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We are delighted to share with you our seventh Journal Club and highlight some of the most interesting papers published recently [...].

Abstract Background In the last years, several efforts have been made to classify colorectal cancer (CRC) into well-defined molecular subgroups, representing the intrinsic inter-patient heterogeneity, known as Consensus Molecular Subtypes (CMSs). Methods In this work, we performed a meta-analysis of CRC patients stratified into four CMSs. We identified a negative correlation between a high level of anaplastic lymphoma kinase (ALK) expression and relapse-free survival, exclusively in CMS1 subtype. Stemming from this observation, we tested cell lines, patient-derived organoids and mice with potent ALK inhibitors, already approved for clinical use. Results ALK interception strongly inhibits cell proliferation already at nanomolar doses, specifically in CMS1 cell lines, while no effect was found in CMS2/3/4 groups. Furthermore, in vivo imaging identified a role for ALK in the dynamic formation of 3D tumor spheroids. Consistently, ALK appeares constitutively phosphorylated in CMS1, and it signals mainly through the AKT axis. Mechanistically, we found that CMS1 cells display several copies of ALKAL2 ligand and ALK-mRNAs, suggesting an autocrine loop mediated by ALKAL2 in the activation of ALK pathway, responsible for the invasive phenotype. Consequently, disruption of ALK axis mediates the pro-apoptotic action of CMS1 cell lines, both in 2D and 3D and enhanced cell-cell adhesion and e-cadherin organization. In agreement with all these findings, the ALK signature encompassing 65 genes statistically associated with worse relapse-free survival in CMS1 subtype. Finally, as a proof of concept, the efficacy of ALK inhibition was demonstrated in both patient-derived organoids and in tumor xenografts in vivo. Conclusions Collectively, these findings suggest that ALK targeting may represent an attractive therapy for CRC, and CMS classification may provide a useful tool to identify patients who could benefit from this treatment. These findings offer rationale and pharmacological strategies for the treatment of CMS1 CRC.

Graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). Rabbit anti-T lymphocyte globulin (ATLG) in addition to calcineurin inhibitors and antimetabolites is a suitable strategy to prevent GVHD in several transplant settings. Randomized studies already demonstrated its efficacy in terms of GVHD prevention, although the effect on relapse remains the major concern for a wider use. Tailoring of ATLG dose on host characteristics is expected to minimize its side effects (immunological reconstitution, relapse, and infections). Here, day -6 to day +15 pharmacokinetics of active ATLG serum level was first assayed in an explorative cohort of 23 patients by testing the ability of the polyclonal serum to bind antigens on human leukocytes. Significantly lower levels of serum active ATLG were found in the patients who developed GVHD (ATLG_AUCCD45: 241.52 ± 152.16 vs. 766.63 +/- 283.52 (μg*day)/ml, p = 1.46e-5). Consistent results were obtained when the ATLG binding capacity was assessed on CD3+ and CD3+/CD4+ T lymphocytes (ATLG_AUCCD3: 335.83 ± 208.15 vs. 903.54 ± 378.78 (μg*day)/ml, p = 1.92e-4; ATLG_AUCCD4: 317.75 ± 170.70 vs. 910.54 ± 353.35 (μg*day)/ml, p = 3.78e-5. Concomitantly, at pre-infusion time points, increased concentrations of CD69+ extracellular vesicles (EVs) were found in patients who developed GVHD (mean fold 9.01 ± 1.33; p = 2.12e-5). Consistent results were obtained in a validation cohort of 12 additional ATLG-treated HSCT patients. Serum CD69+ EVs were mainly represented in the nano (i.e. 100 nm in diameter) EV compartment and expressed the leukocyte marker CD45, the EV markers CD9 and CD63, and CD103, a marker of tissue-resident memory T cells. The latter are expected to set up a host pro-inflammatory cell compartment that can survive in the recipient for years after conditioning regimen and contribute to GVHD pathogenesis. In summary, high levels of CD69+ EVs are significantly correlated with an increased risk of GVHD, and they may be proposed as a tool to tailor ATLG dose for personalized GVHD prevention.

BackgroundInfusion of second generation autologous CD19-targeted chimeric antigen receptor (CAR) T cells in patients with R/R relapsed/refractory B-cell lymphoma (BCL) is affected by inflammatory complications, such as Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS). Current literature suggests that the immune profile prior to CAR-T infusion modifies the chance to develop ICANS.MethodsThis is a monocenter prospective study on 53 patients receiving approved CAR T-cell products (29 axi-cel, 24 tisa-cel) for R/R-BCL. Clinical, biochemical, and hematological variables were analyzed at the time of pre-lymphodepletion (pre-LD). In a subset of 21 patients whose fresh peripheral blood sample was available, we performed cytofluorimetric analysis of leukocytes and extracellular vesicles (EVs). Moreover, we assessed a panel of soluble plasma biomarkers (IL-6/IL-10/GDF-15/IL-15/CXCL9/NfL) and microRNAs (miR-146a-5p, miR-21-5p, miR-126-3p, miR-150-5p) which are associated with senescence and inflammation.ResultsMultivariate analysis at the pre-LD time-point in the entire cohort (n=53) showed that a lower percentage of CD3+CD8+ lymphocytes (38.6% vs 46.8%, OR=0.937 [95% CI: 0.882-0.996], p=0.035) and higher levels of serum C-reactive protein (CRP, 4.52 mg/dl vs 1.00 mg/dl, OR=7.133 [95% CI: 1.796-28], p=0.005) are associated with ICANS. In the pre-LD samples of 21 patients, a significant increase in the percentage of CD8+CD45RA+CD57+ senescent cells (median % value: 16.50% vs 9.10%, p=0.009) and monocytic-myeloid derived suppressor cells (M-MDSC, median % value: 4.4 vs 1.8, p=0.020) was found in ICANS patients. These latter also showed increased levels of EVs carrying CD14+ and CD45+ myeloid markers, of the myeloid chemokine CXCL-9, as well of the MDSC-secreted cytokine IL-10. Notably, the serum levels of circulating neurofilament light chain, a marker of neuroaxonal injury, were positively correlated with the levels of senescent CD8+ T cells, M-MDSC, IL-10 and CXCL-9. No variation in the levels of the selected miRNAs was observed between ICANS and no-ICANS patients.DiscussionOur data support the notion that pre-CAR-T systemic inflammation is associated with ICANS. Higher proportion of senescence CD8+ T cells and M-MDSC correlate with early signs of neuroaxonal injury at pre-LD time-point, suggesting that ICANS may be the final event of a process that begins before CAR-T infusion, consequence to patient clinical history.

Abstract Enzyme replacement therapy (ERT) is a mainstay of treatment for Anderson–Fabry disease (AFD), a pathology with negative effects on the heart and kidneys. However, no reliable biomarkers are available to monitor its efficacy. Therefore, we tested a panel of four microRNAs linked with cardiac and renal damage in order to identify a novel biomarker associated with AFD and modulated by ERT. To this end, 60 patients with a definite diagnosis of AFD and on chronic ERT, and 29 age- and sex-matched healthy individuals, were enrolled by two Italian university hospitals. Only miR-184 met both conditions: its level discriminated untreated AFD patients from healthy individuals (c-statistic = 0.7522), and it was upregulated upon ERT (P

Metastasis is responsible for the majority of cancer‐related deaths. Particularly, challenging is the management of metastatic cancer of unknown primary site (CUP), whose tissue of origin (TOO) remains undetermined even after extensive investigations and whose therapy is rather unspecific and poorly effective. Molecular approaches to identify the most probable TOO of CUPs can overcome some of these issues. In this study, we applied a predetermined set of 89 microRNAs (miRNAs) to infer the TOO of 53 metastatic cancers of unknown or uncertain origin. The miRNA expression was assessed with droplet digital PCR in 159 samples, including primary tumors from 17 tumor classes (reference set) and metastases of known and unknown origin (test set). We combined two different statistical models for class prediction to obtain the most probable TOOs: the nearest shrunken centroids approach of Prediction Analysis of Microarrays (PAMR) and the least absolute shrinkage and selection operator (LASSO) models. The molecular test was successful for all formalin‐fixed paraffin‐embedded samples and provided a TOO identification within 1 week from the biopsy procedure. The most frequently predicted origins were gastrointestinal, pancreas, breast, lung, and bile duct. The assay was applied also to multiple metastases from the same CUP, collected from different metastatic sites: The predictions showed a strong agreement, intrinsically validating our assay. The final CUPs' TOO prediction was compared with the clinicopathological hypothesis of primary site. Moreover, a panel of 13 miRNAs proved to have prognostic value and be associated with overall survival in CUP patients. Our study demonstrated that miRNA expression profiling in CUP samples could be employed as diagnostic and prognostic test. Our molecular analysis can be performed on request, concomitantly with standard diagnostic workup and in association with genetic profiling, to offer valuable indications about the possible primary site, thereby supporting treatment decisions.

BackgroundT cells engineered to target CD19 antigen on neoplastic B cells represent the most striking example of CAR-T cell therapy. The success rate of this therapy is affected by several limitations: target antigen loss, and/or acquisition of a senescent/exhausted phenotype by CAR and non-CAR T cells.Case presentationWe report on a patient affected by refractory Diffuse Large B-cell Lymphoma who was resistant to CAR T-cell therapy and to two cycles post CAR-T of pembrolizumab (PBZ) due to the evolution into a B-cell Hodgkin-like lymphoma. Owing to the CD30 expression and the Hodgkin-like phenotype, the patient was ultimately treated with Brentuximab-Vedotin and finally underwent remission. Upon PBZ treatment, 100% of circulating CAR-T+ cells showed a persistent CD8+ senescent/exhausted phenotype, while an increase in the percentage of senescent cells was found in the non-CAR CD8+ T cells compartment.ConclusionsPBZ is not able to reinvigorate exhausted CAR+ T cells and to confer durable clinical response. We hypothesize that the phenomenon is due to the senescent phenotype of CAR+ T cells, which did not allow PBZ-induced reactivation and proliferative rescue. The phenomenon, together with the loss of CAR-T target CD19 and the shift of non-CAR CD8+ T cells towards a senescent phenotype likely contributed to set up an immune landscape with poor antitumor capacity.

Na virada do século XXI, no campo do saber erudito e no ideário do cidadão comum, revela-se um movimento de resistência à deterioração da arquitetura, da paisagem da cidade e da qualidade de vida do cidadão. Nesse percurso intelectual, ocorrem importantes redefinições conceituais que perpassam o conjunto da sociedade indo da gestão pública e academia até as atividades e expressões ditas do cotidiano da cidade. Nesse contexto, o texto fala de uma nova forma de ser arquiteto e fazer arquitetura e argumenta que isso pressupõem a reformulação do ensino da arquitetura, assim como, uma reinterpretação da política e da forma de se fazer pertencer à cidade. No início desse século, portanto, ao mesmo tempo que se caracteriza uma certa crise na profissão de arquiteto, parece se consolidar um novo território de atuação profissional fruto da reincorporação da atividade de projetar ao campo da política, entendida aqui, como manifestação plural, pois saturada de temas do cotidiano urbano.

Small cell neuroendocrine carcinoma is most frequently found in the lung (SCLC), but it has been also reported, albeit with a very low incidence, in the ovary. Here, we analyze a case of primary small cell carcinoma of the ovary of pulmonary type (SCCOPT), a rare and aggressive tumor with poor prognosis, whose biology and molecular features have not yet been thoroughly investigated. The patient affected by SCCOPT had a residual tumor following chemotherapy which displayed pronounced similarity with neuroendocrine tumors and lung cancer in terms of its microRNA expression profile and mTOR-downstream activation. By analyzing the metabolic markers of the neoplastic lesion, we established a likely glycolytic signature. In conclusion, this in-depth characterization of SCCOPT could be useful for future diagnoses, possibly aided by microRNA profiling, allowing clinicians to adopt the most appropriate therapeutic strategy.

Cancers of unknown primary (CUPs) comprise a heterogeneous group of rare metastatic tumors whose primary site cannot be identified after extensive clinical–pathological investigations. CUP patients are generally treated with empirical chemotherapy and have dismal prognosis. As recently reported, CUP genome presents potentially druggable alterations for which targeted therapies could be proposed. The paucity of tumor tissue, as well as the difficult DNA testing and the lack of dedicated panels for target gene sequencing are further relevant limitations. Here, we propose that circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) could be used to identify actionable mutations in CUP patients. Blood was longitudinally collected from two CUP patients. CTCs were isolated with CELLSEARCH® and DEPArrayTM NxT and Parsortix systems, immunophenotypically characterized and used for single-cell genomic characterization with Ampli1TM kits. Circulating cell-free DNA (ccfDNA), purified from plasma at different time points, was tested for tumor mutations with a CUP-dedicated, 92-gene custom panel using SureSelect Target Enrichment technology. In parallel, FFPE tumor tissue was analyzed with three different assays: FoundationOne CDx assay, DEPArray LibPrep and OncoSeek Panel, and the SureSelect custom panel. These approaches identified the same mutations, when the gene was covered by the panel, with the exception of an insertion in APC gene. which was detected by OncoSeek and SureSelect panels but not FoundationOne. FGFR2 and CCNE1 gene amplifications were detected in single CTCs, tumor tissue, and ccfDNAs in one patient. A somatic variant in ARID1A gene (p.R1276∗) was detected in the tumor tissue and ccfDNAs. The alterations were validated by Droplet Digital PCR in all ccfDNA samples collected during tumor evolution. CTCs from a second patient presented a pattern of recurrent amplifications in ASPM and SEPT9 genes and loss of FANCC. The 92-gene custom panel identified 16 non-synonymous somatic alterations in ccfDNA, including a deletion (I1485Rfs∗19) and a somatic mutation (p. A1487V) in ARID1A gene and a point mutation in FGFR2 gene (p.G384R). Our results support the feasibility of non-invasive liquid biopsy testing in CUP cases, either using ctDNA or CTCs, to identify CUP genetic alterations with broad NGS panels covering the most frequently mutated genes.

Ao analisarmos o último meio século da produção arquitetônica recifense, constatamos uma importante transformação na cultura arquitetônica local, que pode ser sintetizada através da identificação de três momentos: a estética da reentrância, a estética da “fantasmagoria” e a estética da elasticidade. Esses três períodos da produção arquitetônica, longe de se distinguirem por meros aspectos formais, expressam importantes transformações na forma de pensar e fazer o objeto arquitetônico, ao ponto de questionar o que podemos chamar de tradição arquitetônica local – o que inclui tanto a prática arquitetônica como a maneira de pensar e ensinar projeto nas escolas de arquitetura. Conforme veremos, essas transformações não resultam, unicamente, de decisões internas ao campo da arquitetura, mas se articulam e respondem a solicitações econômicas, sociais e tecnológicas, e, via de regra, justamente por em alguns momentos questionarem a tradição arquitetônica e provocarem uma importante alteração urbanística na paisagem da cidade, trazem algum tipo de mal-estar aos que parecem ser seus principais protagonistas: os arquitetos. Por fim, e com base nas respostas que os próprios arquitetos dão as transformações estéticas dos edifícios, e principalmente, pela constatação da emergência de uma nova sensibilidade urbana, pergunta-se se já podemos anunciar uma nova fase na história da arquitetura recifense que poderia ser chamada de “estética da arquitetura urbana ou estética da cidade”?

Background: Programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1) inhibitors represent novel therapeutic options for advanced non-small cell lung cancer (NSCLC). However, approximately 50% of patients do not benefit from therapy and experience rapid disease progression. PD-L1 expression is the only approved biomarker of benefit to anti-PD-1/PD-L1 therapy. However, its weakness has been evidenced in many studies. More recently, tumor mutational burden (TMB) has proved to be a suitable biomarker, but its calculation is difficult to obtain for all patients. Methods: We tested specific NSCLC genetic alterations as potential immunotherapy biomarkers. Tumor DNA was obtained from advanced NSCLC patients treated with anti-PD-1 monoclonal antibody nivolumab ( n = 44) or pembrolizumab ( n = 3). The mutational status of 22 genes was assessed by targeted next-generation sequencing and the association with survival was tested in uni- and multivariate models. The association between gene mutations and clinical benefit was also investigated. Results: The most frequently mutated genes were TP53 (49%), KRAS (43%), ERBB2 (13%), SMAD4 (13%), DDR2 (13%), STK11 (9%), ERBB4 (6%), EGFR (6%), BRAF (6%), and MET (6%). We confirmed that KRAS mut patients have a better response to PD-1 inhibitors, showing a longer progression-free survival (PFS) and overall survival (OS) than KRAS wt patients. In addition, we observed that patients with ERBB -family mutations, including EGFR, ERBB2 , and ERBB4 all failed to respond to PD-1 antibodies, independently of KRAS status. Conclusions: This study suggests that the analysis of KRAS and ERBB -family gene mutational status is valuable when assessing the clinical practice for the selection of NSCLC patients to treat with PD-1 inhibitors.

Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an extraordinary source of smallRNAs, including microRNAs (miRNAs). Contrary to other RNA molecules, miRNAs are stable, nuclease-resistant and quantifiable even in low quality samples. The accurate assessment of miRNA levels in archival samples is of great interest for many pathological conditions, including cancer. In human tumors, microRNA expression is type-specific and can be used as diagnostic, prognostic or response-to-treatment biomarker. In this study, we provide a method for multiple miRNA quantification in 96-well plates, using EvaGreen-based droplet digital PCR technology and miRCURY LNA miRNA assays. This approach allows the absolute quantification of a customizable panel of miRNAs at the same time and under identical experimental conditions, to be used for diagnostic or prognostic applications.

We are delighted to share with you our seventh Journal Club and highlight some of the most interesting papers published recently [...]

A case study of a deliberate change in the design of a new Eucalyptus plantation, aimed at alleviating water impacts, was carried out in an experimental catchment located in the center part of the State of São Paulo, Brazil. It involved the identification of saturated areas in the catchment, based essentially on topographic analysis, as a tool to help in zoning of the new forest plantation, with the objective of improving the flow of water to downstream users, as well as to avoid water quality changes. The design involved the allocation of part of the identified saturated areas as water conservation areas, as well as a change in the spacing of the planting. Measurements of tree growth at the age of two years of the new plantation reveal that the forest productivity of the new plantation design, in terms of projected annual wood increment at the end of the rotation, will be similar to the old plantation scheme, despite the loss of planted area. Preliminary results of the continuous monitoring of the catchment water balance appear to indicate that the objective of increasing the catchment water yield may possibly also be achieved.

Unlabelled: Several studies showed the reliability of the sentinel lymph node (SN) technique in the evaluation of the N parameter in breast cancer so much to induce surgeons to limit the axillary dissection to the biopsy of the SN alone (SNB) in case this is negative to the extemporaneous examination. After a period of focusing on the identification technique, biopsy and histological examination of the SN (October 97-January 98) always followed by a complete dissection of the three axillary node levels (ALND), we started a study to evaluate the reliability of a limited dissection of the 1st level of the axilla (FLND) in women with T < 3 cm, N0-1a, M0, that did not undergo any neoadjuvant treatment and in which the SN resulted free from metastases. We started this phase of the study in February 1998 till May 2001. In the present paper we show the results related to this period., Materials and Methods: We enrolled 256 women with T < 3 cm, N0-1a, M0. In 49 cases we used vital dye, in 23 dye + radioguided surgery (RGS) and in 184 RGS only. The extemporaneous histological examination of the SN has been performed with thin sections, dyed with EE. When SN was negative to the intraoperative examination, we limited the dissection to the 1st level of the axilla, except that in 3 patients, with SN located to the 2nd level, in which we did an ALND. The FLND has been performed in 17 cases with a minimally invasive technique. The definitive histological examination of the SN always included the immunohistochemistry. If the SN was positive, usually underestimated to the intraoperative examination, the patients had an adjuvant chemotherapy., Results: In 203/207 patients (98.1%) SN was found to the pre-operative lymphoscintigraphy. During surgery the SN was identified in 46/49 (94%) using the vital dye, in 22/23 (96%) using the vital dye + RGS and in 176/179 (98.3%) using RGS. To the extemporaneous histological examination SN was negative in 140, metastatic in 101; to the histological definitive results of the SN we noticed 6 false negative, since others lymph nodes than SN were positive (4 cases) or for evidence of micrometastases at the immunohistochemistry which were not detected at the extemporaneous examination (2 cases). On 107 cases of N+ the SN was the only metastatic lymph node in 42 (39.3%). The false negative percentage was 5.6% and the diagnostic accuracy of the SNB was 97.5%. In the group treated with FLND we only noticed two cases of light lymphedema (1.4%)., Conclusions: Our results are in concordance with the international literature and they induced us, from June 2001, to begin a new phase of the study in which we limit the dissection of the axilla to the SN only, if not metastatic, in women with T1 breast carcinoma.

Authors report the results of an experimental application of one of the more recent techniques of liver transplantation, which has the preparation ex situ as a basilar step of the procedure. 18 pig were operated on, from september 1991 to march 1993; they were divided in two groups: the first (group A) underwent an auxiliary segmental autotransplantation of left lobe to heterotopic subdiaphragmatic location; in group B an orthotopic segmental autotransplantation of right lobe after ex situ preparation of the liver according the technique of Pichlmayr was performed. The results show that a bigger application of ex situ surgery may help to find a resolution to the problem of the small number of liver donation.