Human papillomavirus genotyping in high-grade vaginal intraepithelial neoplasia: A multicentric Italian study.

This study aimed to analyze the human papillomavirus (HPV) genotype distribution in a large cohort of high-grade vaginal intraepithelial neoplasia (VaIN) (vaginal HSIL, VaIN2/3) patients from two Italian referral centers. We included all patients with histologically confirmed VaIN2/3 from the Department of Surgical Sciences, Sant'Anna Hospital, University of Torino, Torino, Italy, and Ospedale Maggiore della Carità, Novara, Italy, between 2003 and 2022. After the histological evaluation of formalin-fixed paraffin-embedded samples, we performed HPV genotyping with VisionArray HPV Chip 1.0. We detected HPV DNA in 94.4% of VaIN2/3 (168/178), with HPV 16 as the most prevalent genotype, accounting for 51.8% of all infections, 41.2% of VaIN2 and 77.6% of VaIN3 cases. Other frequent genotypes were HPV 58 (8.3%, 10.9% of VaIN2 and 2.0% of VaIN3), HPV 73 (5.4%, 5.0% of VaIN2 and 6.1% of VaIN3), and HPV 31 (5.4%, 6.7% of VaIN2 and 2.0% of VaIN3). 73.2% of VaIN2/3 had a single HPV genotype infection and 26.8% a multiple infection (20.8% a double infection, 4.8% a triple infection, and 1.2% a quadruple infection). Single infection was more frequently present in VaIN3 than VaIN2 (81.6% vs. 69.8%). 69.1% of single infections and 73.3% of multiple infections had one or more genotypes covered by nine-valent HPV vaccine. HPV vaccination is expected to have a large impact on reducing the incidence of vaginal intraepithelial neoplasia.

Genetic and phenotypic attributes of splenic marginal zone lymphoma.

Splenic marginal zone B-cell lymphoma (SMZL) is a heterogeneous clinico-biological entity. The clinical course is variable, multiple genes are mutated with no unifying mechanism, and essential regulatory pathways and surrounding microenvironments are diverse. We sought to clarify the heterogeneity of SMZL by resolving different subgroups and their underlying genomic abnormalities, pathway signatures, and microenvironment compositions to uncover biomarkers and therapeutic vulnerabilities. We studied 303 SMZL spleen samples collected through the IELSG46 multicenter international study (NCT02945319) by using a multiplatform approach. We carried out genetic and phenotypic analyses, defined self-organized signatures, validated the findings in independent primary tumor metadata and in genetically modified mouse models, and determined correlations with outcome data. We identified 2 prominent genetic clusters in SMZL, termed NNK (58% of cases, harboring NF-κB, NOTCH, and KLF2 modules) and DMT (32% of cases, with DNA-damage response, MAPK, and TLR modules). Genetic aberrations in multiple genes as well as cytogenetic and immunogenetic features distinguished NNK- from DMT-SMZLs. These genetic clusters not only have distinct underpinning biology, as judged by differences in gene-expression signatures, but also different outcomes, with inferior survival in NNK-SMZLs. Digital cytometry and in situ profiling segregated 2 basic types of SMZL immune microenvironments termed immune-suppressive SMZL (50% of cases, associated with inflammatory cells and immune checkpoint activation) and immune-silent SMZL (50% of cases, associated with an immune-excluded phenotype) with distinct mutational and clinical connotations. In summary, we propose a nosology of SMZL that can implement its classification and also aid in the development of rationally targeted treatments.

Genetic mechanisms of HLA-I loss and immune escape in diffuse large B cell lymphoma.

Fifty percent of diffuse large B cell lymphoma (DLBCL) cases lack cell-surface expression of the class I major histocompatibility complex (MHC-I), thus escaping recognition by cytotoxic T cells. Here we show that, across B cell lymphomas, loss of MHC-I, but not MHC-II, is preferentially restricted to DLBCL. To identify the involved mechanisms, we performed whole exome and targeted HLA deep-sequencing in 74 DLBCL samples, and found somatic inactivation of B2M and the HLA-I loci in 80% (34 of 42) of MHC-INEG tumors. Furthermore, 70% (22 of 32) of MHC-IPOS DLBCLs harbored monoallelic HLA-I genetic alterations (MHC-IPOS/mono), indicating allele-specific inactivation. MHC-INEG and MHC-IPOS/mono cases harbored significantly higher mutational burden and inferred neoantigen load, suggesting potential coselection of HLA-I loss and sustained neoantigen production. Notably, the analysis of >500,000 individuals across different cancer types revealed common germline HLA-I homozygosity, preferentially in DLBCL. In mice, germinal-center B cells lacking HLA-I expression did not progress to lymphoma and were counterselected in the context of oncogene-driven lymphomagenesis, suggesting that additional events are needed to license immune evasion. These results suggest a multistep process of HLA-I loss in DLBCL development including both germline and somatic events, and have direct implications for the pathogenesis and immunotherapeutic targeting of this disease.

SIRT1 is an actionable target to restore p53 function in HPV-associated cancer therapy.

BACKGROUND: Our aim was to evaluate the efficacy and anti-cancer action of a precision medicine approach involving a novel SIRT1-dependent pathway that, when disrupted, leads to the restoration of a functional p53 in human papillomavirus (HPV)-transformed cells. METHODS: The anticancer potential of inhibiting SIRT1 was evaluated by examining the effects of the specific SIRT1 inhibitor EX527 (also known as Selisistat) or genetic silencing, either individually or in conjunction with standard chemotherapeutic agents, on a range of HPV+ cancer cells and a preclinical mouse model of HPV16-induced cancer. RESULTS: We show that SIRT1 inhibition restores a transcriptionally active K382-acetylated p53 in HPV+ but not HPV- cell lines, which in turn promotes G0/G1 cell cycle arrest and inhibits clonogenicity specifically in HPV+ cells. Additionally, EX527 treatment increases the sensitivity of HPV+ cells to sublethal doses of standard genotoxic agents. The enhanced sensitivity to cisplatin as well as p53 restoration were also observed in an in vivo tumorigenicity assay using syngeneic C3.43 cells harbouring an integrated HPV16 genome, injected subcutaneously into C57BL/6J mice. CONCLUSIONS: Our findings uncover an essential role of SIRT1 in HPV-driven oncogenesis, which may have direct translational implications for the treatment of this type of cancer.

Active Akt signaling triggers CLL toward Richter transformation via overactivation of Notch1.

Richter's transformation (RT) is an aggressive lymphoma that occurs upon progression from chronic lymphocytic leukemia (CLL). Transformation has been associated with genetic aberrations in the CLL phase involving TP53, CDKN2A, MYC, and NOTCH1; however, a significant proportion of RT cases lack CLL phase-associated events. Here, we report that high levels of AKT phosphorylation occur both in high-risk CLL patients harboring TP53 and NOTCH1 mutations as well as in patients with RT. Genetic overactivation of Akt in the murine Eµ-TCL1 CLL mouse model resulted in CLL transformation to RT with significantly reduced survival and an aggressive lymphoma phenotype. In the absence of recurrent mutations, we identified a profile of genomic aberrations intermediate between CLL and diffuse large B-cell lymphoma. Multiomics assessment by phosphoproteomic/proteomic and single-cell transcriptomic profiles of this Akt-induced murine RT revealed an S100 protein-defined subcluster of highly aggressive lymphoma cells that developed from CLL cells, through activation of Notch via Notch ligand expressed by T cells. Constitutively active Notch1 similarly induced RT of murine CLL. We identify Akt activation as an initiator of CLL transformation toward aggressive lymphoma by inducing Notch signaling between RT cells and microenvironmental T cells.

Aggressive Pelvic Angiomyxoma in a Patient with Twin Pregnancy: Diagnosis, Treatment, and Clinical Complications in Light of the Literature.

(1) Background: Aggressive angiomyxoma is a mesenchymal cancer that is rare during pregnancy. It is a neoplasm that relapses and infiltrates the nearest structures. Our aim is to evaluate the management and outcomes of an observed case, in light of the current literature. (2) Methods: We observed this condition at the "Maggiore della Carità" Hospital in Novara (Italy) in a patient with an initial twin pregnancy and a suspected pelvic mass. The words "angiomyxoma" and "pregnancy" were searched on the main online scientific search sources (PubMed, Google Scholar, Scopus, WES, and Embase, etc.). (3) Results: The patient underwent surgery with a complicated follow-up, but recent negative controls. We analyzed the literature about the topic and found only 24 similar clinical cases. (4) Conclusions: Considering the current literature, it is useful to assess an aggressive angiomyxoma in the differential diagnosis of soft masses in pregnant women. The treatment of choice is surgical excision, and vaginal delivery is feasible. The therapeutic decision depends on each case.

Clonally unrelated Richter syndrome are truly de novo diffuse large B-cell lymphomas with a mutational profile reminiscent of clonally related Richter syndrome.

Richter syndrome (RS) is mostly due to the direct transformation of the chronic lymphocytic leukaemia (CLL) clone, as documented by the same immunoglobulin heavy-chain variable region (IGHV) rearrangement in both CLL and RS cells. In rare cases characterized by a better outcome, the RS clone harbours a different IGHV rearrangement compared to the CLL phase. We investigated the CLL phase of clonally unrelated RS to test whether the RS clone was already identifiable prior to clinicopathologic transformation, albeit undetectable by conventional approaches. CLL cells of eight patients with unrelated RS were subjected to an ultra-deep next-generation sequencing (NGS) approach with a sensitivity of 10-6 . In 7/8 cases, the RS rearrangement was not identified in the CLL phase. In one case, the RS clone was identified at a very low frequency in the CLL phase, conceivably due to the concomitance of CLL sampling and RS diagnosis. Targeted resequencing revealed that clonally unrelated RS carries genetic lesions primarily affecting the TP53, MYC, ATM and NOTCH1 genes. Conversely, mutations frequently involved in de novo diffuse large B-cell lymphoma (DLBCL) without a history of CLL were absent. These results suggest that clonally unrelated RS is a truly de novo lymphoma with a mutational profile reminiscent, at least in part, of clonally related RS.

Elastin MIcrofibriL INterfacer1 (EMILIN-1) is an alternative prosurvival VLA-4 ligand in chronic lymphocytic leukemia.

CD49d, the α4 chain of the VLA-4 integrin, is a negative prognosticator in chronic lymphocytic leukemia (CLL) with a key role in CLL cell-microenvironment interactions mainly occurring via its ligands VCAM-1 and fibronectin. In the present study, we focused on EMILIN-1 (Elastin-MIcrofibriL-INterfacer-1), an alternative VLA-4 ligand whose role has been so far reported only in non-hematological settings, by investigating: i) the distribution of EMILIN-1 in CLL-involved tissues; ii) the capability of EMILIN-1 to operate, via its globular C1q (gC1q) domain, as additional adhesion ligand in CLL; iii) the functional meaning of EMILIN-1 gC1q/VLA-4 interactions in CLL. EMILIN-1 is widely present in the CLL-involved areas of bone marrow biopsies (BMBs) without difference between CD49d negative and positive cases, displaying at least three different expression patterns: "fibrillar", "dot-like" and "mixed". The lack in CLL-BMB of neutrophil elastase, whose proteolytic activity degrades EMILIN-1 and impairs EMILIN-1 function, suggests full functional EMILIN-1 in CLL independently of its expression pattern. Functionally, EMILIN-1 gC1q domain promotes adhesion of CLL cells through specific interaction with VLA-4, and releases pro-survival signals for CLL cells, as demonstrated by enhanced ERK and AKT phosphorylation and impairment of in-vitro-induced apoptosis. EMILIN-1/VLA-4 interaction can efficiently contribute to the maintenance of the neoplastic clone in CLL.

ICOSL Stimulation by ICOS-Fc Accelerates Cutaneous Wound Healing In Vivo.

BACKGROUND: ICOS and its ligand ICOSL are immune receptors whose interaction triggers bidirectional signals that modulate the immune response and tissue repair. AIM: The aim of this study was to assess the in vivo effects of ICOSL triggering by ICOS-Fc, a recombinant soluble form of ICOS, on skin wound healing. METHODS: The effect of human ICOS-Fc on wound healing was assessed, in vitro, and, in vivo, by skin wound healing assay using ICOS-/- and ICOSL-/- knockout (KO) mice and NOD-SCID-IL2R null (NSG) mice. RESULTS: We show that, in wild type mice, treatment with ICOS-Fc improves wound healing, promotes angiogenesis, preceded by upregulation of IL-6 and VEGF expression; increases the number of fibroblasts and T cells, whereas it reduces that of neutrophils; and increases the number of M2 vs. M1 macrophages. Fittingly, ICOS-Fc enhanced M2 macrophage migration, while it hampered that of M1 macrophages. ICOS-/- and ICOSL-/- KO, and NSG mice showed delayed wound healing, and treatment with ICOS-Fc improved wound closure in ICOS-/- and NSG mice. CONCLUSION: These data show that the ICOS/ICOSL network cooperates in tissue repair, and that triggering of ICOSL by ICOS-Fc improves cutaneous wound healing by increasing angiogenesis and recruitment of reparative macrophages.

Multiregional sequencing and circulating tumour DNA analysis provide complementary approaches for comprehensive disease profiling of small lymphocytic lymphoma.

We aimed at molecularly dissecting the anatomical heterogeneity of small lymphocytic lymphoma (SLL), by analysing a cohort of 12 patients for whom paired DNA from a lymph node biopsy and circulating cells, as well as plasma-circulating tumour DNA (ctDNA) was available. Notably, the analyses of the lymph node biopsy and of circulating cells complement each other since a fraction of mutations (20·4% and 36·4%, respectively) are unique to each compartment. Plasma ctDNA identified two additional unique mutations. Consistently, the different synchronous sources of tumour DNA complement each other in informing on driver gene mutations in SLL harbouring potential prognostic and/or predictive value.

DUSP12 acts as a novel endogenous protective signal against hepatic ischemia-reperfusion damage by inhibiting ASK1 pathway.

Ischemia-reperfusion injury (IRI) consequent to major liver surgery is a still unmet clinical problem. The activation of endogenous systems of hepatoprotection can prevent the damaging effects of ischemia-reperfusion (IR) as shown by the phenomenon known as 'ischemic preconditioning'. The identification of endogenous signal mediators of hepatoprotection is of main interest since they could be targeted in future therapeutic interventions. Qiu et al. recently reported in Clin. Sci. (Lond.) (2020) 134(17), 2279-2294, the discovery of a novel protective molecule against hepatic IR damage: dual-specificity phosphatase 12 (DUSP12). IR significantly decreased DUSP12 expression in liver whereas DUSP12 overexpression in hepatocytes protected IRI and DUSP12 deletion in DUSP12 KO mice exacerbated IRI. The protective effects of DUSP12 depended on apoptosis signal-regulating kinase 1 (ASK1) and acted through the inhibition of the ASK1-dependent kinases c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). These results enlighten DUSP12 as a novel intermediate negative regulator of the pro-inflammatory and pro-apoptotic ASK1/JNK-p38 MAPK pathway activated during hepatic IR and identify DUSP12 as potential therapeutic target for IRI.

Mitotically Active Cellular Fibroma of the Ovary Recurring After the Longest Interval of Time (16 yr): A Challenging Case With Systematic Literature Review.

Cellular fibromas represent ~10% of ovarian fibromas. Mitotically active cellular fibromas show mild nuclear atypia but ≥4 mitoses/10 high-power fields: the clinical course is usually uneventful but literature review is lacking. A 34-yr-old woman underwent left oophorectomy for a 9-cm ovarian mitotically active cellular fibroma at another hospital. The tumor was cellular (spindle cells in fascicular and storiform patterns) revealing mild atypia and 4 nonatypical mitoses/10 high-power fields without necrotic areas. After 16 yr, the tumor recurred as a 5-cm peritoneal nodule on the anterior sigmoid wall near the sigmoid-rectal junction. Frozen section revealed a spindle cell tumor invading the intestinal tunica muscularis propria: a gastrointestinal stromal tumor was favored as previous history was unavailable at that time. Intestinal resection was performed: no residual tumor was found. The patient was followed-up for 8 yr without further recurrences. The peritoneal nodule showed 2 mitoses/10 high-power fields and pericellular reticulin staining. The tumor was variably positive for vimentin/bcl-2/melan-A/CD56/ER/PR/α-inhibin/CD10/calretinin, focally positive for desmin, negative for pan-cytokeratin/actin/EMA/CD34/HMB45/CD117/CD99/S100/synaptophysin. The Ki67-index was ~9%. To our systematic literature review, 7 additional recurrent cases were reported. We describe a mitotically active cellular fibroma recurring after the longest interval of time. Extensive sampling of difficult cases should exclude malignant areas. Moderate nuclear atypia, tumor rupture, adhesions to pelvic/abdominal organs, infarction with extraovarian involvement, and incomplete excision may lead to relapse but there are conflicting data: prolonged follow-up can be suggested in these cases.

4D Electromagnetic Navigation Bronchoscopy for the Sampling of Pulmonary Lesions: First European Real-Life Experience.

PURPOSE: The use of Electromagnetic navigation bronchoscopy (ENB) for the diagnosis of pulmonary peripheral lesions is still debated due to its variable diagnostic yield; a new 4D ENB system, acquiring inspiratory and expiratory computed tomography (CT) scans, overcomes respiratory motion and uses tracked sampling instruments, reaching higher diagnostic yields. We aimed at evaluating diagnostic yield and accuracy of a 4D ENB system in sampling pulmonary lesions and at describing their influencing factors. METHODS: We conducted a three-year retrospective observational study including all patients with pulmonary lesions who underwent 4D ENB with diagnostic purposes; all the factors potentially influencing diagnosis were recorded. RESULTS: 103 ENB procedures were included; diagnostic yield and accuracy were, respectively, 55.3% and 66.3%. We reported a navigation success rate of 80.6% and a diagnosis with ENB was achieved in 68.3% of cases; sensitivity for malignancy was 61.8%. The majority of lesions had a bronchus sign on CT, but only the size of lesions influenced ENB diagnosis (p < 0.05). Transbronchial needle aspiration biopsy was the most used tool (93.2% of times) with the higher diagnostic rate (70.2%). We reported only one case of pneumothorax. CONCLUSION: The diagnostic performance of a 4D ENB system is lower than other previous navigation systems used in research settings. Several factors still influence the reachability of the lesion and therefore diagnostic yield. Patient selection, as well as the multimodality approach of the lesion, is strongly recommended to obtain higher diagnostic yield and accuracy, with a low rate of complications.

Genetic Predisposition to Myelodysplastic Syndromes: A Challenge for Adult Hematologists.

Myelodysplastic syndromes (MDS) arising in the context of inherited bone marrow failure syndromes (IBMFS) differ in terms of prognosis and treatment strategy compared to MDS occurring in the adult population without an inherited genetic predisposition. The main molecular pathways affected in IBMFS involve telomere maintenance, DNA repair, biogenesis of ribosomes, control of proliferation and others. The increased knowledge on the genes involved in MDS pathogenesis and the wider availability of molecular diagnostic assessment have led to an improvement in the detection of IBMFS genetic predisposition in MDS patients. A punctual recognition of these disorders implies a strict surveillance of the patient in order to detect early signs of progression and promptly offer allogeneic hematopoietic stem cell transplantation, which is the only curative treatment. Moreover, identifying an inherited mutation allows the screening and counseling of family members and directs the choice of donors in case of need for transplantation. Here we provide an overview of the most recent data on MDS with genetic predisposition highlighting the main steps of the diagnostic and therapeutic management. In order to highlight the pitfalls of detecting IBMFS in adults, we report the case of a 27-year-old man affected by MDS with an underlying telomeropathy.

Primary seminoma arising in the posterior mediastinum: a diagnostic challenge.

Primary mediastinal germ cell tumors are a rare finding, and one third of them are seminomas. Seminomas are found in the anterior mediastinum, whereas they are extremely rare within the posterior compartment. Most clinicians would not consider a primary seminoma in the differential diagnosis of a posterior mediastinal mass, as only two cases have been reported in literature. Here we present the case of a 57-year-old male with a primary seminoma arising in the left posterior mediastinum. He was asymptomatic and the mass was an incidental finding. Positron emission tomography (PET) revealed a small area with an avid tracer uptake. Transthoracic needle aspiration led to a non-diagnostic result. Due to the strong suspect of malignancy, a surgical excision was chosen to obtain a diagnosis. He underwent complete excision, and pathology report demonstrated a mediastinal seminoma. Subsequent further staging did not reveal any other location of the disease. Given the complete excision of the primary tumor, active surveillance was the treatment of choice. The patient is free of disease 48 months after diagnosis.

Precision Medicine in Systemic Mastocytosis.

Mastocytosis is a rare hematological neoplasm characterized by the proliferation of abnormal clonal mast cells (MCs) in different cutaneous and extracutaneous organs. Its diagnosis is based on well-defined major and minor criteria, including the pathognomonic dense infiltrate of MCs detected in bone marrow (BM), elevated serum tryptase level, abnormal MCs CD25 expression, and the identification of KIT D816V mutation. The World Health Organization (WHO) classification subdivides mastocytosis into a cutaneous form (CM) and five systemic variants (SM), namely indolent/smoldering (ISM/SSM) and advanced SM (AdvSM) including aggressive SM (ASM), SM associated to hematological neoplasms (SM-AHN), and mast cell leukemia (MCL). More than 80% of patients with SM carry a somatic point mutation of KIT at codon 816, which may be targeted by kinase inhibitors. The presence of additional somatic mutations detected by next generation sequencing analysis may impact prognosis and drive treatment strategy, which ranges from symptomatic drugs in indolent forms to kinase-inhibitors active on KIT. Allogeneic stem cell transplant (SCT) may be considered in selected SM cases. Here, we review the clinical, diagnostic, and therapeutic issues of SM, with special emphasis on the translational implications of SM genetics for a precision medicine approach in clinical practice.

Absinthin, an agonist of the bitter taste receptor hTAS2R46, uncovers an ER-to-mitochondria Ca2+-shuttling event.

Type 2 taste receptors (TAS2R) are G protein-coupled receptors first described in the gustatory system, but have also been shown to have extraoral localizations, including airway smooth muscle (ASM) cells, in which TAS2R have been reported to induce relaxation. TAS2R46 is an unexplored subtype that responds to its highly specific agonist absinthin. Here, we first demonstrate that, unlike other bitter-taste receptor agonists, absinthin alone (1 µm) in ASM cells does not induce Ca2+ signals but reduces histamine-induced cytosolic Ca2+ increases. To investigate this mechanism, we introduced into ASM cells aequorin-based Ca2+ probes targeted to the cytosol, subplasma membrane domain, or the mitochondrial matrix. We show that absinthin reduces cytosolic histamine-induced Ca2+ rises and simultaneously increases Ca2+ influx into mitochondria. We found that this effect is inhibited by the potent human TAS2R46 (hTAS2R46) antagonist 3ß-hydroxydihydrocostunolide and is no longer evident in hTAS2R46-silenced ASM cells, indicating that it is hTAS2R46-dependent. Furthermore, these changes were sensitive to the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP); the mitochondrial calcium uniporter inhibitor KB-R7943 (carbamimidothioic acid); the cytoskeletal disrupter latrunculin; and an inhibitor of the exchange protein directly activated by cAMP (EPAC), ESI-09. Similarly, the ß2 agonist salbutamol also could induce Ca2+ shuttling from cytoplasm to mitochondria, suggesting that this new mechanism might be generalizable. Moreover, forskolin and an EPAC activator mimicked this effect in HeLa cells. Our findings support the hypothesis that plasma membrane receptors can positively regulate mitochondrial Ca2+ uptake, adding a further facet to the ability of cells to encode complex Ca2+ signals.

Circulating tumor DNA reveals genetics, clonal evolution, and residual disease in classical Hodgkin lymphoma.

The rarity of neoplastic cells in the biopsy imposes major technical hurdles that have so far limited genomic studies in classical Hodgkin lymphoma (cHL). By using a highly sensitive and robust deep next-generation sequencing approach for circulating tumor DNA (ctDNA), we aimed to identify the genetics of cHL in different clinical phases, as well as its modifications on treatment. The analysis was based on specimens collected from 80 newly diagnosed and 32 refractory patients with cHL, including longitudinal samples collected under ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) chemotherapy and longitudinal samples from relapsing patients treated with chemotherapy and immunotherapy. ctDNA mirrored Hodgkin and Reed-Sternberg cell genetics, thus establishing ctDNA as an easily accessible source of tumor DNA for cHL genotyping. By identifying STAT6 as the most frequently mutated gene in ∼40% of cases, we refined the current knowledge of cHL genetics. Longitudinal ctDNA profiling identified treatment-dependent patterns of clonal evolution in patients relapsing after chemotherapy and patients maintained in partial remission under immunotherapy. By measuring ctDNA changes during therapy, we propose ctDNA as a radiation-free tool to track residual disease that may integrate positron emission tomography imaging for the early identification of chemorefractory patients with cHL. Collectively, our results provide the proof of concept that ctDNA may serve as a novel precision medicine biomarker in cHL.

Biological and clinical implications of BIRC3 mutations in chronic lymphocytic leukemia.

BIRC3 is a recurrently mutated gene in chronic lymphocytic leukemia (CLL) but the functional implications of BIRC3 mutations are largely unexplored. Furthermore, little is known about the prognostic impact of BIRC3 mutations in CLL cohorts homogeneously treated with first-line fludarabine, cyclophosphamide, and rituximab (FCR). By immunoblotting analysis, we showed that the non-canonical nuclear factor-κB pathway is active in BIRC3-mutated cell lines and in primary CLL samples, as documented by the stabilization of MAP3K14 and by the nuclear localization of p52. In addition, BIRC3-mutated primary CLL cells are less sensitive to flu-darabine. In order to confirm in patients that BIRC3 mutations confer resistance to fludarabine-based chemoimmunotherapy, a retrospective multicenter cohort of 287 untreated patients receiving first-line FCR was analyzed by targeted next-generation sequencing of 24 recurrently mutated genes in CLL. By univariate analysis adjusted for multiple comparisons BIRC3 mutations identify a poor prognostic subgroup of patients in whom FCR treatment fails (median progression-free survival: 2.2 years, P<0.001) similar to cases harboring TP53 mutations (median progression-free survival: 2.6 years, P<0.0001). BIRC3 mutations maintained an independent association with an increased risk of progression with a hazard ratio of 2.8 (95% confidence interval 1.4-5.6, P=0.004) in multivariate analysis adjusted for TP53 mutation, 17p deletion and IGHV mutation status. If validated, BIRC3 mutations may be used as a new molecular predictor to select high-risk patients for novel frontline therapeutic approaches.

Anti-inflammatory Activity of Absinthin and Derivatives in Human Bronchoepithelial Cells.

Bitter taste receptors (hTAS2R) are expressed ectopically in various tissues, raising the possibility of a pharmacological exploitation. This seems of particular relevance in airways, since hTAS2Rs are involved in the protection of the aerial tissues from infections and in bronchodilation. The bis-guaianolide absinthin (1), one of the most bitter compounds known, targets the hTAS2R46 bitter receptor. Absinthin (1), an unstable compound, readily turns into anabsinthin (2) with substantial retention of the bitter properties, and this compound was used as a starting material to explore the chemical space around the bis-guaianolide bitter pharmacophore. Capitalizing on the chemoselective opening of the allylic lactone ring, the esters 3 and 4, and the nor-azide 6 were prepared and assayed on human bronchoepithelial (BEAS-2B) cells expressing hTAS2R46. Anti-inflammatory activity was evaluated by measuring the expression of MUC5AC, iNOS, and cytokines, as well as the production of superoxide anion, qualifying the methyl ester 3 as the best candidate for additional studies.