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CT-PET scan showing clinical response of Pt#2.

Supplementary Table I: IFN-DC release criteria and median values registered among patient-derived IFN-DC (min-max range); Supplementary Table 2: Patients MHC-I and MHC-II alleles and IGHV epitope sequences

Purpose:This study was aimed at evaluating the feasibility, safety, immunologic and clinical responses in patients with follicular lymphoma treated with monocyte-derived dendritic cells generated in the presence of IFNα and GM-CSF (IFN-DC) in combination with low doses of rituximab.Patients and Methods:Firstly, we analyzed in vitro and in vivo the immunologic properties of IFN-DC against follicular lymphoma. Thus, we performed a phase I trial in 8 patients with refractory and relapsed follicular lymphoma based on sequential intranodal injections of low-dose of rituximab and unloaded IFN-DC and report the safety, clinical, and immunologic results of the enrolled patients.Results:Preclinical studies indicated that IFN-DC can synergize with rituximab leading to increased cytotoxicity and T-cell tumor infiltration. The clinical evaluation showed that the combined treatment was totally safe. The overall response rate was 50%, PET-negative complete response rate 37%, and remission is still ongoing in 2/4 of responding patients (median follow-up 26 months, range 11–47). Notably, following the combined therapy all patients showed induction/enhancement of T-cell responses by CD107 degranulation or IFNγ ELISPOT assay against patient-specific tumor IGHV sequences.Conclusions:These results represent the proof-of-principle on the effectiveness of unloaded IFN-DC in inducing durable clinical responses and promoting induction of tumor-specific peripheral T cells, thus suggesting the occurrence of an effective endogenous antitumor vaccination. The overall findings indicate that some unique properties of IFN-DC can be successfully exploited to induce/enhance antitumor responses, thus representing a valuable antitumor strategy for novel and more effective combination therapies in patients with cancer.

CT-PET scan showing clinical response of Pt#3

Iraq's health care system has gradually declined after several decades of wars, terrorism, and UN economic sanctions. The Oncology Unit at Children's Welfare Teaching Hospital (CWTH) in Baghdad was lacking basic facilities and support. To address this shortcoming, a humanitarian and educational partnership was established between CWTH and Sapienza University of Rome (SUR).We investigated the outcomes of 80 online and 16 onsite educational sessions and 142 teleconsultation sessions from 2006 to 2014. We also determined the outcomes of pathology reviews by SUR of 1216 tissue specimens submitted by CWTH from 2007 until 2019 for second opinions. The primary outcomes were discordance, concordance, and changes among clinical diagnoses and pathology review findings. The measures included the frequency of teleconsultation and tele-education sessions, the topics discussed in these sessions, and the number of pathology samples requiring second opinions.A total of 500 cases were discussed via teleconsultations during the study period. The median patient age was 7 years (range, 24 days to 16·4 years), and the cases comprised 79 benign tumors, 299 leukemias, 120 lymphomas, and 97 solid tumors. The teleconsultation sessions yielded 27 diagnostic changes, 123 confirmed diagnoses, and 13 equivocal impacts. The pathology reviews by SUR were concordant for 996 (81·9%) cases, discordant for 186 (15·3%), and inconclusive for 34 (2·8%). The major cause of discordance was inadequate immunohistochemical staining. The percentage of discordance markedly decreased over time (from 40% to 10%). The cause of the improvement is multifactorial: training of two CWTH pathologists at SUR, better immunohistochemical staining, and the ongoing clinical and pathologic telemedicine activities. The partnership yielded 12 publications, six posters, and three oral presentations by CWTH investigators.The exchange of knowledge and expertise across continental boundaries meaningfully improved the diagnoses and management of pediatric cancer at CWTH.

PURPOUSE: to assess the clinicopathologic features of a poorly defined subset of diffuse large b-cell lymphoma, which is characterized by the secretion of an IgM paraprotein (IgMs-DLBCL). EXPERIMENTAL DESIGN: multicentre, retrospective, and comparative study to analyse with an integrated diagnostic approach the IgMs-DLBCL subset.RESULTS: Overall, 650 DLBCL were enrolled: 102 had an associated serum IgM and 56 other paraproteins (OP), the remaining 492 cases were the reference group (REF). IgMs-DLBCL were characterized by older age, advanced stage, dissemination to extra-nodal organs, elevated LDH and poor PS. As a result, the majority of IgMs had a high IPI, NCC-IPI and CNS-IPI score (p

Purpose: This study was aimed at evaluating the feasibility, safety, immunologic and clinical responses in patients with follicular lymphoma treated with monocyte-derived dendritic cells generated in the presence of IFNα and GM-CSF (IFN-DC) in combination with low doses of rituximab. Patients and Methods: Firstly, we analyzed in vitro and in vivo the immunologic properties of IFN-DC against follicular lymphoma. Thus, we performed a phase I trial in 8 patients with refractory and relapsed follicular lymphoma based on sequential intranodal injections of low-dose of rituximab and unloaded IFN-DC and report the safety, clinical, and immunologic results of the enrolled patients. Results: Preclinical studies indicated that IFN-DC can synergize with rituximab leading to increased cytotoxicity and T-cell tumor infiltration. The clinical evaluation showed that the combined treatment was totally safe. The overall response rate was 50%, PET-negative complete response rate 37%, and remission is still ongoing in 2/4 of responding patients (median follow-up 26 months, range 11–47). Notably, following the combined therapy all patients showed induction/enhancement of T-cell responses by CD107 degranulation or IFNγ ELISPOT assay against patient-specific tumor IGHV sequences. Conclusions: These results represent the proof-of-principle on the effectiveness of unloaded IFN-DC in inducing durable clinical responses and promoting induction of tumor-specific peripheral T cells, thus suggesting the occurrence of an effective endogenous antitumor vaccination. The overall findings indicate that some unique properties of IFN-DC can be successfully exploited to induce/enhance antitumor responses, thus representing a valuable antitumor strategy for novel and more effective combination therapies in patients with cancer.

Myopericytoma is a benign neoplasm presenting cells with different shapes, from oval to spindle, and myoid showing with perivascular growth, which frequently originates from the skin and soft tissues of distal extremities, trunk, head, and neck regions. These tumors rarely have been reported to occur in visceral sites. There is only one case of myopericytoma showing pulmonary involvement with multiple nodules. Although most myopericytomas behave in a benign manner, some cases of malignant myopericytoma arising in both superficial soft tissue and visceral locations have been described. We describe two cases of pulmonary tumors with myopericytoma-like features.

Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is the most common entity of mature T-cell neoplasms. PTCL-NOS generally has an aggressive behavior and is often refractory to standard therapy. Only a few cases of PTCL with aberrant expression of B-cell antigens have been reported so far. This phenotypic aberrancy may lead to misdiagnosis as B-cell non-Hodgkin lymphomas and eventual inappropriate patient management, whereas in an accurately diagnosed PTCL, the presence of CD20 may appear as an appealing therapeutic target. In this setting, response to anti-CD20 monoclonal antibody in combination with chemotherapy has been poorly explored. We describe the case of a 59-year-old male diagnosed by a pathological and molecular approach as PTCL-NOS with aberrant co-expression of the B-cell antigens CD20 and CD79a, which proved non-responsive to the addition of rituximab to standard polychemotherapy. This case highlights that the presence of CD20 in PTCL may be misleading in the diagnosis and also act as a lure for the clinician to adopt a rituximab-based treatment, the effectiveness of which is undefined as the molecular mechanisms underlying B-cell marker expression in PTCL.

Cytokine production is essential for follicular dendritic cell (FDC) maintenance and organization of germinal centres. In follicular lymphoma, FDCs are often disarrayed and may lack antigens indicative of terminal differentiation. We investigated the in situ distribution of cells producing lymphotoxin-beta (LTB), lymphotoxin-alpha (LTA), and tumour necrosis factor-alpha (TNFA) transcripts in human reactive lymph nodes and in follicular lymphomas with follicular or diffuse growth pattern. LTB was the cytokine most abundantly produced in germinal centres. LTB was present in nearly 90% of germinal centre cells whereas LTA and TNFA were detected in 30 and 50%, respectively. Moreover, the amount of LTB expressed in reactive germinal centre cells was 80-fold higher than that of LTA and 20-fold higher than that of TNFA. LTB-positive cells were more numerous in the germinal centre dark zone, whereas expression of the FDC proteins CD21, CD23, VCAM, and CXCL13 was more intense in the light zone. Tumour cells of follicular lymphomas produced less LTB than reactive germinal centre cells. The results of the in situ study were confirmed by RT-PCR; LTB was significantly more abundant in reactive lymph nodes than in follicular lymphoma, with the lowest values detected in predominantly diffuse follicular lymphoma. In neoplastic follicles, low production of LTB by tumour B cells was associated with weaker expression of CD21+/CD23+ by FDCs. Our findings detail for the first time the distribution of LTA-, LTB-, and TNFA-producing cells in human reactive germinal centres and in follicular lymphoma. They suggest the possibility that impaired tumour-cell LTB production may represent a determinant of FDC phenotype loss and for defective follicular organization in follicular lymphoma.

Background Early onset (≤50y) colorectal carcinomas (EO-CRCs) are increasing in incidence according to epidemiological data. We investigated clinical-pathological, molecular features and outcomes of 62 left sided EO-CRCs (EOLS-CRCs) and compared them to a group of late onset (≥65) LS-CRCs (LOLS-CRCs). Materials and methods Samples were evaluated for pathological features and microsatellite instability (MSI). Overall survival (OS), disease free survival (DFS) and disease specific survival were evaluated in both groups. Results Five out 62 (8%) EOLS-CRCs showed MSI phenotype. Interestingly these cases were aged 26–39y. Most EOLS-CRCs present at advanced stage and this was statistically significant when compared to LOLS-CRCs. OS was better in EOLS-CRCs whilst DFS showed a worst profile in EOLS-CRCs either in low and high stages even though young patients were treated more often with adjuvant chemotherapy compared to older ones at the same disease stage. Conclusions Most EOLS-CRCs are sporadic non Lynch, microsatellite stable (MSS) CRCs. Our data show that when compared with LOLS-CRCs the early group represents an aggressive disease with worst outcome underlining a possible different carcinogenic pathway.

BACKGROUND: In 2014 we identified a new subset of DLBCL, defined as "IgM-secreting" (Cox MC & Di Napoli A , PLOS One 2014). This was characterised by poor prognostic features and outcome as well as frequent central nervous (CNS) system localizations. Furthermore, IgM-secretion, was an independent prognostic factor in multivariate analysis. Here we report on the largest series of IgM-secreting-DLBCL, from a multicentre Italian study. METHODS: The observational and biological study was approved by the Ethical Committee of the AUO Sant'Andrea, Italy. Enrolment criteria were: DLBCL with an associated IgM paraprotein diagnosed between 1st January 2010 and 31st December 2018 (IgM-secreting). Data were collected both prospectively and retrospectively from 17 Centres participating in the study. In addition, histopathology samples were centrally revised for immunohistochemistry (IHC) and FISH analyses. The control group (CTRL) consisted in a series of consecutive DLBCL, without an associated IgM-paraprotein (diagnosed between 01/01/2013 and 30/06/2016, enrolled in the Lymphoma Registry of the Lazio region (ReLLi Network). Last follow-up was carried out on 31st December 2019. RESULTS: 569 DLBCL cases were enrolled: 102 (17.9%) were IgM-secreting; 48 (8.4%) had a non-IgM paraprotein (IgA, IgG, or other), and 414 (72.7%) had no associated paraprotein (CTRL). IgM-secreting cases within the consecutive DLBCL patients enrolled in the ReLLi Registry were 41/466 (8.8%, 95CI 6.4-11.7%) while non IgM-paraprotein DLBCL cases were 11/466 (2.4%, 95CI 1.2-4.2%). The median level of IgM paraprotein was 17gr/L (range: 60 (p=.001); 2] advanced stage (pUNL (p=.008) ; 5] ≥2 Extra-nodal sites involved (p60, B-symptoms, bulky disease, IPI >low risk and IgM-secreting IgM showed a worse survival (all with p CONCLUSION: Our data confirm that IgM-secreting DLBCL: 1) represents a sizable proportion of non-DH DLBCL; 2) have poor prognostic features and 3) have mostly a non-GC phenotype. Furthermore, IgM secretion appears to be an independent prognostic factor for both PFS and OS. Studies to define the biological features of this new subset are ongoing. Disclosures Cantonetti: Mundipharma: Consultancy; Takeda: Consultancy; Vifor: Consultancy; Roche: Consultancy. Re:BerGenBio ASA: Research Funding. Abruzzese:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bms: Honoraria.

Stage II colon cancer (CC) represents a challenging scenario for the choice of adjuvant chemotherapy; here, histologic factors need to be weighed up to establish the risk of recurrence. Tumor budding (TB) has recently been indicated as a confident predictor of clinical outcome in CC. Likewise, the presence of poorly differentiated clusters (PDCs) in a tumor has been pointed out as a leading criterion of a tumor grading system. Our aim was to evaluate in patients with stage II CC the relationship between these features and clinical outcome. The study included 174 cases of stage II CC; histopathologic parameters such as TB, PDCs, microsatellite instability (MSI), and CDX2 expression were analyzed. There were 107 (70.9%), 32 (21.2%), and 12 (7.9%) TB scored 1, 2, and 3 respectively; 113 (72.9%), 30 (19.4%), and 12 (7.7%) tumors showed grade 1, 2, and 3 PDCs respectively. A high-MSI was detected in 32 cases (18.4%) while CDX2 was negative in 20 (11.5%) tumor samples. In the whole study population, only the TB was found to be associated with disease-specific survival (P = 0.01). No parameter apart from age (P = 0.04) was a significant prognostic factor for overall survival (P

Expression of the latent membrane protein-1 (LMP1) of Epstein-Barr virus (EBV) was investigated in 153 cases of EBV+ classic Hodgkin lymphoma (cHL); 120 cases were pediatric patients (< 14years of age) from Iraq, and 33 cases were adult patients from Italy. We describe for the first time the presence of LMP1 protein in EBV-encoded RNA (EBER)-negative follicular dendritic cells (FDCs) of reactive germinal centers (GC) associated with EBV+ cHL. Presence of LMP1+ GCs was independent of geographic region and age of patients. Variable numbers of reactive GCs were present in 22.2% of cases (34 of 153), whereas LMP1 staining of FDCs was present in about a third of cases (10 of 34) with reactive GC. Most cases with LMP1+ GC were mixed-cellularity (MC) subtype, but some nodular sclerosis (NS) was also present. GC cells with LMP1+ FDCs were surrounded by numerous EBV-infected cells which were positive for EBER, LMP1, and CD30. Double immunolocalization analysis revealed that LMP1 was associated with CD63, an exosomal marker, and with CD21. The possibility is discussed that peri-follicular EBV-infected cells release LMP1 protein, perhaps through exosomes, and that the protein is then captured by FDCs and is presented to EBER-negative GC B cells.

An accurate diagnosis of clinically distinct subgroups of aggressive mature B cell lymphomas is crucial for the choice of proper treatment. Presently, precise recognition of these disorders relies on the combination of morphological, immunophenotypical, and cytogenetic/molecular features. The diagnostic workup in such situations implies the application of costly and time-consuming analyses, which are not always required, since an intensified treatment option is reasonably reserved to fit patients. The Italian Group of Haematopathology proposes herein a practical algorithm for the diagnosis of aggressive mature B cell lymphomas based on a stepwise approach, aimed to select cases deserving molecular analysis, in order to optimize time and resources still assuring the optimal management for any patient.

An accurate diagnosis of clinically distinct subgroups of aggressive mature B cell lymphomas is crucial for the choice of proper treatment. Presently, precise recognition of these disorders relies on the combination of morphological, immunophenotypical, and cytogenetic/molecular features. The diagnostic workup in such situations implies the application of costly and time-consuming analyses, which are not always required, since an intensified treatment option is reasonably reserved to fit patients. The Italian Group of Haematopathology proposes herein a practical algorithm for the diagnosis of aggressive mature B cell lymphomas based on a stepwise approach, aimed to select cases deserving molecular analysis, in order to optimize time and resources still assuring the optimal management for any patient.

Sinonasal melanomas are rare neoplasms with poor prognosis that may harbor KIT and NRAS genes mutations. Molecular alterations represent possible targets of tailored therapeutic approaches. We describe the case of a 74-year-old patient with primary melanoma of the nasal cavity. Mutational analysis of KIT demonstrated a point missense mutation D820G in exon 17. This represents, to our knowledge, the first case of sinonasal melanoma harboring this specific KIT mutation. Although KIT mutations confer sensibility to thyrosine-kinase inhibitor, it has been proved that this is strongly dependent on the region in which this alteration occurs. Thus it seems very important to perform an accurate gene mutational analysis to provide a biological rationale to the tailored therapy.

Epstein-Barr virus (EBV)-related lymphoproliferative disorders are relatively common in Iraqi children. Burkitt lymphoma (BL) accounted for 40% of lymphoma cases. The mean age of 125 BL cases was 5.9 ± 3.1 years, and the male-to-female ratio was 3.6:1. Clinical presentation was abdominal in 66% and head and neck in 34%. Bone marrow involvement was higher (P 0.001) in children with head and neck disease. Tumor cells had MYC translocation (96%) and were CD20

Epstein-Barr virus (EBV)-related lymphoproliferative disorders are relatively common in Iraqi children. Burkitt lymphoma (BL) accounted for 40% of lymphoma cases. The mean age of 125 BL cases was 5.9 ± 3.1 years, and the male-to-female ratio was 3.6:1. Clinical presentation was abdominal in 66% and head and neck in 34%. Bone marrow involvement was higher (P

The regulatory role of dopamine (DA) in endocrine, cardiovascular and renal functions has been extensively studied and used for clinical purposes. More recently DA has been indicated as a regulatory molecule for immune cells and malignant cell proliferation. We assessed the expression and the functional role DA, DA receptors, and transporters in primary small cell lung cancer (SCLC). By HPLC DA plasma levels were more elevated in SCLC patients in comparison with NSCLC patients and healthy controls. SCLC cell expressed DA D1- and D2-like receptors and membrane and vesicular transporters at protein and mRNA levels. We also investigated the effects of independent D1- or D2-like receptor stimulation on SCLC cell cultures. DA D1 receptor agonist SKF38393 induced the increase of cAMP levels and DARPP-32 protein expression without affecting SCLC growth rate. Cell treatment with the DA D1 receptor antagonist SCH23390 inhibited SKF38393 effects. In contrast, the DA D2 receptor agonist quinpirole (10 μM) counteracted, in a dose and time dependent way, SCLC cell proliferation, it did not affect cAMP levels and decreased phosphorylated AKT that was induced by DA D2 receptor antagonist sulpiride. However, in only one SCLC line, stimulation of DA D2 receptor failed to inhibit cell proliferation in vitro. This effect was associated to the existence of rs6275 and rs6277 polymorphisms in the D2 gene. These results gave more insight into DA control of lung cancer cell behavior and suggested the existence of different SCLC phenotypes.

Hodgkin lymphoma (HL) associated with Warthin tumor (WT) is extremely rare, accounting for only 3 cases of classical HLs. Here, we report for the first time the occurrence of a nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) involving the lymphoid stroma of a WT of the parotid gland. Pathogenesis of WT is controversial, with both a nodal and a parenchymal possible origin. On the other hand, extranodal involvement by HLs is uncommon. In our case, the coexistence of a WT and of a NLPHL within its stroma and in cervical lymph node emphasizes the importance of a careful evaluation of the lymphoid tissue in WT in order to exclude the possibility of an associated lymphoid malignancy.

Objectives: Carcinomas of the left colon represent a neoplasm of older patients (late onset), but epidemiologic evidence has been showing an increasing incidence in patients 50 years or younger (early onset). In this study, we investigate pathologic and molecular features of early- and late-onset carcinoma of the left colon. Methods: We selected 22 patients 50 years or younger and 21 patients 70 years or older with left-sided colorectal carcinoma (CRC). All samples were evaluated for pathologic features, microsatellite instability, and KRAS and BRAF mutations. Moreover, both groups were analyzed to identify CpG island methylator phenotype features and assessed with restriction landmark genome scanning (RLGS) to unveil differential DNA methylation patterns. Results: Early-onset patients had advanced pathologic stages compared with late-onset patients (P = .0482). All cases showed a microsatellite stable profile and BRAF wild-type sequence. Early-onset patients (43%) more frequently had mutations at KRAS codon 12 compared with late-onset patients (14%) (P =.0413). RLGS showed that patients younger than 50 years who had CRC had a significantly lower percentage of methylated loci than did patients 70 years or older (P = .04124), and differential methylation of several genomic loci was observed in the two groups. Conclusions: Our results suggest that left-sided CRCs may present differential patterns of aberrant DNA methylation when they are separated by age.

The importance of T cell-dependent immune responses in achieving long-term cure of chemoimmunotherapy-treated cancer patients is underscored by the recently described "vaccinal effect" exerted by therapeutic mAbs. In accordance, pre- and post-therapy peripheral blood lymphopenia represents a well-established negative prognostic factor in DLBCL. We analyzed the phenotypic and functional (IFNγ production, and Granzyme B (GrzB) cytotoxic granule marker expression) profile of peripheral blood T lymphocyte subsets ("conventional" CD4+ and CD8+, FOXP3+CD25bright Treg, and "innate-like" CD56+) in DLBCL patients at diagnosis, and assessed the long-term impact of R-CHOP chemoimmunotherapy, in a prospective study. At diagnosis, DLBCL patients showed lower lymphocyte counts, due to selective decrement of CD4+ T (including Treg) and B lymphocytes. While all T cell subsets transiently decreased during therapy, CD4+ T cell and Treg remained significantly lower than controls, up to 1 year after R-CHOP. Phenotypically skewed profile of CD4+ and CD8+ T cell subsets associated with higher frequencies of IFNγ+ and GrzB+ cells at diagnosis, that transiently decreased during therapy, and re-attained persistently elevated levels, till up to 1 year after therapy. Differently, the pre-therapy elevated levels of circulating monocytes, and of plasma IL-6 and IL-10 rapidly normalized upon R-CHOP. In sum, we describe a quantitatively and functionally altered status of the peripheral blood T cell compartment in DLBCL patients at diagnosis, that persists long-term after tumor eradication, and it is only transiently perturbed by R-CHOP chemoimmunotherapy. Moreover, data suggest the association of selected T cell functional features with DLBCL phenotype, and with therapy outcome.

e20628 Background: Hyperprogressive disease (HPD) incidence ranges from 8% to 21% in patients treated with anti-PD-1/PD-L1 mAbs in NSCLC and is associated with poor survival. Previously published data underlined a link between HPD across different cancers types and specific genetic alterations, such as MDM2 amplification and EGFR aberrations. We present a single-center cohort of patients with NSCLC and PD-L1 > 50% treated with 1st-line pembrolizumab. We performed NGS, IHC and FISH analysis to evaluate genetic correlations with the clinical phenotype. Methods: Clinical data from 20 patients with diagnosis of advanced NSCLC treated with 1stline immunotherapy pembrolizumab were retrospectively collected. HPD was defined by Time to Treatment Failure ≤2 months and raising in Tumor Burden ≥50% compared with basal CT-scan. MDM2 amplification was investigated by FISH on FFPE tissue sections using the MDM2/CON12 break apart FISH Probe. Positive cases were defined as those with > 10% positive tumor cells. We performed IHC for MDM2 protein on FFPE tissue sections. The staining was semiquantitatively graded for the intensity as: 0, negative; 1+, weak positive; 2+, moderately positive; 3+, strongly positive, and for the extent as 0– < 1% (negative), 1–50% (focal), and > 50% (diffuse). We also performed NGS analysis (FoundationOne CDX, Foundation Medicine Inc.) on 324 preidentified genes. Results: We identified 5 cases of HPD; all five cases showed MDM2amplification by FISH analysis and a focal protein expression by IHC with the strongest nuclear staining observed in the cases showing a higher degree of MDM2 amplification (8/9 dots) and a weaker expression in those with a lower MDM2amplification (4/5 dots). NGS analysis showed MDM2amplification in 1/5 HPD patient and a loss of CDKN2A/B in 4/5 patients. None of the non-HPD patients had IHC expression of MDM2 or amplification of the gene. Among the non-HPD patients no genetic alterations regarding MDM2 and/or CDKN2A/B were found on NGS analysis. Conclusions: Our data suggest a potential role of CDKN2A/B gene loss and alteration of MDM2 on the establishment of HPD in NSCLC patients treated with immunotherapy. Because the HPD logic is not yet clear, more data is needed to better understand the link between this genomic signature and the development of HPD.

Background Classical Hodgkin lymphoma (cHL) in children is often associated with EBV infection, more commonly in developing countries. Procedure Here we describe the histological, immunohistochemical, and molecular features of 57 cases of HL affecting Iraqi children under 14 years of age. Results Histologically, 51 cases were classified as cHL of Mixed Cellularity and Nodular Sclerosis subtypes (MC = 69%; NS = 31%), and 6 cases as Nodular Lymphocyte Predominant HL (NLP-HL). EBV infection of H/RS cells was demonstrated in 44 of 51 cases of cHL (86%), and was more common in MC than in NS (97% vs. 63%; P = 0.0025). The immunophenotypic profile of H/RS cells was similar in MC and NS, and was not influenced by EBV infection; H/RS cells were consistently positive for PAX-5 and to a lesser degree for other B cell markers including CD20/CD79a, OCT-2, and BOB-1. Clonal IGH rearrangements were detected in 14 of 38 cHL (37%), with no significant difference between MC and NS cases, and with no association with the EBV status. Oligoclonal/monoclonal TCRγ rearrangements were present in 28 of 38 cases (74%), suggestive of restricted T cell responses. Conclusions Our findings indicate that cHL occurring in Iraqi children is characterized by immunohistochemical and molecular features undistinguishable from those present in cHL occurring elsewhere in the world. Moreover, the high incidence of EBV-infected H/RS cells and frequent occurrence of restricted T cell responses might be indicative of a defective local immune response perhaps related to the very young age of the children. Pediatr Blood Cancer 2013;60:2068–2072. © 2013 Wiley Periodicals, Inc.

We used optimized immunohistochemistry (IHC) with the D5F3 antibody for detection of tumours in a prospective study of 307 pulmonary adenocarcinomas. Cases positive by IHC (1+, 2+, 3+) were further investigated by fluorescent in situ hybridization (FISH). Of 307 cases, 22 (7.2%) were moderately intensely positive (2+/3+); 18 of these (82%) were also positive by FISH. Of the four IHC-positive/FISH-negative cases, one was unsuitable for FISH and three had abnormalities of the ALK gene. All cases with weak reactivity with D5F3 (1+) were FISH-negative. The FISH positive/IHC-positive cases with moderately intense reactivity had the typical clinicopathologic features of ALK-positive patients (younger age, p

Primary rectal MALT lymphoma is a very rare entity among extranodal MALT lymphomas and its therapeutic management has not been standardized. Different approaches including surgery, chemotherapy and radiotherapy have been proposed in the last decades. There have been reports on complete responses after anti-Helicobacter pylori therapy, also in patients without serological or histological evidence of Helicobacter pylori infection. In our patient we obtained a complete response with anti-Helicobacter pylori therapy and a disease-free survival of 34 months. Endoscopic ultrasound was useful for diagnosis and follow-up. Although the mechanisms that determined this response remain a matter of debate, anti-Helicobacter pylori therapy can be considered as first-line therapy in stage IE, nonbulky primary rectal MALT lymphoma. Endoscopic ultrasound could play a relevant role in the management of this rare condition.

To investigate prevalence and age-distribution of ALK- or ROS1-translocated adenocarcinomas in patients ≤50 years of age.Paraffin sections of pulmonary adenocarcinoma were analyzed for ALK (637 cases) and ROS1 (376 cases) translocations using FISH, and for EGFR mutations (789 cases) using mutant-specific Real-Time PCR.ALK or ROS1 fusions were detected in 55 of 637 cases (8.6%). When patients were stratified for age, it was found that six of six cases (100%) of lung adenocarcinoma diagnosed in patients30 years of age were translocated for ALK (4 cases) or ROS1 (2 cases). With the increase of age, there was a gradual decrease in the percentage of positive cases. In fact, ALK-translocated or ROS1-translocated cases were 5 of 17 cases (29%) in the 31-40 years age-group, 6 of 46 cases (13%) in the 41-50 years age-group, and 38 of 568 cases (7.0%) in patients older than 50 years. The six patients30 years of age (5F/1M), including two pediatric patients (≤18 years old), presented with stage IV disease, were never or light smoker, and had no family history of pulmonary tumours. Four of the six patients, were treated with crizotinib and had an objective response.Our findings provide evidence that ALK or ROS1 translocations are crucial events in tumourigenesis of pulmonary adenocarcinoma of very young patients, including pediatric patients.

Purpose: We have developed a novel DC population differentiated from human monocytes in the presence of GM-CSF and IFN-alpha (IFN-DC), which is highly efficient in internalizing tumour cell antigens and in the cross-priming of CD8+T cells, promoting efficient anti-lymphoma response. Preclinical results prompted us to start a clinical trial of IFN-DC combined with low-dose Rituximab(RTX) in patients with Follicular lymphoma (FL), which is an indolent, immune-responsive, but still incurable cancer. Patients and Methods: In October 2014 we started a phase I clinical trial (EudraCT: 2013-003158-25) testing safety, immunogenicity and systemic clinical responses of low-dose RTX combined with autologous IFN-DC, administered by intra-nodal injections. Patients features: age 18-75y, indolent FL, relapsed or refractory (R/R), stage III-IV low tumour burden, with at least 1 superficial pathologic lymph node. One leukapheresis was scheduled at study entry for harvesting mononuclear cells. Eight cycles of RTX (5-20mg) and IFN-DC (30±10x106) were planned: the first 4 every two weeks, followed by other 4 cycles administered monthly. All injections were delivered with the aid of an ultrasound guide. Blood test for immune-responses and autoimmunity markers were scheduled at 0,+72d, +132 days ; +9; +18;+24 months. Results: as of the 31st July 2018, 8 patients have completed treatment and have been evaluated for response (median follow-up=24 months; range=8-45 mo). Median age of the study population was 60 years (range 27-76). All subjects had disseminated disease and enlarged lymph nodes or lesions greater than 1.5 cm at multiple sites available for local injections and monitoring for distant response. All patients were R/R after previous systemic therapy (median lines=2, range1-4) and 3/8 (37.5%) after auto-transplantation. The treatment was very well tolerated and manageable. No relevant adverse events were observed. Four out of 8 (50%) patients had objective response (OR) with 3/8(37.5%) attaining complete remission (CR); 2/8 (25%) are in stable disease at +8 and +25 months; 2/8 (25%), who did not achieve OR, progressed at +8 and +14 respectively. One patient progressed 4 months after achieving CR (Figure1). In all patients who achieved OR or who are in stable disease, induction of a tumour specific immune response was observed. Conclusions: local RTX and IFN-DC combined therapy was effective in eliciting in 50% of R/R FL with low tumour burden systemic and durable responses, which were associated with induction of specific anti-FL T cell responses. Disclosures No relevant conflicts of interest to declare.

700 Background: The standard of care for locally advanced rectal cancer (RC) is neoadjuvant chemoradiotherapy (CRT) with 5FU aimed to tumor downstaging prior to radical resection but there is a wide spectrum of responses to it, from none to complete. The aim of this project is to validate top candidate genes previously identified by microarray studies as prognostic and predictive classifiers of locally advanced RC, using the NanoString nCounter Platform. Methods: Two cohorts of RC patients were identified according to tumor regression grade (TRG) of AJCC Staging Manual (7th) system: 1) good prognosis: patients that after neoadjuvant CRT obtained TRG0 (complete response); 2) poor prognosis: patients with TRG1 (moderate response), patients with TRG2 (minimum response rate) and patients with TRG3 (poor response). Pre-treatment biopsies tissues from ten TRG0 patients (ID TRG0bio) and thirteen TRG1-3 patients (ID TRG1-3bio) were macro-dissected from FFPE sections, RNA isolated and used for expression profiling of a 305 genes custom code set consisting of 12 normalizer genes, 101 prognostic genes (markers of stemness, invasiveness, proliferation, drug-resistance), 192 predictive genes (involved in response to 5-FU, to radiation therapy and in the response to CRT). All samples were normalized using the geometric mean of the housekeeper genes. P-values were calculated by student’s t-test and was consider significance if was less than 0.05. Gene-specific RNA expression profiles were compared using Spearman's correlation. The heat map was generated using MeV 4.9.0. Results: When we compared TRG0bio to TRG1-3bio tissues, among the 305 genes assayed, changes in expression levels of 18 genes (SSB, GAPDH, TXNDC9, DUT, PKM, STAT1,SLC28A3, DAG1, TYMS, FERMT1, ARNT,SLC6A6, SMAD3, SCRN1, POU5F1, GNG4, PDRG1, ATP5E) were statistically significant. Conclusions: Results suggest that TRG0 RC is characterized by distinct molecular events compared TRG1-3 disease. Next steps will be: to amplify sample size, to understand signaling pathways of top differentially expressed genes and to validate prospectively our gene signature.

OBJECTIVES T2238C ANP (atrial natriuretic peptide) gene variant has been associated with increased cardiovascular risk in humans and with a significant pharmacogenomic effect on cardiovascular disease outcome in hypertensive patients. We investigated the impact of T2238C ANP gene variant on oxidative stress production, cell proliferation and migration, angiogenesis and vascular remodeling in human umbilical vein endothelial cells in vitro. METHODS Differentially expressed genes in human umbilical vein endothelial cells exposed to either wild-type (TT2238) or mutant (CC2238) alpha-ANP were characterized by real time-PCR-macroarray analysis using human oxidative stress, angiogenesis and matrix arrays. Reactive oxygen species (ROS) production was determined by dihydroethidium and by evaluation of dichlorofluorescein content. NADPH oxidase gp91phox subunit was investigated by western blotting. Endothelial cell proliferation, migration and tube formation were characterized both in the presence and in the absence of NADPH oxidase inhibition. RESULTS Compared with TT2238, CC2238 alpha-ANP altered the redox state balance of the cells in a more significant manner, favoring ROS production and reducing antioxidative stress response. Gene expression of molecules involved in atherogenesis and vascular remodeling was enhanced. In contrast to TT2238 peptide, CC2238 was unable to stimulate cell proliferation and it markedly inhibited endothelial cell tube formation. NADPH oxidase inhibition restored the cell proliferative properties under CC2238 peptide exposure. CONCLUSION CC2238 alpha-ANP led to ROS accumulation and increased expression of genes related to atherosclerosis and vascular remodeling in human umbilical vein endothelial cells. As a consequence of NADPH-derived ROS, blunted endothelial cell proliferation and impaired endothelial cell tube formation were observed. These in-vitro effects may link the T2238C alpha-ANP variant to enhanced susceptibility to vascular damage in vivo.

The regulatory role of dopamine (DA) in endocrine, cardiovascular and renal functions has been extensively studied and used for clinical purposes. More recently DA has been indicated as a regulatory molecule for immune cells and malignant cell proliferation. We assessed the expression and the functional role DA, DA receptors, and transporters in primary small cell lung cancer (SCLC). By HPLC DA plasma levels were more elevated in SCLC patients in comparison with NSCLC patients and healthy controls. SCLC cell expressed DA D1- and D2-like receptors and membrane and vesicular transporters at protein and mRNA levels. We also investigated the effects of independent D1- or D2-like receptor stimulation on SCLC cell cultures. DA D1 receptor agonist SKF38393 induced the increase of cAMP levels and DARPP-32 protein expression without affecting SCLC growth rate. Cell treatment with the DA D1 receptor antagonist SCH23390 inhibited SKF38393 effects. In contrast, the DA D2 receptor agonist quinpirole (10 μM) counteracted, in a dose and time dependent way, SCLC cell proliferation, it did not affect cAMP levels and decreased phosphorylated AKT that was induced by DA D2 receptor antagonist sulpiride. However, in only one SCLC line, stimulation of DA D2 receptor failed to inhibit cell proliferation in vitro. This effect was associated to the existence of rs6275 and rs6277 polymorphisms in the D2 gene. These results gave more insight into DA control of lung cancer cell behavior and suggested the existence of different SCLC phenotypes.

Natural Killer (NK) cells are a key component of tumor immunosurveillance and thus play an important role in rituximab-dependent killing of lymphoma cells via an antibody-dependent cellular cytotoxicity (ADCC) mechanism. We evaluated the phenotypic and functional assets of peripheral blood NK cell subsets in 32 newly-diagnosed diffuse large B-cell lymphoma (DLBCL) patients and in 27 healthy controls. We further monitored long-term modifications of patient NK cells for up to 12 months after rituximab-based immunochemotherapy. At diagnosis, patients showed a higher percentage of CD56dim and CD16+ NK cells, and a higher frequency of GrzB+ cells in CD56dim, CD56bright, and CD16+ NK cell subsets than healthy controls. Conversely, DLBCL NK cell killing and interferon γ (IFNγ) production capability were comparable to those derived from healthy subjects. Notably, NK cells from refractory/relapsed patients exhibited a lower "natural" cytotoxicity. A marked and prolonged therapy-induced reduction of both "natural" and CD16-dependent NK cytotoxic activities was accompanied by the down-modulation of CD16 and NKG2D activating receptors, particularly in the CD56dim subset. However, reduced NK cell killing was not associated with defective lytic granule content or IFNγ production capability. This study firstly describes tumor-associated and therapy-induced alterations of the systemic NK cell compartment in DLBCL patients. As these alterations may negatively impact rituximab-based therapy efficacy, our work may provide useful information for improving immunochemotherapeutic strategies.

This study investigates the expression of thrombospondin-1 in papillary carcinoma of the thyroid and the role of Met–HGF interaction in TSP-1 regulation. In tissue sections, immunostaining for TSP-1 was associated with the fibrous tumour stroma, and showed areas of marked intensity adjacent to the basal membrane of tumour cells. Investigation of TSP-1 RNA expression showed that, in 10 of 14 cases, TSP-1 mRNA levels were significantly lower in tumour tissue (20–100% reduction; mean = 55% ± 20; p = 0.001) than in the corresponding normal thyroid. Since it has been reported that HGF can downregulate the expression of TSP-1 mRNA, TSP-1 mRNA levels were measured in 7 primary cultures, established from thyroid papillary carcinomas (TPC), and in 1 TPC cell line prior to, or after, stimulation with HGF. A marked decrease in TSP-1 mRNA levels was observed after HGF stimulation in 6/7 primary cultures (60–100% decrease (mean = 79 ± 15%; p = 0.006) and in the TPC cell line; moreover, the decrease in TSP-1 mRNA in cell extracts was associated with a decrease in TSP-1 protein in culture supernatants. The HGF activity was dose dependent and the downregulation lasted for at least 48 h after stimulation. The high-level expression of Met protein, the high-affinity receptor for HGF, in most cases of papillary carcinoma of the thyroid is consistent with the possibility that HGF–Met interaction plays a crucial role in regulating the expression of TSP-1 in this tumour type. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Hashimoto's thyroiditis (HT) represents the most common cause of hypothyroidism and nonendemic goiter, but its clinical and pathological heterogeneity opens the question if this disease should be more properly considered as a spectrum of different thyroid conditions rather than as a single nosological entity. In this study, we analysed 133 cases of HT for the expression of galectin-3, a lectin molecule involved in malignant transformation, apoptosis and cell cycle control. An unexpected expression of galectin-3 was demonstrated in a subset of HT together with the presence of HBME-1, c-met and cyclin-D1 that are also involved in malignant transformation and deregulated cell growth. Furthermore, a loss of allelic heterozygosity in a specific cancer-related chromosomal region was demonstrated in some HT harbouring galectin-3-positive follicular cells, by using laser capture microdissection. On the basis of the morphological and molecular findings we identified four subsets of HT: (a) HT with classic features of chronic autoimmune thyroiditis; (b) HT associated to hyperplastic/adenomatous lesions; (c) HT harbouring thyroid cancer precursors; (d) HT associated to unequivocal thyroid microcarcinomas. Our findings provide a well-substantiated morphological and molecular demonstration that HT may include a spectrum of different thyroid conditions ranging from chronic autoimmune thyroiditis to thyroiditis triggered by specific immune-response to cancer-related antigens.

Hypomethylation has been reported to be responsible for the activation of several oncogenes. The possibility that hypomethylation is involved in the regulation of MET transcription was investigated through the analysis of the methylation status of one CpG island containing 43 CpGs in six cases of papillary carcinoma, in the corresponding normal thyroid tissue, and in two cases of hyperplastic goitre. Evidence of methylation was not found in any of the analysed CpG.

Met protein, the high affinity receptor for hepatocyte growth factor (HGF), was highly expressed by the tumour cells of 64 well-differentiated papillary carcinomas of the thyroid. The p145 mature form and the p170 precursor form of the protein were both isolated from the tumours. Enhanced expression of Met protein was associated with a 9.5 ± 5-fold increase in MET RNA transcript levels, suggesting increased transcription of the gene. In the same tumours, the levels of RNA transcripts for hypoxia inducible factor-1 (HIF-1), a potent stimulator of met gene transcription, were 4.5 ± 3-fold higher than those present in the surrounding normal thyroid tissues. HIF-1 is generally induced by hypoxia. Histological features suggestive of a hypoxia were observed in 37 of 50 tumours and included coagulative necrosis, psammoma bodies, cystic changes, intratumoural haemorrhage, and hyalinization of the fibrous stroma. Immunostaining for Met protein was particularly intense in some cells located at the tumour periphery which were characterized by an invasive phenotype. Microdissection of tumour cell nests from the invading front revealed that the levels of RNA transcripts for MET/HIF were higher than in the centre of the tumour in four of nine cases. Taken together, the findings of this study suggest that HIF-1, perhaps driven by hypoxia, may be one of the factors leading to the increased transcription of met gene in papillary carcinoma and that this event is often more pronounced at the tumour periphery. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Chemotherapy for cancer could have negative effects on HIV-1 dynamic in addition to the effects on immunological status. At the moment few data are available about the effects of chemotherapy on systemic HIV-1 replication, but the effects on the central nervous system, considered an independent compartment for viral replication, has never been investigated. We studied 19 HIV-1-infected patients with non-Hodgkin lymphoma (NHL) treated concomitantly with chemotherapy and highly active antiretroviral therapy (HAART) to evaluate HIV-1 replication and assess virological response to HAART in cerebrospinal fluid during chemotherapy. No patients were diagnosed with lymphoma involvement of the central nervous system. In 18 of 19 patients an HIV-1 load below 200 copies/ml was obtained in cerebrospinal fluid (CSF) during treatment. A correlation between plasma and CSF HIV-1 RNA levels was present at baseline, and was confirmed at the fourth cycle of chemotherapy. A significant decline in the mean difference between plasma and CSF HIV-1 RNA load was observed when comparing the value at the first cycle of chemotherapy with subsequent cycles. HAART even during chemotherapy allows us to obtain an effective control of HIV-1 infection in CSF in patients affected by NHL. In HIV-infected patients with NHL, the contemporaneous administration of HAART and chemotherapy is advisable to obtain a suppression of HIV-1 replication in CNS compartment during the potentially immunosuppressing effect of cancer treatment.

The up-regulation of "tissue" transglutaminase (TG2) gene has been shown to occur in various pathologies and can lead to severe liver injury; however, its role in the onset of liver damage has not yet been clarified. To address this issue, we have used two experimental settings: carbon tetrachloride (CCl(4))-induced liver injury in wild-type and TG2 knockout mice; and liver biopsies obtained from a large cohort of hepatitis C virus (HCV)-infected patients. Mice lacking TG2 failed to clear the hepatic necrotic tissue formed in response to prolonged CCl(4) exposure (5 weeks) and 60% of them died before the end of the treatment. By contrast, wild-type mice were able to recover after the toxic insult. CCl(4)-treated TG2 null mice showed a derangement of the hepatic lobular architecture and a progressive accumulation of extracellular matrix (ECM) components and inflammatory cells which were not observed in the liver of control animals. Consistent with this protective role, we observed that TG2 levels were much higher (up to 15-fold) during the initial stages of liver fibrosis in HCV-infected individuals (METAVIR = F2) compared with uninfected controls, in which the enzyme protein localized in the hepatocytes facing the periportal infiltrate. By contrast, the enzyme levels decreased in the advanced stages (METAVIR = F3 and F4) and their localization was limited to the ECM. Our data demonstrate that TG2 plays a protective role in the liver injury by favoring tissue stability and repair.