Breast implant associated anaplastic large cell lymphoma: Evidence for an efficient diagnostic workup.

INTRODUCTION: During the last few years it has been shown that an anaplastic T cell lymphoma can develop as a rare and late sequelae of implant-based breast reconstruction. This malignancy was recognized in the 2017 by WHO and named breast implant associated anaplastic large T cell lymphoma (BIA-ALCL). BIA-ALCL usually presents as abundant effusion around the implant, thus, in addition to cytology smears, its diagnosis also requires immunohistochemistry, T cells clonality and cytometry. Due to the increasing attention of clinicians, it is likely that the number of the BIA-ALCL suspected cases will grow in the future, implying the necessity of a reliable and cost-effective diagnostic procedure. METHODS: To achieve this goal, we retrospectively analyzed the results of laboratory investigations performed at our Institute (Fondazione IRCCS Istituto Nazionale dei Tumori Milan, Italy) on 44 effusions obtained from 31 women suspected for BIA-ALCL. RESULTS: Through cytology, eight out of 44 effusions showed the presence of BIA-ALCL cells. Lymphoma cells were than confirmed in seven samples by immunohistochemistry and/or T cell clonality and/or cytometry. Overall, cytology showed 100% sensitivity, 97% specificity and positive and negative predictive values of 87.5% and 100% respectively. Further analyses were particularly useful in effusions showing small percentages of BIA-ALCL cells. Moreover, an extended cytometric profile that can be applied when fast confirmation of the cytologic result is required was also identified. CONCLUSIONS: Our results evidenced a central role of cytopathology in the management of BIA-ALCL suspected effusions and suggested that further laboratory investigations might be applied only in cases showing atypical/activated lymphoid cells through cytology.

Systemic lupus erythematosus reactivation after chemoimmunotherapy in preexisting autoimmune disease.

The use of immune checkpoint inhibitors (ICIs) offers new possibilities in modern treatment of many types of cancers. Few data regarding safety and efficacy of ICIs are available, and are mainly from retrospective studies and case reports rather than from clinical trials, in the context of preexisting autoimmune disease, mainly due to the risk of severe toxicity. We present an unexpected life-threatening reactivation of systemic lupus erythematosus after one dose of chemo-immunotherapy with pembrolizumab for oligometastatic non-small-cell lung cancer. We analyze data coming from the published literature in this setting and discuss the risk-benefit balance of immunotherapy in patients with preexisting severe autoimmune disease.

Dose-adjusted EPOCH and rituximab for the treatment of double expressor and double-hit diffuse large B-cell lymphoma: impact of TP53 mutations on clinical outcome.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, including one-third of cases overexpressing MYC and BCL2 proteins (double expressor lymphoma, DEL) and 5-10% of patients with chromosomal rearrangements of MYC, BCL2 and/or BCL-6 (double/triple-hit lymphomas, DH/TH). TP53 mutations are detected in 20- 25% of DEL. We report the efficacy of dose-adjusted EPOCH and rituximab (DA-EPOCH-R) in a series of 122 consecutive patients, including DEL (n=81, 66%), DEL-MYC (n=9, 7%), DEL-BCL2 (n=13, 11%), or high-grade lymphomas (DH/TH) (n=19, 16%). Central nervous system (CNS) prophylaxis included intravenous methotrexate (n=66), intrathecal chemotherapy (IT) (n=40) or no prophylaxis (n=16). Sixty-seven patients (55%) had highintermediate or high International Prognostic Index (IPI) and 30 (25%) had high CNS-IPI. The 2-year progression-free survival (PFS) and overall survival (OS) for the entire study population were 74% and 84%, respectively. There was a trend for inferior OS for DH/TH (2-year OS: 66%, P=0.058) as compared to all the others. The outcome was significantly better for the IPI 0-2 versus IPI 3-5 (OS: 98% vs. 72%, P=0.002). DA-EPOCH-R did not overcome the negative prognostic value of TP53 mutations: 2-year OS of 62% versus 88% (P=0.036) were observed for mutated as compared to wild-type cases, respectively. Systemic CNS prophylaxis conferred a better 2-year OS (94%) as compared to IT or no prophylaxis (76% and 65%, respectively; P=0.008). DA-EPOCH-R treatment resulted in a favorable outcome in patients with DEL and DEL with single rearrangement, whereas those with multiple genetic alterations such as DEL-DH/TH and TP53 mutated cases still have an inferior outcome.

Fifteen-year follow-up of relapsed indolent non-Hodgkin lymphoma patients vaccinated with tumor-loaded dendritic cells.

We previously published the results of a pilot study showing that vaccination with tumor-loaded dendritic cells (DCs) induced both T and B cell response and produced clinical benefit in the absence of toxicity in patients with relapsed, indolent non-Hodgkin lymphoma (iNHL). The purpose of the present short report is to provide a 15-year follow-up of our study and to expand the biomarker analysis previously performed. The long-term follow-up highlighted the absence of particular or delayed toxicity and the benefit of active immunization with DCs loaded with autologous, heat-shocked and UV-C treated tumor cells in relapsed iNHL (5-year and 10-year progression-free survival (PFS) rates: 55.6% and 33.3%, respectively; 10-year overall survival (OS) rate: 83.3%). Female patients experienced a better PFS (p=0.016) and a trend towards a better OS (p=0.185) compared with male patients. Of note, we observed a non-negligible fraction of patients (22%) who experienced a long-lasting complete response. In a targeted gene expression profiling of pre-treatment tumor biopsies in 11 patients with available formalin-fixed, paraffin-embedded tissue, we observed that KIT, ATG12, TNFRSF10C, PBK, ITGA2, GATA3, CLU, NCAM1, SYT17 and LTK were differentially expressed in patients with responder versus non-responder tumors. The characterization of peripheral monocytic cells in a subgroup of 14 patients with available baseline blood samples showed a higher frequency of the subset of CD14++CD16+ cells (intermediate monocytes) in patients with responding tumors. Since in patients with relapsed iNHL the available therapeutic options are often incapable of inducing a long-lasting complete remission and can be sometimes characterized by intolerable toxicity, we think that the encouraging results of our long-term follow-up analysis represent a stimulus to further investigate the role of active vaccination in this specific setting and in earlier lines of therapy and to explore novel combinatorial strategies encompassing other innovative immunotherapy agents, such as immune-checkpoint inhibitors.

A three-gene signature based on MYC, BCL-2 and NFKBIA improves risk stratification in diffuse large B-cell lymphoma.

Recent randomized trials focused on gene expression-based determination of the cell of origin in diffuse large B-cell lymphoma could not show significant improvements by adding novel agents to standard chemoimmunotherapy. The aim of this study was the identification of a gene signature able to refine current prognostication algorithms and applicable to clinical practice. Here we used a targeted gene expression profiling panel combining the Lymph2Cx signature for cell of origin classification with additional targets including MYC, BCL-2 and NFKBIA, in 186 patients from 2 randomized trials (discovery cohort) (NCT00355199 and NCT00499018). Data were validated in 3 independent series (2 large public datasets and a real-life cohort). By integrating the cell of origin, MYC/BCL-2 double expressor status and NFKBIA expression, we defined a 3-gene signature combining MYC, BCL-2 and NFKBIA (MBN-signature), which outperformed the MYC/BCL-2 double expressor status in multivariate analysis, and allowed further risk stratification within the germinal center B-cell/unclassified subset. The high-risk (MBN Sig-high) subgroup identified the vast majority of double hit cases and a significant fraction of Activated B-Cell-derived diffuse large B-cell lymphomas. These results were validated in 3 independent series including a cohort from the REMoDL-B trial, where, in an exploratory ad hoc analysis, the addition of bortezomib in the MBN Sig-high subgroup provided a progression free survival advantage compared with standard chemoimmunotherapy. These data indicate that a simple 3-gene signature based on MYC, BCL-2 and NFKBIA could refine the prognostic stratification in diffuse large B-cell lymphoma, and might be the basis for future precision-therapy approaches.

CDKN2A deletion is a frequent event associated with poor outcome in patients with peripheral T-cell lymphoma not otherwise specified (PTCL-NOS).

Nodal peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) remains a diagnosis encompassing a heterogenous group of PTCL cases not fitting criteria for more homogeneous subtypes. They are characterized by a poor clinical outcome when treated with anthracycline-containing regimens. A better understanding of their biology could improve prognostic stratification and foster the development of novel therapeutic approaches. Recent targeted and whole exome sequencing studies have shown recurrent copy number abnormalities (CNAs) with prognostic significance. Here, investigating 5 formalin-fixed, paraffin embedded cases of PTCL-NOS by whole genome sequencing (WGS), we found a high prevalence of structural variants and complex events, such as chromothripsis likely responsible for the observed CNAs. Among them, CDKN2A and PTEN deletions emerged as the most frequent aberration, as confirmed in a final cohort of 143 patients with nodal PTCL. The incidence of CDKN2A and PTEN deletions among PTCL-NOS was 46% and 26%, respectively. Furthermore, we found that co-occurrence of CDKN2A and PTEN deletions is an event associated with PTCL-NOS with absolute specificity. In contrast, these deletions were rare and never co-occurred in angioimmunoblastic and anaplastic lymphomas. CDKN2A deletion was associated with shorter overall survival in multivariate analysis corrected by age, IPI, transplant eligibility and GATA3 expression (adjusted HR =2.53; 95% CI 1.006-6.3; p=0.048). These data suggest that CDKN2A deletions may be relevant for refining the prognosis of PTCL-NOS and their significance should be evaluated in prospective trials.

Fluorescence in situ hybridization (FISH) provides estimates of minute and interstitial BAP1, CDKN2A, and NF2 gene deletions in peritoneal mesothelioma.

The aim of this study was to assess the performance of fluorescence in situ hybridization (FISH) in identifying the copy number profiles of the three key peritoneal mesothelioma tumor suppressor genes BAP1, CDKN2A, and NF2, with particular emphasis on minute homozygous deletions, a copy number abnormality recently unveiled at the 3p21 (BAP1) chromosomal region using high-throughput methods. FISH was performed on 75 formalin-fixed-paraffin-embedded peritoneal mesotheliomas and recognized two types of monoallelic loss (monosomy, and hemizygous deletion) and two types of biallelic loss (canonical homozygous deletion with a complete loss of FISH signal and homozygous deletion with diminished signal). Diminished FISH signals revealed deletions occurring within the genomic region covered by the gene-specific probe and affected all three tumor suppressors. BAP1 homozygous deletions with diminished signal outnumbered canonical homozygous deletions (13 vs 3): conversely, canonical homozygous deletions were prevalent for CDKN2A (2 vs 14). Diminished signal homozygous deletion was the only pattern of biallelic loss observed for NF2 (2 cases). Hemizygous deletion mainly affected BAP1 (21 vs 6), while monosomy was prevalent for CDKN2A (14 vs 7) and particularly for NF2 where it accounts for all monoallelic losses. FISH/immunohistochemistry (BAP1, CDKN2A, and MTAP) correlation showed that all homozygous deletions, including those with diminished signals, resulted in a null BAP1 and CDKN2A immunophenotype but only canonical CDKN2A homozygous deletions resulted in MTAP loss of expression. BAP1 hemizygous deletion, but not monosomy, was also invariably associated with loss of protein expression whereas neither type of CDKN2A monoallelic loss correlated with p16 or MTAP immunohistochemistry. Array comparative genomic hybridization performed on a spontaneously emerging peritoneal mesothelioma cell line provided support for the interpretation of the FISH patterns and allowed us to extend the number of chromatin remodeling factors involved in mesothelioma to SETD7 and PCGF5, two previously unreported genes.

Molecular Signatures for Combined Targeted Treatments in Diffuse Malignant Peritoneal Mesothelioma.

Background-There are currently no effective therapies for diffuse malignant peritoneal mesothelioma (DMPM) patients with disease recurrence. In this study, we investigated the biology of DMPM by analyzing the EGFR family, Axl, and MET, in order to assess the presence of cross-talk between these receptors, suggesting the effectiveness of combined targeted treatments in DMPM. Method-We analyzed a series of 22 naïve epithelioid DMPM samples from a single institute, two of which showed higher-grade malignancy ("progressed"). EGFR, HER2, HER3, Axl, and MET activation and expression were investigated by biochemical analysis, real-time PCR immunofluorescence, immunohistochemistry, next-generation sequencing, miRNA, and mRNA in situ hybridization. Results-In most DMPMs, a strong EGFR activation was associated with HER2, HER3, Axl, and MET co-activation, mediated mainly by receptor heterodimerization and autocrine-paracrine loops induced by the expression of their cognate ligands. Axl expression was downregulated by miRNA34a. Mutations in MET Sema domain were exclusively found in two "progressed" DMPMs, and the combined Axl and MET inhibition reduced cellular motility in a DMPM cell line obtained from a "progressed" DMPM. Conclusion-The results indicate that the coordinated activity of multiple cross-talks between RTKs is directly involved in the biology of DMPM, suggesting the combined inhibition of PIK3 and mTOR as an effective strategy that may be easily implemented in clinical practice, and indicating that the combined inhibition of EGFR/HER2 and HER3 and of Axl and MET deserves further investigation.

Clinical and molecular characteristics of lymphoplasmacytic lymphoma not associated with an IgM monoclonal protein: A multicentric study of the Rete Ematologica Lombarda (REL) network.

Lymphoplasmacytic lymphoma (LPL) is usually associated with a serum IgM paraprotein, corresponding to Waldenström's Macroglobulinemia (WM). Cases presenting with IgG or IgA, or without a monoclonal protein are extremely rare. We analyzed clinical characteristics, frontline treatment, and the outcome of 45 patients with non-IgM LPL, and compared them with a control group of WM patients. The median age was similar, with significantly higher prevalence of females in non-IgM LPL, than in WM patients (60% vs 39%, P = .016). Patients with non-IgM LPL more frequently presented with lymphadenopathies (53% vs 15%, P < .001), splenomegaly (22% vs 8%, P = .015) or extranodal involvement (20% vs 8%, P = .05). In non-IgM LPL a serum monoclonal protein and bone marrow infiltration were less common than in WM patients (69% and 84% of cases respectively, P < .001 for both comparisons). The MYD88 (L265P) mutation was found in 8/19 patients using allele-specific polymerase chain reaction. A CXCR4 mutation was found in 4/17 cases using Sanger. In 16 patients we performed targeted next-generation sequencing of genes MYD88, CXCR4, ARID1-A, KMT2D, NOTCH2, TP53, PRDM1, CD79B, TRAF3, MYBBP1A, TNFAIP3. Seven patients (44%) had a MYD88 mutation (S219C in one), four (25%) a CXCR4 mutation, three (19%) a KMT2D mutation, one (6%) a TP53 mutation and one (6%) a TRAF3 mutation. With a median follow-up of 55.7 months, 36 non-IgM LPL patients (80%) were treated. Non-IgM LPL patients received more frequently anthracycline-containing regimens, as compared with WM patients, who mainly received alkylating-based therapies. Five-year overall survival (OS) was 84%, similar to that of WM patients.

PD-L1, LAG3, and HLA-DR are increasingly expressed during smoldering myeloma progression.

Symptomatic multiple myeloma (MM) is a plasma cell neoplasm that represents the final stage of a continuum of clinical conditions that start from monoclonal gammopathy of unknown significance (MGUS), then transits in the more advance, but still asymptomatic, smoldering MM (SMM), with a final evolution in symptomatic MM. To investigate SMM microenvironment modifications, we studied 16 patients diagnosed at our hospital. Eight of them (group A) developed MM within 2 years from diagnosis while the others (group B) had stable SMM. Samples were bone marrow biopsies at diagnosis and after 2 years (± 4 months) and were analyzed by immunohistochemical analysis. Firstly, we found a significant increase in both CD4+ cells (11 vs 17%, p < 0.01) and CD8+ cells (15 vs 18%, p < 0.01) between diagnosis and at follow-up samples (whole cohort). This was associated to an increase in the CD4+/CD8+ ratio (0.74 vs 0.93, p < 0.01). Secondly, we discovered an increased expression of T cell inhibitory molecules during SMM evolution. In fact, plasma cell PD-L1 and microenvironment cell LAG3 expression increased from 1 to 12% (p = 0.03) and 4 to 10% (p = 0.04), respectively, from diagnosis to follow-up. Also, plasma cells and microenvironment cells HLA-DR expression augmented during SMM evolution from 7 to 10% (p = 0.04) and 29 to 39% (p = 0.01), respectively. When comparing group A vs group B, we found an increased CD68-KP1+ cell infiltration in favor of group B at diagnosis (23 vs 28%, p = 0.01) and a greater plasma cell infiltration at follow-up (50 vs 26%, p < 0.01). Our findings suggest how immune escape mechanisms appear earlier during multiple myeloma evolution, and that LAG3 could be a possible immunologic target in this setting.

Pervasive mutations of JAK-STAT pathway genes in classical Hodgkin lymphoma.

Dissecting the pathogenesis of classical Hodgkin lymphoma (cHL), a common cancer in young adults, remains challenging because of the rarity of tumor cells in involved tissues (usually <5%). Here, we analyzed the coding genome of cHL by microdissecting tumor and normal cells from 34 patient biopsies for a total of ∼50 000 singly isolated lymphoma cells. We uncovered several recurrently mutated genes, namely, STAT6 (32% of cases), GNA13 (24%), XPO1 (18%), and ITPKB (16%), and document the functional role of mutant STAT6 in sustaining tumor cell viability. Mutations of STAT6 genetically and functionally cooperated with disruption of SOCS1, a JAK-STAT pathway inhibitor, to promote cHL growth. Overall, 87% of cases showed dysregulation of the JAK-STAT pathway by genetic alterations in multiple genes (also including STAT3, STAT5B, JAK1, JAK2, and PTPN1), attesting to the pivotal role of this pathway in cHL pathogenesis and highlighting its potential as a new therapeutic target in this disease.

Multicystic mesothelioma: Operative and long-term outcomes with cytoreductive surgery and hyperthermic intra peritoneal chemotherapy.

BACKGROUND: Multicystic peritoneal mesothelioma (MCPM) is an extremely rare disease with 40-50% rate of recurrence after surgical debulking. Due to the recurrent nature of the disease, the option of cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) was offered for this condition. In the present study we aimed to describe the outcomes of this strategy in a single center cohort. METHODS: We retrospectively reviewed a prospectively collected database of all patients with MCPM that underwent the combined procedure in our center. Clinical presentation, operative procedures and outcomes were reviewed. RESULTS: Between August 1997 and October 2017, 19 patients with MCPM underwent 20 cytoreduction and HIPEC procedures in our center. The majority of the patients were females (n = 17, 89%), and mean age was 42, as reported in other series. Disease extent, as measured by mean peritoneal carcinomatosis index (PCI) was 15.5 ± 9.9 and total number of procedures performed 6.7 ± 2.6. Major complications occurred in 3 (15%) patients, with no perioperative mortality. After a median of follow-up of 69 months (range 4-220) all patients were alive and 4 patients had recurrence (21%). Patients with high PCI (defined by median PCI) had shorter recurrence free survival (RFS) than patient with low PCI (mean RFS = 106.4 ± 6.6 for high PCI vs. 125.6 ± 34.1 for low PCI, p = 0.03). CONCLUSION: Cytoreduction and HIPEC offer low recurrence rate and prolonged mean RFS for patients with MCPM. The combined procedure can be offered with acceptable morbidity in specialized centers.

Epithelioid peritoneal mesothelioma: a hybrid phenotype within a mesenchymal-epithelial/epithelial-mesenchymal transition framework.

The aim of this study was to reconsider the biological characteristics of epithelioid malignant peritoneal mesothelioma (E-MpM) in the light of new concepts about epithelial mesenchymal transition and mesenchymal epithelial reverse transition (EMT/MErT) and the role of epigenetic reprogramming in this context. To this end we profiled surgical specimens and derived cells cultures by a number of complementary approaches i.e. immunohistochemistry, immunofluorescence, in situ hybridization, biochemistry, pluripotent stem cell arrays, treatments with cytokines, growth factors and specific inhibitors.The analyses of the surgical specimens showed that i) EZH2 is expressed throughout the spectrum of MpM, ii) that E-MpM (including the high-grade undifferentiated form) are characterised by c-MYC and miRNA 17-5p expression, and iii) that progression to sarcomatoid MpM is dictated by EMT regulators. They also showed that E-MpM expressed c-MET and are enriched in E- and P-cadherins- and VEGFR2-expressing CSCs, thus strongly supporting a role for MErT reprogramming in endowing E-MpM tumour cells with stemness and plasticity, and hence with a drug resistant phenotype. The cell culture-based experiments confirmed the stemness traits and plasticity of E-MpM, and support the view that EZH2 is a druggable target in this tumor.

The Role of Ki-67 and Pre-cytoreduction Parameters in Selecting Diffuse Malignant Peritoneal Mesothelioma (DMPM) Patients for Cytoreductive Surgery (CRS) and Hyperthermic Intraperitoneal Chemotherapy (HIPEC).

BACKGROUND: We conducted a prognostic analysis of preoperative parameters and Ki-67 determination to develop selection criteria for cytoreductive surgery (CRS) and HIPEC in patients with diffuse malignant peritoneal mesothelioma (DMPM). METHODS: DMPM patients treated with CRS and HIPEC at NCI of Milan participated in this study. Multivariate analysis was conducted using Cox proportional hazard model and conditional inference tree method to select independent predictors of overall survival (OS) from the followings pre-cytoreduction parameters: age, sex, ECOG performance status, Charlson comorbidity index, previous systemic chemotherapy, CA-125, histological subtype (epithelioid vs. biphasic/sarcomatoid), Ki-67 (determined with immunohistochemistry), and peritoneal cancer index (PCI). RESULTS: A total of 117 patients (male/female: 67/50) with median age of 60.5 (range 22-75) years were included. Eighty-three patients had ECOG performance status = 0, median Charlson comorbidity index was 4 (range 2-9), and 102 cases had epithelioid subtype. Median Ki-67 was 5 % (range 1-60). Ninety-four (80.3 %) cases were optimally cytoreduced. The Cox analysis identified Ki-67, PCI, and histological subtype as independent prognosticators of OS. Conditional inference tree method identified three prognostic subsets: (I) Ki-67 ≤ 9 %; (II) Ki-67 > 9 % and PCI ≤ 17; and (III) Ki-67 > 9 % and PCI > 17. The median OS for subsets I, II, and III were, 86.6, 63.2, and 10.3 months, respectively. CONCLUSIONS: Ki-67 is a powerful prognosticator that allows, along with PCI, and histological subtype, a good prediction of OS in patients with DMPM. Patients with Ki-67 > 9 % and PCI > 17 are unlikely to benefit from the procedure and should be considered for other treatment protocols.

Immunohistochemical Evaluation of Minichromosome Maintenance Protein 7 (MCM7), Topoisomerase IIα, and Ki-67 in Diffuse Malignant Peritoneal Mesothelioma Patients Using Tissue Microarray.

PURPOSE: Immunohistochemistry and tissue microarray (TMA) were used to perform a prognostic analysis of markers related to cell proliferation in diffuse malignant peritoneal mesothelioma (DMPM). METHODS: Clinicopathologic data were extracted from a prospectively collected database containing cases of peritoneal mesothelioma treated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy in the National Cancer Institute of Milan from 1995 to 2013. Eighty-one DMPM patients were recruited and their tissue samples were used to construct TMAs. We evaluated the immunoexpressions of markers related to cell proliferation-topoisomerase IIα, minichromosome maintenance protein 7 (MCM7), and Ki-67-and then conducted a multivariate Cox model to identify the predictors of overall survival (OS) and progression-free survival (PFS) among the following parameters: age, sex, Eastern Cooperative Oncology Group (ECOG) performance status, baseline serum albumin, Charlson Comorbidity Index, previous systemic chemotherapy, histological subtype (epithelioid vs. biphasic/sarcomatoid), peritoneal cancer index, completeness of cytoreduction (CC), and proliferative biological markers. RESULTS: The rates of high/intermediate immunoreactivity were 95 % for topoisomerase IIα and 90 % for MCM7, and the median Ki-67 labeling index was 5 %. The independent predictors of OS were baseline serum albumin >3.5 g/dl, CC, and Ki-67 >5 %, whereas those for PFS were an ECOG performance status of 0, baseline serum albumin >3.5 g/dl, Charlson Comorbidity Index >3, previous systemic chemotherapy, morbidity G3-5, and Ki-67 >5 %. The remaining biological markers were not associated with outcome. CONCLUSIONS: Ki-67 was found to be a new powerful determinant of outcome. Patients with a Ki-67 labeling index >5 % carry a very poor prognosis and do not benefit from the combined procedure. Further studies should be conducted to confirm the present data.

HSPH1 inhibition downregulates Bcl-6 and c-Myc and hampers the growth of human aggressive B-cell non-Hodgkin lymphoma.

We have shown that human B-cell non-Hodgkin lymphomas (B-NHLs) express heat shock protein (HSP)H1/105 in function of their aggressiveness. Here, we now clarify its role as a functional B-NHL target by testing the hypothesis that it promotes the stabilization of key lymphoma oncoproteins. HSPH1 silencing in 4 models of aggressive B-NHLs was paralleled by Bcl-6 and c-Myc downregulation. In vitro and in vivo analysis of HSPH1-silenced Namalwa cells showed that this effect was associated with a significant growth delay and the loss of tumorigenicity when 10(4) cells were injected into mice. Interestingly, we found that HSPH1 physically interacts with c-Myc and Bcl-6 in both Namalwa cells and primary aggressive B-NHLs. Accordingly, expression of HSPH1 and either c-Myc or Bcl-6 positively correlated in these diseases. Our study indicates that HSPH1 concurrently favors the expression of 2 key lymphoma oncoproteins, thus confirming its candidacy as a valuable therapeutic target of aggressive B-NHLs.

Alternate clonal dominance in richter transformation presenting as extranodal diffuse large B-cell lymphoma and synchronous classic Hodgkin lymphoma.

OBJECTIVES: Richter transformation (RT) represents the rare occurrence of a secondary aggressive lymphoma in the setting of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). METHODS: Here we describe the peculiar case of a patient with trisomy 12+ and atypical (CD5+, CD23-) CLL/SLL who developed a two-step RT with complex morphologic and molecular features. RESULTS: Molecular analysis of a CLL/SLL population detected two different immunoglobulin rearrangement patterns corresponding to a main peak and a minor peak. Transformation took place both as gastric diffuse large B-cell lymphoma and as a synchronous bone marrow classic Hodgkin lymphoma with the same immunoglobulin rearrangement pattern corresponding to the minor peak detected in CLL/SLL at diagnosis. During chemotherapy, progression occurred as axillary nodal involvement by a CD5+ high-grade lymphoma with an immunoglobulin rearrangement pattern corresponding to the main CLL peak. CONCLUSIONS: In this case, the elaborate clinical and molecular picture may be correlated to an alternate dominance of two distinct clonal populations probably influenced by therapeutic and environmental factors.

Alternative activation of human plasmacytoid DCs in vitro and in melanoma lesions: involvement of LAG-3.

Plasmacytoid dendritic cells (pDCs) at tumor sites are often tolerogenic. Although pDCs initiate innate and adaptive immunity upon Toll-like receptor (TLR) triggering by pathogens, TLR-independent signals may be responsible for pDC activation and immune suppression in the tumor inflammatory environment. To identify molecules that are potentially involved in alternative pDC activation, we explored the expression and function of lymphocyte activation gene 3 (LAG-3) in human pDCs. In this report, we showed the expression of LAG-3 on the cell surface of a subset of circulating human pDCs. LAG-3+ pDCs exhibited a partially mature phenotype and were enriched at tumor sites in samples from melanoma patients. We found that LAG-3 interacted with major histocompatibility complex class II (MHC-II) to induce TLR-independent activation of pDCs with limited IFNα and enhanced IL-6 production. This in vitro cytokine profile of LAG-3-activated pDCs paralleled that of tumor-associated pDCs analyzed ex vivo. By confocal microscopy, LAG-3+ pDCs detected in melanoma-invaded lymph nodes (LNs) stained positive for IL-6 and preferentially localized near melanoma cells. These results suggest that LAG-3-mediated activation of pDCs takes place in vivo at tumor sites, and it is in part responsible for directing an immune-suppressive environment.

Transcriptional profiling of melanoma sentinel nodes identify patients with poor outcome and reveal an association of CD30(+) T lymphocytes with progression.

Sentinel lymph nodes set the stance of the immune system to a localized tumor and are often the first site to be colonized by neoplastic cells that metastasize. To investigate how the presence of neoplastic cells in sentinel lymph nodes may trigger pathways associated with metastatic progression, we analyzed the transcriptional profiles of archival sentinel node biopsy specimens obtained from melanoma patients. Biopsies from positive nodes were selected for comparable tumor infiltration, presence or absence of further regional node metastases, and relapse at 5-year follow-up. Unsupervised analysis of gene expression profiles revealed immune response to be a major gene ontogeny represented. Among genes upregulated in patients with progressing disease, the TNF receptor family member CD30/TNFRSF8 was confirmed in biopsy specimens from an independent group of patients. Immunohistochemical analysis revealed higher numbers of CD30(+) lymphocytes in nodes from progressing patients compared with nonprogressing patients. Phenotypic profiling demonstrated that CD30(+) lymphocytes comprised a broad population of suppressive or exhausted immune cells, such as CD4(+)Foxp3(+) or PD1(+) subpopulations and CD4(-)CD8(-) T cells. CD30(+) T lymphocytes were increased in peripheral blood lymphocytes of melanoma patients at advanced disease stages. Our findings reinforce the concept that sentinel nodes act as pivotal sites for determining progression patterns, revealing that the presence of CD30(+) lymphocytes at those sites associate positively with melanoma progression.