In vivo intratumoral Epstein-Barr virus replication is associated with XBP1 activation and early-onset post-transplant lymphoproliferative disorders with prognostic implications.

Post-transplant lymphoproliferative disorders are life-threatening complications following hematopoietic or solid organ transplantation. They represent a spectrum of mostly EBV-driven lymphoplasmacytic proliferations. While the oncogenic effect of EBV is related to latent infection, lytic infection also has a role in lymphomagenesis. In vitro, EBV replication is linked to plasma cell differentiation and XBP1 activation, although this phenomenon has never been addressed in vivo. We analyzed for the first time latent and lytic intratumoral EBV infection in a series of 35 adult patients with a diagnosis of post-transplant lymphoproliferative disorder (26M/9F, median age 54 years). A complete EBV study was performed including the analysis of the latent EBER, latent membrane protein-11, and EBV nuclear antigens as well as the immediate-early BZLF1/ZEBRA and early BMRF1/EADE31 lytic genes. XBP1 activation was assessed by nuclear protein expression. EBV infection was observed in 28 (80%) cases being latency II and III the most frequently observed 22 (79%). Intratumoral EBV replication was detected in 17 (60%) cases. Among these, XBP1 activation was observed in 11/12 evaluable cases associated with strong cytoplasmic immunoglobulin expression consistent with plasma cell differentiation. Intriguingly, the combination of latency III infection and EBV replication identified a high-risk subgroup of patients with significantly shorter survival (overall survival at 1 year 18% vs 48%) and early-onset (median of 7 vs 26 months) post-transplant lymphoproliferative disorder. Moreover, these patients appear to be more heavily immunosuppressed, so they exhibit lower rates of rejection and graft vs host disease but higher rates of cytomegalovirus reactivation. In conclusion, EBV replication is associated with plasma cell differentiation and XBP1 activation with prognostic implications. Both latency III and lytic EBV infection are related to aggressive and early-onset post-transplant lymphoproliferative disorder. These results suggest that immunohistochemical study of latent and lytic EBV genes in the clinical practice may help to select higher-risk patients to new therapies including antiviral treatments.

Multiplex RNA-based detection of clinically relevant MET alterations in advanced non-small cell lung cancer.

MET inhibitors have shown activity in non-small-cell lung cancer patients (NSCLC) with MET amplification and exon 14 skipping (METΔex14). However, patient stratification is imperfect, and thus, response rates have varied widely. Here, we studied MET alterations in 474 advanced NSCLC patients by nCounter, an RNA-based technique, together with next-generation sequencing (NGS), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR), exploring correlation with clinical benefit. Of the 474 samples analyzed, 422 (89%) yielded valid results by nCounter, which identified 13 patients (3%) with METΔex14 and 15 patients (3.5%) with very-high MET mRNA expression. These two subgroups were mutually exclusive, displayed distinct phenotypes and did not generally coexist with other drivers. For METΔex14, 3/8 (37.5%) samples positive by nCounter tested negative by NGS. Regarding patients with very-high MET mRNA, 92% had MET amplification by FISH and/or NGS. However, FISH failed to identify three patients (30%) with very-high MET RNA expression, among which one received MET tyrosine kinase inhibitor treatment deriving clinical benefit. Our results indicate that quantitative mRNA-based techniques can improve the selection of patients for MET-targeted therapies.

Implementation of an NGS panel for clinical practice in paraffin-embedded tissue samples from locally advanced and metastatic melanoma patients.

Aim: Single biomarker diagnostic test of BRAFV600 locus in metastatic melanoma is mandatory for treatment decision; however, multiple-gene based techniques, such as targeted next-generation sequencing (NGS) are being used to maximize the number of patients that can benefit from a targeted therapy. The main objective of this study is to investigate whether an NGS panel could be adopted in routine clinical care for advanced melanoma. Methods: Patients diagnosed with advanced melanoma at our center from 2017 to 2019 were included. Presence of genetic alterations was performed using two methods: real-time polymerase chain reaction-based Idylla test (Biocartis) and NGS with the oncomine solid tumor DNA kit (Thermo Fisher Scientific). Total genomic DNA was extracted from formalin-fixed and paraffin embedded samples for sequencing. Results: A total of 155 samples were evaluated for molecular analysis but 40 samples (25.8%) were inadequate for sequencing. The clinical utility of BRAFV600 real-time polymerase chain reaction and targeted-NGS was compared in 29 samples and a very good concordance was observed (Kappa = 0.89, 95% confidence interval 0.68 ± 1.05). An oncogenic mutation by NGS was found in 75 samples (65%)-53% of whom were candidates for personalized therapies. The most prevalent mutated genes were BRAF (39%), TP53 (23%), and NRAS (14%). Other genes identified at lower incidence (< 5%) were: PIK3CA, ERBB4, CTNNB1, STK11, FGFR1, SMAD4, KRAS, FGFR3, PTEN and AKT. Co-occurrence of oncogenic mutations was detected in 40% of the samples. Among the mutations identified, TP53 was significantly more prevalent in men (men 31.8% versus women 12.2%, P = 0.03) and NRAS in women (men 9.1% versus women 24.4%, P = 0.03). Conclusions: Targeted-NGS testing is a feasible technique to implement in the routine clinical practice. Based on our results, NGS has provided more information on target-genes than RT-PCR technique, maximizing the benefit for patients with advanced melanoma.

TP53 mutation and tumoral PD-L1 expression are associated with depth of invasion in desmoplastic melanomas.

BACKGROUND: Desmoplastic melanoma (DM) is a rare subtype of spindle cell malignant melanoma characterized by frequent local recurrences and hematogenous spread, but without molecular classification. The aim of the study was to investigate in a DM series the incidence of relevant gene alterations in cancer, the programmed death-ligand 1 (PD-L1) expression status and the association with clinicopathological features and melanoma progression. METHODS: A total of 38 patients were included. Clinical follow-up and the histopathological features of all cases were retrospectively collected. PD-L1 expression by immunohistochemistry (IHC) and BRAF genomic alterations by real-time PCR were determined in 34 samples. Additionally, a molecular analysis by next-generation sequencing was performed in 25 DMs. RESULTS: Tumors occurred predominantly in men (76%) and in the head and neck region (50%). Most tumors were pure DMs (66%), containing less than 10% of conventional melanoma. Overall, 48% of our cohort harbored TP53 mutations, most of them showing a molecular signature associated with ultraviolet (UV)-oncogenesis, and 29%, BRAF mutations. A positive correlation between TP53 with depth of invasion (P=0.005) and presence of elastosis (P=0.002) was found. High-expression of PD-L1 in tumor cells was observed in 38% of cases and correlated with depth of tumoral infiltration (P=0.003), TP53 (P=0.016), PD-1 (P<0.001) and tumor-infiltrating lymphocytes (TILS) (P<0.001). PD-L1 expression in immune cells correlated with PD-1 (P=0.006), tumoral PD-L1 expression (P=0.029) and TP53 mutation (P=0.002). Survival correlated with depth of invasion (P=0.003), stage of tumors (P=0.015), positive sentinel lymph node (P=0.004), lymph node metastasis (P=0.024) and distant metastasis (P<0.001). CONCLUSIONS: Our results suggest that progressed DMs with deep tumoral infiltration frequently harbor TP53 mutations, PD-L1 expression and present a high inflammatory response, probably related to adaptive immune resistance in this tumor-type.

Prospective Evaluation of Single Nucleotide Variants by Two Different Technologies in Paraffin Samples of Advanced Non-Small Cell Lung Cancer Patients.

Targeted therapies are a new paradigm in lung cancer management. Next-generation sequencing (NGS) techniques have allowed for simultaneous testing of several genes in a rapid and efficient manner; however, there are other molecular diagnostic tools such as the nCounter® Vantage 3D single nucleotide variants (SNVs) solid tumour panel which also offer important benefits regarding sample input and time-to-response, making them very attractive for daily clinical use. This study aimed to test the performance of the Vantage panel in the routine workup of advanced non-squamous non-small cell lung cancer (NSCLC) patients and to validate and compare its outputs with the Oncomine Solid Tumor (OST) panel DNA kit, the standard technique in our institution. Two parallel multiplexed approaches were performed based on DNA NGS and direct digital detection of DNA with nCounter® technology to evaluate SNVs. A total of 42 advanced non-squamous NSCLC patients were prospectively included in the study. Overall, 95% of samples were successfully characterized by both technologies. The Vantage panel accounted for a sensitivity of 95% and a specificity of 82%. In terms of predictive values, the probability of truly presenting the SNV variant when it is detected by the nCounter panel was 82%, whereas the probability of not presenting the SNV variant when it is not detected by the platform was 95%. Finally, Cohen's Kappa coefficient was 0.76, indicating a substantial correlation grade between OST and Vantage panels. Our results make nCounter an analytically sensitive, practical and cost-effective tool.

Assessment of SOX11 expression in routine lymphoma tissue sections: characterization of new monoclonal antibodies for diagnosis of mantle cell lymphoma.

The diagnosis of mantle cell lymphoma (MCL) can be difficult, especially when no t(11;14) translocation and cyclin D1 overexpression can be detected. In such cases, the transcription factor SOX11 represents an important diagnostic marker, as it is expressed in most MCLs and, in particular, in all cyclin D1-negative MCLs reported so far. A reliable anti-SOX11 antibody is therefore a very useful tool for routine diagnosis. Here, we characterize the new monoclonal anti-SOX11 antibodies, suitable for Western blot assay and immunohistochemistry (IHC) on formalin-fixed paraffin-embedded tissue; we tested them on a large series of primary lymphoid tumors and compared these results with those of other routinely used antibodies. Moreover, we show that IHC results depend on transcription levels of SOX11, which suggests that posttranscriptional and posttranslational modifications do not significantly affect cutoff levels for IHC detection of SOX11.

Nonhepatosplenic γδ T-cell lymphomas represent a spectrum of aggressive cytotoxic T-cell lymphomas with a mainly extranodal presentation.

γδ T cells represent a minor T-cell subset that is mainly distributed in mucosal surfaces. Two distinct lymphomas derived from these cells have been recognized: hepatosplenic γδ T-cell lymphoma (HSTL) and primary cutaneous γδ T-cell lymphoma (PCGD-TCL). However, whether other anatomic sites may also be involved and whether they represent a spectrum of the same disease are not well studied. The lack of T-cell receptor (TCR)ß expression has been used to infer a γδ origin when other methods are not available. We studied 35 T-cell tumors suspected to be γδ TCL using monoclonal antibodies reactive with TCR δ or γ in paraffin sections. We were able to confirm γδ chain expression in 22 of 35 cases. We identified 8 PCGD-TCLs, 6 HSTLs, and 8 γδ TCLs without hepatosplenic or cutaneous involvement involving mainly extranodal sites. Two such cases were classified as enteropathy-associated T-cell lymphoma, type II. The other γδ TCL presented in the intestine, lung, tongue, orbit, and lymph node. In addition, we observed 13 cases with mainly extranodal involvement that lacked any TCR expression ("TCR silent"). In all cases, a natural killer cell origin was excluded. In conclusion, the lack of TCRß expression does not always predict γδ-T-cell derivation, as TCR silent cases may be found. The recognition of γδ TCL presenting in extranodal sites other than skin and liver/spleen expands the clinical spectrum of these tumors. However, non-HSTL γδ TCL do not seem to represent a single entity. The relationship of these tumors with either HSTL or PCGD-TCL requires further study.

Predeposit self-transfusion (PDS) in a hepatobiliopancreatic surgery (HBPS) unit: preliminary data.

Hepatobiliary pancreatic surgery (HBPS) has high morbility and mortality and frequently requires blood transfusion. Allogeneic transfusion may cause adverse sequelae. Predeposit self-transfusiOn (PDS) minimizes allogeneic blood transfusion and avoids most adverse reactions. We present the preliminary data of our PDS experience (with recombinant human erythropoieting, r-HuEPO) in HBPS during the first year. We studied our first-year HBPS-PDS program by a retrospective review of the case histories and transfusion records in our Blood Bank. Sex, weight, underlying disease, packed red cell units (PRCUs) requested, drawn, and transfused, and hospital and ICU stays were analyzed. Nine patients were admitted in the PDS program. Of desired blood units, 83% was obtained, successfully in 77.8% of patients, and 63.2% were transfused with autologous blood transfusion. Only three patients needed allogeneic blood (33.3%). All complications occurred in patients who received allogeneic units. Also, we found stays were three times longer in those patients. PDS could be a valid and safe alternative for patients undergoing elective HBPS because it decreases allogeneic blood requirements, reduces overall complications, and also reduces hospital and ICU stays.