Diffuse Large B Cell Lymphoma Arising in Patients with Preexisting Hodgkin Lymphoma.

The metachronic onset of diffuse large B-cell lymphoma (DLBCL) after classic Hodgkin lymphoma (cHL) is a rare event affecting patients' outcomes. However, although several studies have investigated the prognostic role of this event, little is known about a hypothetical common origin of the two different neoplastic cells. AIMS: To investigate a possible relationship between DLBCL and cHL, in this retrospective study of 269 patients with newly diagnosed cHL treated at Bari University Hospital (Italy) between 2007 and 2020, we analyzed data from 4 patients (3 male and 1 female) with cHL who subsequently developed DLBCL. METHODS: Gene expression profile analysis, assessed by NanoString Lymphoma Subtype Assay, was performed to identify the cell of origin in the DLBCL cases, in addition to Hans's algorithm. RESULTS: Using Hans's algorithm, all DLBCL cases showed a germinal center-B-Cell subtype. The gene expression profile evaluated by the NanoString Lymphoma Subtype Assay revealed two cases of the GCB molecular subtype, while the others were unclassified. After first-line chemotherapy, 1 patient achieved complete remission, 3 were non-responders (2 died of lymphoma within 6 months, whereas the other achieved complete remission after autologous and allogeneic stem cell transplantation and is still alive). CONCLUSIONS: The origin of the second neoplastic cell in patients with DLBCL with a previous history of cHL remains controversial, although the different immunophenotypic characteristics suggest that it may mainly arise de novo in a subject with a possible individual predisposition to develop lymphoid neoplasms.

NR1H3 (LXRα) is associated with pro-inflammatory macrophages, predicts survival and suggests potential therapeutic rationales in diffuse large b-cell lymphoma.

The role of macrophages (Mo) and their prognostic impact in diffuse large B-cell lymphomas (DLBCL) remain controversial. By regulating the lipid metabolism, Liver-X-Receptors (LXRs) control Mo polarization/inflammatory response, and their pharmacological modulation is under clinical investigation to treat human cancers, including lymphomas. Herein, we surveyed the role of LXRs in DLBCL for prognostic purposes. Comparing bulk tumors with purified malignant and normal B-cells, we found an intriguing association of NR1H3, encoding for the LXR-α isoform, with the tumor microenvironment (TME). CIBERSORTx-based purification on large DLBCL datasets revealed a high expression of the receptor transcript in M1-like pro-inflammatory Mo. By determining an expression cut-off of NR1H3, we used digital measurement to validate its prognostic capacity on two large independent on-trial and real-world cohorts. Independently of classical prognosticators, NR1H3high patients displayed longer survival compared with NR1H3low cases and a high-resolution Mo GEP dissection suggested a remarkable transcriptional divergence between subgroups. Overall, our findings indicate NR1H3 as a Mo-related biomarker identifying patients at higher risk and prompt future preclinical studies investigating its mouldability for therapeutic purposes.

Ovarian Stem Cells (OSCs) from the Cryopreserved Ovarian Cortex: A Potential for Neo-Oogenesis in Women with Cancer-Treatment Related Infertility: A Case Report and a Review of Literature.

Cancer treatment related infertility (CTRI) affects more than one third of young women undergoing anti-cancer protocols, inducing a premature exhaustion of the ovarian reserve. In addition to ovarian suppression by GnRHa, oocyte and cortex cryopreservation has gained interest in patients with estrogen-sensitive tumors for whom the hormonal burst to prompt the multiple follicular growth could provide a further pro-life tumor pulsing. On the other hand, cortex reimplantation implies a few drawbacks due to the unknown consistency of the follicles to be reimplanted or the risk of reintroducing malignant cells. The capability of ovarian stem cells (OCSs) from fresh ovarian cortex fragments to differentiate in vitro to mature oocytes provides a tool to overcome these drawbacks. In fact, since ovarian cortex sampling and cryopreservation is practicable before gonadotoxic treatments, the recruitment of OSCs from defrosted fragments could provide a novel opportunity to verify their suitability to be expanded in vitro as oocyte like cells (OLCs). Here, we describe in very preliminary experiments the consistency of an OSC population from a single cryopreserved ovarian cortex after thawing as well as both their viability and their suitability to be further explored in their property to differentiate in OLCs, thus reinforcing interest in stemness studies in the treatment of female CTRI.

The Effect of the Tumor Microenvironment on Lymphoid Neoplasms Derived from B Cells.

Lymphomas are characteristic tumors surrounded by an inflammatory microenvironment. The cells of the microenvironment are essential for the growth and survival of neoplastic cells and are recruited through the effect of cytokines/chemokines. Lymphomas include heterogeneous groups of neoplasms infiltrating various lymphoid structures which may arise from B lymphocytes, T lymphocytes, and natural killer (NK) cells at various stages of their differentiation state. In this review article, we analyze the literature data concerning the involvement of the tumor microenvironment (TME) in the progression of lymphomas and the recent advances in the analysis of microenvironment components in the most common forms: some mature B cell lymphoma neoplasms and classic Hodgkin lymphomas. The complex crosstalk between the TME and tumor cells led to the discovery of many mechanisms usable as molecular-targeted therapy through the control of diverse elements of the TME, varying from inhibitors of angiogenic cytokines and their receptors to the regulation of cells' activities and the novel immune checkpoint inhibitors.

Electronic case report forms generation from pathology reports by ARGO, automatic record generator for onco-hematology.

The unstructured nature of Real-World (RW) data from onco-hematological patients and the scarce accessibility to integrated systems restrain the use of RW information for research purposes. Natural Language Processing (NLP) might help in transposing unstructured reports into standardized electronic health records. We exploited NLP to develop an automated tool, named ARGO (Automatic Record Generator for Onco-hematology) to recognize information from pathology reports and populate electronic case report forms (eCRFs) pre-implemented by REDCap. ARGO was applied to hemo-lymphopathology reports of diffuse large B-cell, follicular, and mantle cell lymphomas, and assessed for accuracy (A), precision (P), recall (R) and F1-score (F) on internal (n = 239) and external (n = 93) report series. 326 (98.2%) reports were converted into corresponding eCRFs. Overall, ARGO showed high performance in capturing (1) identification report number (all metrics > 90%), (2) biopsy date (all metrics > 90% in both series), (3) specimen type (86.6% and 91.4% of A, 98.5% and 100.0% of P, 92.5% and 95.5% of F, and 87.2% and 91.4% of R for internal and external series, respectively), (4) diagnosis (100% of P with A, R and F of 90% in both series). We developed and validated a generalizable tool that generates structured eCRFs from real-life pathology reports.

Hodgkin Lymphoma: A Special Microenvironment.

Classical Hodgkin's lymphoma (cHL) is one of the most particular lymphomas for the few tumor cells surrounded by an inflammatory microenvironment. Reed-Sternberg (RS) and Hodgkin (H) cells reprogram and evade antitumor mechanisms of the normal cells present in the microenvironment. The cells of microenvironment are essential for growth and survival of the RS/H cells and are recruited through the effect of cytokines/chemokines. We summarize recent advances in gene expression profiling (GEP) analysis applied to study microenvironment component in cHL. We also describe the main therapies that target not only the neoplastic cells but also the cellular components of the background.

Psychological Distress in Outpatients With Lymphoma During the COVID-19 Pandemic.

Cancer patients are a population at high risk of contracting COVID-19 and, also of developing severe complications due to the infection, which is especially true when they are undergoing immunosuppressive treatment. Despite this, they had still to go to hospital to receive chemotherapy during lockdown. In this context, we have evaluated the psychological status of onco-hematological outpatients receiving infusion and not deferrable anti-neoplastic treatment for lymphoproliferative neoplasms, with the aim of both measuring the levels of post-traumatic symptoms, depression, and anxiety during the pandemic and also of investigating the perception of risk of potential nosocomial infection. The Impact of Event Scale-Revised (IES-R) and the Hospital Anxiety and Depression Scale (HADS) were administered to all patients. Moreover, patients were investigated about their worries regarding the impact of COVID-19 on their lives as onco-hematologic patients. Since the 2nd to the 29th April 2020 (during the first phase of the lockdown period in Italy), 77 outpatients were prospectively evaluated. They were diagnosed with non-Hodgkin's lymphoma, classical Hodgkin lymphoma, and Chronic lymphocytic leukemia/Small lymphocytic lymphoma. The mean age was 56.6 (range 22-85). We found that 36% of patients had anxiety (HADS-A), 31% depression (HADS-D), and 43% were above the cut-off for the HADS-General Scale; 36% fulfilled the diagnostic criteria for post-traumatic stress disorder (PTSD). Women and younger patients were found to be more vulnerable to anxiety and PTSD. The study firstly analyzes the psychological impact of the COVID-19 pandemic on the frail population of patients affected by lymphoproliferative neoplasms, to underly the importance of screening patients for emotional and distress conditions and then offering them psychological support.

The Tumor Microenvironment of DLBCL in the Computational Era.

Among classical exemplifications of tumor microenvironment (TME) in lymphoma pathogenesis, the "effacement model" resembled by diffuse large B cell lymphoma (DLBCL) implies strong cell autonomous survival and paucity of non-malignant elements. Nonetheless, the magnitude of TME exploration is increasing as novel technologies allow the high-resolution discrimination of cellular and extra-cellular determinants at the functional, more than morphological, level. Results from genomic-scale studies and recent clinical trials revitalized the interest in this field, prompting the use of new tools to dissect DLBCL composition and reveal novel prognostic association. Here we revisited major controversies related to TME in DLBCL, focusing on the use of bioinformatics to mine transcriptomic data and provide new insights to be translated into the clinical setting.

Should Tumor Infiltrating Lymphocytes, Androgen Receptor, and FOXA1 Expression Predict the Clinical Outcome in Triple Negative Breast Cancer Patients?

Tumor-infiltrating lymphocytes (TILs) are a valuable indicator of the immune microenvironment that plays the central role in new anticancer drugs. TILs have a strong prognostic role in triple negative breast cancer (TNBC). Little is known about the interaction with the androgen receptor (AR) and forkhead box A1 (FOXA1). We analyzed the relationships between TIL levels, AR, and FOXA1 expression and their clinical significance in TNBC patients. Further, we investigated their interaction with other biomarkers like programmed cell death ligand-1 (PD-L1), breast cancer type 1 susceptibility protein (BRCA1), poly (ADP-Ribose) polymerase 1 (PARP1), and Na+/H+ exchanger regulatory factor 1 (NHERF1). The expression of the proteins was evaluated by immunohistochemistry in 124 TNBC samples. TILs were performed adhering to International TILs Working Group 2014 criteria. Cox proportional hazards models were also used to identify risk factors associated with poor prognosis. Multivariate analysis identified TILs as independent prognostic factor of disease free survival (DFS; p = 0.045). A Kaplan-Meyer analysis revealed that the patients with high TILs had a better DFS compared to patients with low TILs (p = 0.037), and the phenotypes TILs-/AR+ and TILs-/FOXA1- had a worse DFS (p = 0.032, p = 0.001 respectively). AR was associated with FOXA1 expression (p = 0.007), and the tumors FOXA1+ presented low levels of TILs (p = 0.028). A poor DFS was observed for AR+/FOXA1+ tumors compared to other TNBCs (p = 0.0117). Low TILs score was associated with poor patients' survival, and TILs level in combination with AR or FOXA1 expression affected patient's clinical outcome. In addition, AR+/FOXA1+ phenotype identified a specific subgroup of TNBC patients with poor prognosis. These data may suggest new ways of therapeutic intervention to support current treatments.

Hierarchical clustering analysis identifies metastatic colorectal cancers patients with more aggressive phenotype.

A large percentage of metastatic colorectal cancer (mCRC) patients presents metastasis at the time of diagnosis. In the last years, great efforts have been made in the treatment of these patients with the identification of different phenotypes playing a key role in the definition of new systemic therapies. Unsupervised hierarchical clustering analysis (HCA) was performed considering the clinicopathological characteristics of 51 mCRCs. Using immunohistochemistry on tissue microarrays, we assessed the expression of ß-catenin, NHERF1, RASSF1A, TWIST1, HIF-1α proteins in tumors and paired liver metastases. We also analyzed RASSF1A methylation status on the samples of the same patients. HCA distinguished Group 1 and Group 2 characterized by different clinicopathological features. Group 1 was characterized by higher number of positive lymph nodes (p=0.0139), poorly differentiated grade (p<0.0001) and high extent of tumor spread (p=0.0053) showing a more aggressive phenotype compared to Group 2. In both Groups, we found a common "basal" condition with a higher level of nuclear TWIST1 (p<0.0001 and cytoplasmic ß-catenin (p<0.0001) in tumors than in paired liver metastases. Furthermore, the Group 1 was also characterized by RASSF1A hypermethylation (p<0.0001) and nuclear HIF-1α overexpression (p=0.0354) in paired liver metastases than in tumors. In conclusion, HCA identifies mCRC patients with a more aggressive phenotype. Moroever, our results support the important contribution to the progression of the disease of RASSF1A methylation and the oncogenic role of HIF-1α in these patients. These evidences, should provide relevant information concerning the biology of this tumor and, as a consequence, potential new systemic therapeutic approaches.

NHERF1 together with PARP1 and BRCA1 expression as a new potential biomarker to stratify breast cancer patients.

It has been recognized that Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) in breast cancer (BC) acts as a tumor suppressor or as an oncogenic protein, depending on its subcellular localization. This study aims to correlate NHERF1 expression to BRCA1 and PARP1 proteins, to investigate their relationship, and their biological and clinical significance. Using immunohistochemistry on tissue microarrays, we evaluated subcellular NHERF1, BRCA1 and PARP1 expression in 308 BCs including a subgroup (n=80) of triple negative BCs (TNBCs). Herein, we show that nuclear NHERF1 (nNHERF1) expression was significantly associated with nuclear BRCA1 (nBRCA1) expression (p=0.0008), and an association was also found between nuclear PARP1 (nPARP1) and nBRCA1 (p<0.0001). Cytoplasmic NHERF1 (cNHERF1) was correlated to nPARP1 (p<0.0001). Survival analyses showed that the patients with positive nPARP1 and nNHERF1 tended toward a shorter 5-year overall survival (OS) (p=0.057). In TNBCs, the association between nBRCA1 and nPARP1 was maintained (p<0.0001), and an association between nNHERF1 and nPARP1 was observed (p=0.010). Univariate analysis revealed that TNBCs with positive cNHERF1 and nPARP1 had a shorter 5-year OS (p=0.048). Our data suggest that NHERF1 could be a new potential biomarker in combination with PARP1 and BRCA1 expression to stratify BC patients. In particular, in TNBCs, cNHERF1 associated with nPARP1 expression identified a patient subgroup with a shorter survival, for whom it may be useful to develop novel therapeutic strategies.

ß-catenin interaction with NHERF1 and RASSF1A methylation in metastatic colorectal cancer patients.

There is an increasing need to identify new biomarkers in colorectal cancer (CRC) to further characterize this malignancy. ß-catenin plays a central role in the Wnt signaling pathway. It also binds Na+/H+ exchanger regulating factor 1 (NHERF1) and interacts with the RAS-association domain family 1, isoform A (RASSF1A), but the mechanisms of this possible crosstalk are still not fully understood. In this study, we analyzed for the first time the different subcellular expression of ß-catenin, NHERF1, and RASSF1A and their relationships with RASSF1A methylation in the progression of CRC. We assessed immunohistochemical expression and RASSF1A methylation in 51 patients with stage IV colorectal cancer. Biomarker expression analysis was carried out considering the tumor-adjacent normal tissue, the primary tumor, and the paired liver metastases. Regarding the tumor compartment, it was found that cytoplasmic ß-catenin expression was positively correlated to membranous (r = 0.3002, p = 0.0323) and nuclear NHERF1 (r = 0.293, p = 0.0368). In the liver metastases, instead, we found a positive correlation of cytoplasmic and nuclear ß-catenin expression with RASSF1A methylation (r = 0.4019, p = 0.0068 and r = 0.3194, p = 0.0345, respectively).In conclusion, our results showed that ß-catenin was the crucial protagonist in metastatic CRC through different effector proteins involved in this developing process. In tumor tissues, ß-catenin was predominantly associated with NHERF1 in a dynamic context, while interestingly in liver metastases, we noted an increase of its oncogenic function through RASSF1A inactivation.

Interval sentinel lymph nodes in melanoma: a digital pathology analysis of Ki67 expression and microvascular density.

The presence of interval sentinel lymph nodes in melanoma is documented in several studies, but controversies still exist about the management of these lymph nodes. In this study, an immunohistochemical evaluation of tumor cell proliferation and neo-angiogenesis has been performed with the aim of establishing a correlation between these two parameters between positive and negative interval sentinel lymph nodes. This retrospective study reviewed data of 23 patients diagnosed with melanoma. Bioptic specimens of interval sentinel lymph node were retrieved, and immunohistochemical reactions on tissue sections were performed using Ki67 as a marker of proliferation and CD31 as a blood vessel marker for the study of angiogenesis. The entire stained tissue sections for each case were digitized using Aperio Scanscope Cs whole-slide scanning platform and stored as high-resolution images. Image analysis was carried out on three selected fields of equal area using IHC Nuclear and Microvessel analysis algorithms to determine positive Ki67 nuclei and vessel number. Patients were divided into positive and negative interval sentinel lymph node groups, and the positive interval sentinel lymph node group was further divided into interval positive with micrometastasis and interval positive with macrometastasis subgroups. The analysis revealed a significant difference between positive and negative interval sentinel lymph nodes in the percentage of Ki67-positive nuclei and mean vessel number suggestive of an increased cellular proliferation and angiogenesis in positive interval sentinel lymph nodes. Further analysis in the interval positive lymph node group showed a significant difference between micro- and macrometastasis subgroups in the percentage of Ki67-positive nuclei and mean vessel number. Percentage of Ki67-positive nuclei was increased in the macrometastasis subgroup, while mean vessel number was increased in the micrometastasis subgroup. The results of this study suggest that the correlation between tumor cell proliferation and neo-angiogenesis in interval sentinel lymph nodes in melanoma could be used as a good predictive marker to distinguish interval positive sentinel lymph nodes with micrometastasis from interval positive lymph nodes with macrometastasis subgroups.

T cells, mast cells and microvascular density in diffuse large B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is recognized as the most common form of non-Hodgkin lymphoma (NHL), accounting for about 40 % of all cases of NHL. Among the cellular components of the tumor inflammatory infiltrate, T cells and mast cells have been demonstrated to be correlated with tumor angiogenesis. In this report, we have investigated CD3 and tryptase expression and their relationship with microvascular density (MVD) in DLBCL patients. Moreover, we determined the significance of CD3 expression in bulky and non-bulky disease. CD3 expression was significantly lower in bulky disease patients when compared to non-bulky ones. CD3 showed a positive correlation with tryptase and MVD, while multiple regression analysis efficaciously predicted MVD depending on CD3 and tryptase as predictors, supporting a complex interplay between these cells in sustaining tumor angiogenesis in DLBCL patients.

Everolimus restrains the paracrine pro-osteoclast activity of breast cancer cells.

BACKGROUND: Breast cancer (BC) cells secrete soluble factors that accelerate osteoclast (OC) differentiation, leading to the formation of osteolytic bone metastases. In the BOLERO-2 trial, BC patients with bone involvement who received Everolimus had a delayed tumor progression in the skeleton as a result of direct OC suppression through the inhibition of mTOR, in addition to the general suppressor effect on the cancer cells. Here, we explored the effect of Everolimus, as mTOR inhibitor, on the pro-OC paracrine activity of BC cells. METHODS: Both MDA-MB-231 and MCF-7 BC cell lines were incubated with sub-lethal amounts of Everolimus, and their conditioned supernatants were assessed for their capacity to differentiate OCs from PBMC from healthy donors, as well as to interfere with their bone resorbing activity shown on calcium phosphate slices. We also measured the mRNA levels of major pro-OC factors in Everolimus-treated BC cells and their secreted levels by ELISA, and evaluated by immunoblotting the phosphorylation of transcription factors enrolled by pathways cooperating with the mTOR inhibition. Finally, the in vivo pro-OC activity of these cells was assessed in SCID mice after intra-tibial injections. RESULTS: We found that Everolimus significantly inhibited the differentiation of OCs and their in vitro bone-resorbing activity, and also found decreases of both mRNA and secreted pro-OC factors such as M-CSF, IL-6, and IL-1ß, whose lower ELISA levels paralleled the defective phosphorylation of NFkB pathway effectors. Moreover, when intra-tibially injected in SCID mice, Everolimus-treated BC cells produced smaller bone metastases than the untreated cells. CONCLUSIONS: mTOR inhibition in BC cells leads to a suppression of their paracrine pro-OC activity by interfering with the NFkB pathway; this effect may also account for the delayed progression of bone metastatic disease observed in the BOLERO-2 trial.

Physiology of incretins and loss of incretin effect in type 2 diabetes and obesity.

An important role in the regulation of glucose homeostasis is played by incretins, which are gut-derived hormones released in response to nutrient ingestion. In humans, the major incretin hormones are glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic polypeptide (GIP), and together they fully account for the incretin effect (that is, higher insulin release in response to an oral glucose challenge compared to an equal intravenous glucose load). Studies have shown that GLP-1 and GIP levels and actions may be perturbed in disease states, and the loss of incretin effect is likely to contribute importantly to the postprandial hyperglycaemia in type 2 diabetes. However, the specific cause-effect relationship between disease and incretins is still unclear. This review focuses on several key studies elucidating the association of defective incretin action with obesity and T2DM and the effects of metformin and other anti-diabetic agents on the incretin system.