1±10.6; PMF: 25.8±6.5) compared with those with ET or NCs (ET: 10.4±4.6; NCs: 7±3.4). VEGFR-1 expression was increased in Ph- MPNs, particularly in PV and PMF (NCs: 0.07±0.03; ET: 0.15±0.09; PV: 0.31±0.2; PMF: 0.31±0.04). VEGF expression parallelled VEGFR-1 and resulted increased in Ph- MPNs (NCs: 0.08±0.04; ET: 0.13±0.06; PV: 0.29±0.2; PMF: 0.31±0.15) and higher in post-polycythaemic myelofibrosis and in the fibrotic stage of PMF than in the non-fibrotic phases of both diseases. VEGFR-1 protein expression correlated with MVD and VEGF expression in Ph- MPNs. VEGFR-1 and VEGF were expressed by the same bone marrow populations: megakaryocytes, macrophages and immature myeloid precursors showed a moderate to strong immunostaining intensity in both Ph- MPNs and NCs. The erythroid precursors were not immunoreactive.ConclusionsVEGFR-1 and VEGF were increased and co-localised in megakaryocytes, macrophages and myeloid precursors of Ph- MPNs. This finding supports the hypothesis of a VEGF/VEGFR-1 autocrine loop in the neoplastic cells of Ph- MPNs.

Reduced-intensity conditioning regimens have reshaped the clinical presentation of graft-versus-host disease after hematopoietic stem cell transplants. However, histopathologic features of graft-versus-host disease following reduced-intensity conditioning regimens have not been fully characterized. In a series of 112 biopsies (skin, n = 60; gastrointestinal [GI] tract, n = 44; liver, n = 8), we described the morphologic profile of graft-versus-host disease following reduced-intensity conditioning and investigated whether histopathologic changes of graft-versus-host disease following reduced-intensity conditioning have a diagnostic and/or prognostic value. Forty-four patients (49.5%) experienced acute graft-versus-host disease, 2(2.2%) late-onset acute graft-versus-host disease (grade I, n = 13; grade II-IV, n = 33), 24(27.0%) chronic graft-versus-host disease (de novo n = 12, progressive n = 12) and 19 (21.3%) overlap syndrome. In the skin, we observed: (i) phase-nonspecific changes, such as acute graft-versus-host disease features in chronic graft-versus-host disease patients (n = 4/24; 16.6%), (ii) subtle alterations such as superficial fibrosis in widened dermal papillae (n = 8), in acute graft-versus-host disease/late-onset graft-versus-host disease (n = 6/46; 13.0%) or chronic graft-versus-host disease (n = 2/24, 8.3%) patients, and (iii) features of chronic and acute graft-versus-host disease coexisting in the same specimen in overlap syndrome (n = 3/19; 15.7%). In the GI tract, we did not demonstrate peculiar features differing from those commonly observed in the myeloablative setting. By univariate analysis, a reduced overall survival was associated with graft-versus-host disease type (chronic graft-versus-host disease P = .006, acute graft-versus-host disease P = .03), older age (P = .04), and histopathologic diagnosis of "consistent with" + definite graft-versus-host disease (P = .02). Histopathologic diagnosis retained an independent prognostic value by multivariate analysis (P = .01). The present study indicates that pathologists should be aware of the peculiar morphologic changes of cutaneous graft-versus-host disease following reduced-intensity conditioning and further recommends histopathology in the diagnostic workup of graft-versus-host disease in patients undergoing reduced-intensity conditioning regimen. (C) 2011 Elsevier Inc. All rights reserved.

Background Human papilloma viruses (HPV) are the necessary cause of invasive cervical cancer (ICC). Of the many different types identified so far, only a few of them account for the great majority of cases worldwide, with geographical differences in their distribution. Data on the local distribution are now of interest in view of the soon-to-come introduction of HPV type-specific prophylactic vaccines. Results We have investigated HPV type distribution in samples of 48 ICC cases occurred in women living in North-East Italy in the years 1997–1999. Cases were extracted from the Venetian Tumour Registry files, as incident cases whose specimens had been processed in two Pathology Departments. Search and typing were performed by polymerase chain reaction (PCR) using GP5+/GP6+ primers, followed by direct sequencing or reverse dot blot. Three cases were PCR negative using the housekeeping primers and hence excluded. One case was negative by all HPV tests used. HPV 16 was present in 32 (72.7%) cases, as single infection in 28, in mixed infection in 4. Of the 44 positive cases, HPV 16 and HPV 18 accounted for 33 (75%), as single or mixed infections. The other high risk HPV types accounted for 11 (25%) of the remaining infections. Of the 32 HPV 16 positive cases, sequencing of the E6 gene could be performed in 25; the prototype isolate was identified in 7, and the variant T350G in 18; in 4 cases one or more additional mutations were present. Conclusions Our results suggest that HPV 16 has a very high prevalence among women with invasive cervical cancer in Italy; therefore, the use of a prophylactic vaccine for HPV types 16 and 18 could prevent up to 75% of invasive cervical cancers in Italy.

Objective. We evaluated the prevalence of genital human papillomavirus (HPV) types in correlation with cytomorphological findings in patients at different risk for cervical intraepithelial neoplasia living in northeast Italy. Methods. Exfoliated cervicovaginal cells from 943 women, who were divided into three groups, were analyzed by polymerase chain reaction. Results. Overall, HPV prevalence rates were 7%, 38%, and 52%, respectively. The single most frequent type was HPV 16 (18%), followed by types 6, 31, 53, 58, 61, and novel/unidentified (5–7%); other types had a frequency

The effect of antiretroviral therapy on the natural history of human papillomavirus (HPV)-associated genital lesions was evaluated in 201 human immunodeficiency virus (HIV)-infected women who were followed-up for 1-6 years. Gynecologic examinations were performed every 6-12 months. HPV sequences in cervico-vaginal cells, analyzed by polymerase chain reaction and typed by restriction fragment-length polymorphism analysis, were repeatedly detected in 126 women; 29 had transient HPV infection. Genital lesions were found in 137 patients; prevalence was comparable in women who were receiving different antiretroviral regimens. Regression of low-grade lesions was more prevalent among patients receiving highly active antiretroviral therapy than among those receiving other regimens; high-grade lesions regressed in the majority of cases, regardless of antiretroviral therapy. HPV infection persisted in nearly 80% of the cases. In conclusion, our data show that antiretroviral therapy does not prevent the development of HPV-associated lesions and does not eliminate HPV infection; therefore, early and strict gynecologic follow-up of HIV-infected women is warranted.

In situ PCR, amplification of target DNA sequences in fixed cells, is a very useful molecular biology tecnique with potential to combine the high sensitivity of tube PCR with the precise anatomical localization of the targeted bsequences. It allows the study of low copy viral or bacterial DNA. In this study we document the utility of directin situ PCR with single primer pair by applying it to 3 infectious agents in different model systems: Borrelia burgdorferi in 5 Eritema migrans lesions and 55 primitive cutaneous B cell lymphomas, Chlamydia pneumoniae in 200 autoptic atheromasic lesions, and Papilloma virus in 20 CIN 1 (mild cervical dysplasia). In situ PCR seems to be a very promising tecnique; however, the prerequisite for the success of in situ PCR is conditioned by optimal standardization of the key variables which, on the other hand, are influenced by tissue composition.

Background Cervical cancer is preventable through screening, and HIV treatment guidelines recommend that all HIV-infected women receive cervical cancer twice during the year after HIV diagnosis and annually thereafter. Different immunohistochemical markers have been studied to highlight cervical intraepithelial lesions of low and high grade, the most widely used being p16. Recent studies have shown that insulin-like growth factor mRNA binding protein 3 (IMP3) plays a role in the development of invasive squamous cell carcinoma from cervical dysplasia, both in histology and in liquid-based cytology. Methods: We evaluated the clinical significance of the immunohistochemical expression of IMP3 and p16 in histological samples of cervical intraepithelial neoplasia from 56 samples of HIV-positive and 30 samples of HIV-negative patients. Results: A significant difference was found in IMP3 and p16 protein expression between HIV-positive and HIV-negative specimens. All cases of HIV-positive low grade squamous intraepithelial neoplasia (L-SIL) with IMP3 expression progressed in high grade (H)-SIL. However, the HIV-positive patients with IMP3-negative L-SIL remained stable or had a negative follow up. The L-SIL of HIV-negative patients with IMP3 protein expression had an uneventful follow up. IMP3-positive H-SIL recurred with low- or high-grade dysplasia during follow up after conisation in both populations. All IMP3-negative L-SIL and H-SIL had negative pap tests at follow up. Conclusions: In HIV-positive cases, IMP3 showed a higher sensitivity than p16 in identifying patients at risk of progression and recurrence.

The spectrum of CD30+ cutaneous lymphoproliferative disorders is characterized by the histology of a high-grade lymphoma but frequent clinical regression of skin lesions in lymphomatoid papulosis (LyP) and occasional regression in CD30+ large cell lymphomas (LCLs). A recent study shows that apoptosis may be a significant mechanism of regression of LyP (Arch Dermatol 133:828-833, 1997). Therefore, we studied expression of proteins that induce apoptosis, including CD27, CD40, CD95, and nerve growth factor receptor (NGF-R), as well as anti-apoptotic protein bcl-2 in skin lesions from 25 patients within the spectrum of CD30+ cutaneous lymphoma. Our results show consistent expression of CD95 (APO-1/Fas), but rare or absent expression of CD27, CD40, and NGF-R on tumor cells from both regressing LyP lesions and nonregressing CD30+ lymphomas. Bcl-2 was expressed at low levels in LyP and at high levels in pleomorphic CD30+ lymphomas. These results indicate that, in addition to CD30, CD95 expression is preferentially expressed at high levels in all cutaneous CD30+ lymphomas and suggest that CD95 may play a role in the regression of CD30+ skin lesions. Expression of bcl-2 appears to protect tumor cells from apoptosis in CD30+ lymphoproliferative disorders.

We evaluated the microvessel area (an index of angiogenesis) by using the factor VIII-related antigen (factor VIII-RA), and the expression of three components of the subendothelial basement membrane, namely: tenascin, laminin and type IV collagen. The four markers were assessed by immunohistochemical methods in 57 B-cell non-Hodgkin's lymphomas (B-NHL) and 28 benign lymphadenopathies. We found that microvessel area and the basement membrane markers had a different distribution pattern in relation to the histological type and degree of malignancy. In reactive and atypical lymphoid hyperplasias and in follicular low- and intermediate-grade B-NHL, microvessels were distributed within the interfollicular zones. By contrast, diffuse intermediate-grade and high-grade B-NHL were highly vascularized and microvessels were closely related to the neoplastic cells. Microvessel area was significantly associated with tenascin expression in all histological grades. Conversely, it was not correlated to laminin and type IV collagen expression, especially in diffuse intermediate-grade and high-grade B-NHL, where the expression of these markers was poor and fragmented. Our study suggests that angiogenesis and tenascin expression are associated phenomena in B-NHL, and that both increase with the malignancy grade. The prognostic value of angiogenesis and tenascin in B-NHL warrants further assessment in follow-up studies.

Intranuclear p53 accumulation is a common finding in many different human tumors and is associated with a worse prognosis in breast, colon, and lung carcinomas. We immunostained a series of common epithelial ovarian cancers to assess (1) the prevalence of p53 accumulation and its clinicopathologic correlations, and (2) its prognostic implications. The study population comprised 98 patients (83 with follow-up data). A variable degree of p53 immunoreactivity, strictly confined to the neoplastic cells, was detected in 54 tumors (55%). Among these tumors there were 10 low expressors (1% to 10% immunoreactive tumor cells), 16 moderate expressors (10% to 50% immunoreactive cells), and 28 high expressors (>50% immunoreactive cells). The prevalence of p53 immunoreactivity did not show any association with the histologic type of the tumors or with the disease stage at presentation. p53 Accumulation, however, was significantly more prevalent among poorly differentiated tumors ( P = .0059, by analysis of variance). Life table analysis demonstrated that patients with tumors showing moderate and high p53 expression had worse disease-free and adjusted lengths of survival ( P = .0011 and P = .0025, respectively, by Mantel-Cox). The patients with "early stage" disease (stages I and II) and p53 accumulation showed a trend toward shorter length of survival, but this did not reach statistical significance. However, patients with "advanced stage" disease (stages III and IV) and moderate or high p53 accumulation had a poorer prognosis ( P = .0154 and P = .0171, for disease-free and adjusted length of survival, respectively). These results suggest that p53 accumulation occurs more frequently in tumors with aggressive behavior and that p53 immunoreactivity may have a prognostic role in certain subsets of patients with ovarian carcinoma.

Summary Background: Experimental and clinical studies suggest that cyclin Dl is involved in transformation and tumour progression. However, there is little and contradictory data on the clinical significance of cyclin Dl in human invasive breast carcinoma. Patients and methods: We investigated whether the determination of cyclin Dl has prognostic value in a series of 180 patients with node-positive breast carcinoma and treated with adjuvant therapy with a median follow-up exceeding 6 years. We assessed cyclin Dl expression using the CDS-6 monoclonal antibody and a highly sensitive immunohistochemical technique. Results: We found that most of the evaluable tumours (117 of 167; 70.1%) presented nuclear cyclin Dl staining and that its expression was significantly associated with both the hormone receptors (P- 0.009 and P= 0.005 for ER and PgR, respectively). Furthermore, 29 (17%) of 167 tumours had a weak (15 cases) or strong (9 cases) cytoplasmic cyclin Dl staining. In a subgroup of cases we also studied the amplification of the cyclin Dl gene and a moderate agreement between cyclin Dl nuclear overexpression assessed immunohistochemically and the gene amplification was found. In univariate analysis, cyclin Dl nuclear positivity was significantly associated with improved 6-year relapse-free survival (RFS) (P - 0.004), but not with overall survival (OS) (P = 0.12). The results of the Cox multivariate analysis (final model) indicate that cyclin Dl expression (P— 0.0049) as well as the number of involved nodes (P < 0.001) and tumour size (P — 0.036) are significant prognostic indicators for RFS. Only the number of involved nodes retained significance (P < 0.001) for OS in our series. The joint assessment of the variables considered in the final model of the multivariate analyses had a moderate prognostic capability as determined using the Harrell c statistic (c " 0.66 and 0.64 for RFS and OS, respectively). Conclusions: The patients with node-positive breast cancer who have a higher likelihood of gaining benefit from adjuvant therapy are those with tumours with cyclin Dl nuclear expression, small size and less than 3 metastatic nodes. Further studies are needed to verify the prognostic value of cyclin Dl in relation to different adjuvant treatments and to deepen the biological pathways that regulate its activation/ suppression in human breast carcinoma.

To validate the prognostic value of the determination of p53 expression, intratumoral microvessel density (IMD) (a measure of angiogenesis), and the conventional features, we studied 531 patients operated of breast cancer (271 node-positive and 260 node-negative), with a median follow-up exceeding 6 years. IMD was assessed by using the anti-CD31 antibody to identify the microvessels. p53, estrogen receptor (ER) and progesterone receptor (PgR) were determined by immunocytochemistry using the antibodies PAb1801, H-222 Sp2y and KD-68, respectively. The prognostic value of the markers was analyzed by univariate and multivariate statistical analyses. In the overall series p53 expression, IMD, nodal status, ER and PgR were statistically significant prognostic indicators for both relapse-free survival (RFS) and overall survival (OS) in the final multivariate model. Likewise, tumor size and menopausal status were significant prognostic indicators for RFS and OS, respectively. In the subgroup of node-negative patients who did not receive adjuvant therapy only p53, IMD, and tumor size were statistically significant in multivariate analysis. In the subgroup of node-positive patients treated with adjuvant chemotherapy, IMD, the number of involved nodes and PgR were statistically significant in multivariate analysis. In the subgroup of node-positive patients treated with adjuvant tamoxifen, IMD and ER (and the number of involved nodes, only for OS) were statistically significant for both RFS and OS in the final multivariate model. Different markers played a diverse prognostic role in the diverse subgroups studied. Angiogenesis was the sole marker which retained prognostic value in all the sub-groups analyzed. p53 retained significance only in the subgroup of node-negative patients, whilst ER and PgR were statistically significant in the subgroups of node-positive patients treated with adjuvant hormone therapy or chemotherapy, respectively.

In a series of 205 node-negative breast cancers (NNBC), we determined staining by the novel antibody Ki-S1, a marker of tumor cell proliferation, in order to test its association with other prognostic variables and its prognostic significance. Ki-S1 was determined in routinely formalin-fixed paraffin-embedded tumor samples. Ki-S1 gave a nuclear staining in the majority of the carcinomas (188 of 205), with percentages of reacting nuclei ranging from 2% to 90% (median value of 7%). In 107 tumors frozen sections were available to also assess the Ki-67 antibody. Among these, 94 had a nuclear staining of cancer cells ranging from 5% to 80% (median value of 7%). In 46 tumors we also determined the MIB-1 antibody. The percentage of MIB-1 nuclear staining ranged from 1% to 50% (median value of 20%). There was no significant relationship between Ki-S1 and the other two cell kinetic markers. Ki-S1 labeling was significantly associated only with tumor size (p = 0.03). With a median follow-up of 6 years, Ki-S1 had no significant prognostic value for either relapse-free survival (RFS) or overall survival (OS) (Ki-S1 as continuous logarithmic variable; p = 0.86 and p = 0.23, respectively). For RFS the following variables had a significant prognostic value: Ki-67 (or = 10% vs10%; p = 0.037); progesterone receptor (PgR) expression (- vs+/++; p = 0.041); tumor size (pT1 vs pT2-3; p = 0.042) and grading (GI vs GII-III; p = 0.047). For OS, tumor size (p = 0.0044), age (continuous variable; p = 0.0060), and Ki-67 (p = 0.043) were significantly prognostic. In multivariate analysis (final model), only tumor size retained a significant and independent prognostic value for RFS (p = 0.0042). For OS, both tumor size (p = 0.0029) and age (or = 55 years vs55 years; p = 0.041) retained significance in the multivariate model. In conclusion, Ki-S1 does not seem to have prognostic relevance in this series of NNBC. Possible hypotheses to explain this observation are discussed.

Background ERBB2 amplification occurs in 5% of RAS wild-type metastatic colorectal cancer (mCRC) and it has been shown to be a target for treatment with 2 HER2-directed combinations of trastuzumab and lapatinib or trastuzumab and pertuzumab. We present long-term clinical results of trastuzumab and lapatinib (HERACLES-A trial) at 6.7 years (82 months) follow-up and focus on central nervous system (CNS) recurrences. Patients and Methods Patients had histologically confirmed KRAS exon 2 (codons 12 and 13) wild-type and HER2-positive mCRC. HER2 positivity was assessed by immunohistochemistry and in situ hybridization HERACLES diagnostic criteria. Patients were treated with intravenous trastuzumab 4 mg/kg loading dose, then 2 mg/kg once per week, and oral lapatinib 1000 mg per day until disease progression or toxicity. Patients who presented with symptoms or signs of CNS disease received brain computed tomography scan or magnetic resonance imaging. Results A total of 35 patients received trastuzumab and lapatinib and 32 were evaluable for response. One patient (3%) achieved complete response (CR), 8 (25%) partial response, and 13 (41%) stable disease. Therefore, response rate was 28%. Median progression-free survival was 4.7 months (95% confidence interval [CI] 3.7–6.1). Median overall survival was 10.0 months (95% CI 7.9–15.8). One patient achieved sustained CR still maintained at 7 years of follow-up. Progression in the central nervous system (CNS) occurred in 6 (19%) of 32 patients. Conclusions Long-term (6.7 years) follow-up analysis of HERACLES-A supports using of trastuzumab and lapatinib as treatment reference for KRAS wild-type, chemorefractory HER2-positive mCRC. In this subset of patients, prolongation of survival is accompanied by CNS recurrences that will require diagnostic and therapeutic attention in future studies. Clinicaltrials. Gov identifier: NCT 03225937.