Prevalence of Molecular Alterations in a Swiss Cohort of 512 Colorectal Carcinoma Patients by Targeted Next-Generation Sequencing Analysis in Routine Diagnostics.

INTRODUCTION: Colorectal carcinoma (CRC) is among the most common carcinomas in women and men. In the advanced stage, patients are treated based on the RAS status. Recent studies indicate that in the future, in addition to KRAS and NRAS, alterations in other genes, such as PIK3CA or TP53, will be considered for therapy. Therefore, it is important to know the mutational landscape of routinely diagnosed CRC. METHOD: We report the molecular profile of 512 Swiss CRC patients analyzed by targeted next-generation sequencing as part of routine diagnostics at our institute. RESULTS: Pathogenic and likely pathogenic variants were found in 462 (90%) CRC patients. Variants were detected in TP53 (54.3%), KRAS (48.2%), PIK3CA (15.6%), BRAF (13.5%), SMAD4 (10.5%), FBXW7 (7.8%), NRAS (3.5%), PTEN (2.7%), ERBB2 (1.6%), AKT1 (1.5%), and CTNNB1 (0.9%). The remaining pathogenic alterations were found in the genes ATM(n= 1), MAP2K1(n= 1), and IDH2(n= 1). DISCUSSION/CONCLUSIONS: Our analysis revealed the prevalence of potential predictive markers in a large cohort of CRC patients obtained during routine diagnostic analysis. Furthermore, our study is the first of this size to uncover the molecular landscape of CRC in Switzerland.

NFATc2-rearranged sarcomas: clinicopathologic, molecular, and cytogenetic study of 7 cases with evidence of AGGRECAN as a novel diagnostic marker.

NFATc2-rearranged sarcomas (NFATc2-Sarcomas) are infrequent round cell tumors characterized by EWSR1-NFATc2 fusions and FUS-NFATc2 fusions. Although our knowledge on these neoplasms has increased recently, novel diagnostic tools and more comprehensive series are still needed. Here, we describe the features of a series of seven molecularly confirmed NFATc2-Sarcomas (EWSR1-NFATc2, n = 4; FUS-NFATc2, n = 3) and demonstrate the utility of AGGRECAN immunohistochemistry for their identification. Patients were four males and three females, ranging in age from 19 to 66 years (median: 33). All were primary bone tumors (femur, n = 4; tibia, n = 2; ilium, n = 1), frequently infiltrating the surrounding soft tissues. Treatment often consisted of neoadjuvant chemotherapy and surgery. Follow-up was available for six patients (median 18 months, range 5-102 months), three patients died of disease and four patients are currently alive. Histologically, tumors consisted of monotonous round cells growing in lobules and sheets in variable amounts of fibrous to myxoid stroma. Other findings included spindle cells, corded and trabecular architecture, nuclear pleomorphism, cartilaginous differentiation, and osteoid-like matrix. Histological response to neoadjuvant chemotherapy was poor in all resection specimens available for review (n = 4). Tumors were diffusely positive for AGGRECAN and CD99 (7/7), and a subset expressed Pan-Keratin (AE1-AE3; 3/6), S100 (2/6), BCOR (2/6), ETV-4 (2/5), WT1 (2/6), and ERG (2/5). Desmin, NKX3-1, and SATB2 were negative (0/6). Diffuse AGGRECAN staining was also seen in 8/129 round cell sarcomas used for comparison, including mesenchymal chondrosarcoma (7/26) and CIC-sarcoma (1/26). Array-CGH showed complex karyotypes with recurrent deletions of tumor suppressor genes (CDKN2A/B, TUSC7, and DMD) in three FUS-NFATC2 cases and a simpler profile without homozygous losses in one EWSR1-NFATc2 case. Segmental chromosomal gains covering the loci of the fusion genes were detected in both variants. Overall, our study confirms and expands previous observations on NFATc2-sarcomas and supports that AGGRECAN is a useful biomarker of these tumors.

Correction: NFATc2-rearranged sarcomas: clinicopathologic, molecular, and cytogenetic study of 7 cases with evidence of AGGRECAN as a novel diagnostic marker.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular Profile of Gastrointestinal Stromal Tumors in Sixty-Eight Patients from a Single Swiss Institution.

INTRODUCTION: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm of the gastrointestinal tract. It has distinct molecular features and primarily affects the KIT and PDGFRA genes. OBJECTIVE: We wanted to assess the molecular profile of 68 GIST patients who were sequenced consecutively between 2014 and 2019 at our institute of pathology. METHODS: Our cohort comprised 60 primary and 8 metastatic GIST patients; 43 and 57% of the cases, respectively, were analyzed by Sanger sequencing or next-generation sequencing (NGS). RESULTS: Of the 60 primary GIST patients, 47 (78%) showed a KIT mutation; 2 cases showed a double KIT mutation, and 1 of these was a therapy-naive GIST. Nine (15%) patients harbored a PDGFRA mutation, 2 (3%) had a BRAF mutation, 1 (2%) had a PIK3CA mutation, and 1 (2%) did not show any mutation. One BRAF and the PIK3CA mutation have not been described in GIST before. All metastatic GIST harbored exclusively KIT mutations. CONCLUSION: A retrospective analysis of GIST sequenced at our institute revealed incidences of KIT and PDGFRA mutations comparable to those in other cohorts from Europe. Interestingly, we found 2 previously undescribed mutations in the BRAF and PIK3CA genes as well as 1 treatment-naive case with a double KIT mutation in exon 11.

CD10 delineates a subset of human IL-4 producing follicular helper T cells involved in the survival of follicular lymphoma B cells.

In follicular lymphoma (FL), follicular helper T cells (TFH) have been depicted as one of the main components of the malignant B-cell niche and a promising therapeutic target. Although defined by their capacity to sustain FL B-cell growth together with specific gene expression and cytokine secretion profiles, FL-TFH constitute a heterogeneous cell population. However, specific markers reflecting such functional heterogeneity are still lacking. In this study, we demonstrate that CD10 identifies a subset of fully functional germinal center TFH in normal secondary lymphoid organs. Importantly, this subset is amplified in the FL context, unlike in other B-cell lymphomas with a follicular growth pattern. Furthermore, whereas FL-TFH produce high levels of interleukin (IL)-21 and low levels of IL-17 irrespectively of their CD10 expression, CD10(pos) FL-TFH specifically exhibit an IL-4(hi)IFN-γ(lo)TNF-α(hi) cytokine profile associated with a high capacity to sustain directly and indirectly malignant B-cell survival. Altogether, our results highlight the important role of this novel functional subset in the FL cell niche.

Lymphadenectomy Specimens in a Large Retrospective Cohort of Pediatric Patients Reveal No in situ Lymphomas but a Broad Spectrum of Reactive Changes.

OBJECTIVES: While the incidence and prevalence of in situ follicular neoplasia (ISFN) and in situ mantle cell neoplasia (ISMCN) in adults are well documented, little is known about these early (precursor) lesions in pediatric populations. The aim of this study was to analyze so-called 'reactive' lymph nodes harvested for the purpose of staging solid tumors, unexplained lymphadenopathies, or presumed inflammatory processes or in conjunction with other surgical interventions in children and adolescents aged <18 years, with special attention to ISFN and ISMCN. METHODS: Formalin-fixed, paraffin-embedded reactive lymph node samples from an unselected pediatric population from two catchment areas in Switzerland were retrospectively analyzed for the presence of ISFN and ISMCN and specific reactive lymph node patterns. RESULTS: While a diverse range of histopathological patterns of reactive lymph node changes with a particular periodic increase in mycobacterioses could be observed in this pediatric population, not a single case of ISFN or ISMCN was found. CONCLUSIONS: Early histological lymphomagenesis equivalents in the form of in situ lymphomas are exceedingly rare events in children and young adolescents. The spectrum of reactive lymph node changes is large, with differences possibly determined by regional variations in geography, demographics, catchment areas, seasons, and years, respectively.

Strømme Syndrome Is a Ciliary Disorder Caused by Mutations in CENPF.

Strømme syndrome was first described by Strømme et al. (1993) in siblings presenting with "apple peel" type intestinal atresia, ocular anomalies and microcephaly. The etiology remains unknown to date. We describe the long-term clinical follow-up data for the original pair of siblings as well as two previously unreported siblings with a severe phenotype overlapping that of the Strømme syndrome including fetal autopsy results. Using family-based whole-exome sequencing, we identified truncating mutations in the centrosome gene CENPF in the two nonconsanguineous Caucasian sibling pairs. Compound heterozygous inheritance was confirmed in both families. Recently, mutations in this gene were shown to cause a fetal lethal phenotype, the phenotype and functional data being compatible with a human ciliopathy [Waters et al., 2015]. We show for the first time that Strømme syndrome is an autosomal-recessive disease caused by mutations in CENPF that can result in a wide phenotypic spectrum.

Transient myeloproliferative disorder in neonates without Down syndrome: case report and review.

Transient myeloproliferative disorder (TMD) is a clonal proliferation of megakaryoblasts, typically occurring in newborns with Down syndrome. It is believed that TMD occurs in the presence of GATA1 mutation together with trisomy 21. However, a limited number of patients with TMD but without Down syndrome have been reported, all with a blast population with numeric or rarely structural chromosome 21 abnormalities. We present the first case of a newborn boy with a TMD without trisomy 21 and without any of the mentioned molecular or cytogenetic abnormalities. This case report suggests that unknown disease mechanisms may provoke or mimic TMD. This case report is followed by a concise review of the literature discussing the different entities and pathomechanisms of TMD and acute megakaryocytic leukaemia in patients with or without Down syndrome.

Two centres experience of lung cancer resection in patients with advanced non-small cell lung cancer upon treatment with immune checkpoint inhibitors: safety and clinical outcomes.

OBJECTIVES: Recent trials have begun to explore immune checkpoint inhibitors for non-small cell lung cancer in the neoadjuvant setting, but data on tumour response and surgical outcome remain limited. METHODS: Retrospective evaluation of clinical data from patients with non-small cell lung cancer treated with immune checkpoint inhibitors followed by lung resection was performed at 2 large volume institutions (1 North American, 1 European). Data were analysed using Chi-squared, Fisher's and Wilcoxon rank-sum tests where appropriate. RESULTS: Thirty-seven patients were identified from 2017 to 2019. Forty-nine per cent were Stage IIIB and IV. Forty-six per cent received immunotherapy alone and 54% in combination with chemo- and/or radiotherapy. Sixteen per cent of cases were successfully performed minimally invasively. Twenty patients were operated with lobectomy (6 of these with wedges or segments of a neighbouring lobe, 2 with sleeve resections and 1 with a chest wall resection), 4 with bilobectomies, 11 with pneumonectomy (including 5 extrapleural pneumonectomies and 1 atrial resection) and 1 with a wedge resection. Overall, 10 patients (27%) developed postoperative complications and the 90-day mortality was zero. One-year recurrence-free survival was 73% for stage II/IIIA and 55% for stage IIIB/stage IV. The major pathologic response rate was 34%. CONCLUSION: In this retrospective study, lung resection after immunotherapy (alone or in combination) is safe, although often requires complex surgery. Due to increasing number of clinical trials adopting immunotherapy in the neoadjuvant setting, it is likely that this therapy will become part of standard of care. Immunotherapy may also allow surgery to have a role for selected patients with advanced disease.

Blunted sFasL signalling exacerbates TNF-driven neutrophil necroptosis in critically ill COVID-19 patients.

OBJECTIVES: Critically ill coronavirus disease 2019 (COVID-19) patients are characterised by a severely dysregulated cytokine profile and elevated neutrophil counts, impacting disease severity. However, it remains unclear how neutrophils contribute to pathophysiology during COVID-19. Here, we assessed the impact of the dysregulated cytokine profile on the regulated cell death (RCD) programme of neutrophils. METHODS: Regulated cell death phenotype of neutrophils isolated from critically ill COVID-19 patients or healthy donors and stimulated with COVID-19 or healthy plasma ex vivo was assessed by flow cytometry, time-lapse microscopy and cytokine multiplex analysis. Immunohistochemistry of COVID-19 patients and control biopsies were performed to assess the in situ neutrophil RCD phenotype. Plasma cytokine levels of COVID-19 patients and healthy donors were measured by multiplex analysis. Clinical parameters were correlated to cytokine levels of COVID-19 patients. RESULTS: COVID-19 plasma induced a necroptosis-sensitive neutrophil phenotype, characterised by cell lysis, elevated release of damage-associated molecular patterns (DAMPs), increased receptor-interacting serine/threonine-protein kinase (RIPK) 1 levels and mixed lineage kinase domain-like pseudokinase (MLKL) involvement. The occurrence of neutrophil necroptosis MLKL axis was further confirmed in COVID-19 thrombus and lung biopsies. Necroptosis was induced by the tumor necrosis factor receptor 1 (TNFRI)/TNF-α axis. Moreover, reduction of soluble Fas ligand (sFasL) levels in COVID-19 patients and hence decreased signalling to Fas directly increased RIPK1 levels, exacerbated TNF-driven necroptosis and correlated with disease severity, which was abolished in patients treated with glucocorticoids. CONCLUSION: Our results suggest a novel role for sFasL signalling in the TNF-α-induced RCD programme in neutrophils during COVID-19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome.

FOXP1 protein overexpression is associated with inferior outcome in nodal diffuse large B-cell lymphomas with non-germinal centre phenotype, independent of gains and structural aberrations at 3p14.1.

AIMS: To determine the molecular epidemiology and prognostic importance of structural and numeric FOXP1 gene aberrations with respect to BCL-6 gene and to FOXP1 protein expression in 389 diffuse large B-cell lymphomas (DLBCL) from the pre-rituximab era on tissue microarrays. METHODS AND RESULTS: By interphase fluorescence in situ hybridization with colour-labelled bacterial artificial chromosome clones, 12% (27/223) analysable cases showed FOXP1 gains and 1% (2/210) FOXP1 breaks. Seven percent of cases with known BCL-6 and FOXP1 gene status (n = 159) showed an isolated FOXP1 gain, 19% an isolated BCL-6 gain and 18% a trisomy 3. FOXP1 gains (isolated and due to trisomy 3) were more frequent in nodal than extranodal DLBCL and in non-germinal centre B-cell-like (non-GCB) DLBCL than in GCB DLBCL. By immunohistochemistry, FOXP1 protein was more often overexpressed in non-GCB than in GCB cases. FOXP1 overexpression was associated with poor disease-specific survival in all DLBCL, particularly in nodal and non-GCB cases. There was no correlation between FOXP1 gene aberrations and either FOXP1 protein expression or survival. CONCLUSIONS: FOXP1 is recurrently targeted by numeric, and rarely by structural, genetic aberrations in DLBCL. Only the presence of FOXP1 protein, irrespective of its gene status, is decisive for prognosis in DLBCL.

BOB.1, CD79a and cyclin E are the most appropriate markers to discriminate classical Hodgkin's lymphoma from primary mediastinal large B-cell lymphoma.

AIMS: To clarify which immunohistochemical markers could be helpful in distinguishing between classical Hodgkin's lymphoma (cHL) and primary mediastinal B-cell lymphoma (PMBCL) to more narrowly define 'B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and cHL'. METHODS AND RESULTS: Two hundred and 83 cHLs and 51 PMBCLs were analysed on validated tissue microarray platforms with antibodies to BOB.1, CD15, CD20, CD23, CD30, CD79a, cyclin E, LMP-1, MUM1p, p63 and Oct2. The marker cut-off scores were calculated using receiver-operating characteristic curves. Markers with the highest positive predictive value for cHL were: CD15, cyclin E, LMP-1 (all 100%), MUM1p (93%) and CD30 (83%). High sensitivity was achieved only by CD30 (92%) and cyclin E (79%). Nineteen percent of PMBCLs were also positive for CD30, which led to a lower specificity of CD30 as regards cHL (81%) compared with cyclin E (100%). The antibodies with the highest positive predictive value for PMBCL were: CD23 (98%), p63 (96%), BOB.1 (94%) and CD79a (90%), with high sensitivity for BOB.1 (100%), CD79a (89%) and p63 (82%). CONCLUSIONS: The use of at least three of the most accurate immunohistochemical markers, cyclin E, CD79a and BOB.1, may be helpful in the differential diagnosis of cHL and PMBCL.

TP53 and PTEN as driver mutations in Zenker's carcinoma-a clinical presentation.

Zenker carcinoma is still being treated empirically because of the lack of evidence- based guidelines. We report for the first time about the genetic examination of this rare entity. The revealed mutations show genetic similarities with HPV(-)HNSCC which suggests that well-known therapeutic strategies may be applicable for this disease.

Recurrent numerical aberrations of JAK2 and deregulation of the JAK2-STAT cascade in lymphomas.

The Janus kinase 2 (JAK2)-signal transducers and activators of transcription (STAT) pathway plays an important role in hematological malignancies. Mutations and translocations of the JAK2 gene, mapped at 9p24, lead to constitutive activation of JAK2 and its downstream targets. The presence of JAK2 mutations in lymphomas has been addressed in larger cohorts, but there are little systemic data on numerical and structural JAK2 aberrations in lymphoid neoplasms. To study the molecular epidemiology of these aberrations and the consecutive activation of the JAK2-STAT pathway in lymphomas, we examined 527 cases, covering the most common entities, in a tissue microarray by fluorescent in situ hybridization with breakable JAK2 probes, and immunohistochemistry for phosphorylated JAK2 (pJAK2) and its preferred downstream pSTAT3 and pSTAT5. 9p24 gains were detected in 6/17 (35%) primary mediastinal B-cell lymphomas (PMBCLs), 25/77 (33%) Hodgkin's lymphomas (HLs), 3/16 (19%) angioimmunoblastic T-cell lymphomas (AILTs) and 1/5 ALK1(+) anaplastic large cell lymphomas (ALCLs); breaks were observed only in three cases. pJAK2 expression was most prevalent in PMBCL, peripheral T-cell lymphomas and HL. pSTAT3 predominated in ALCLs, HLs, AILTs, PMBCLs and peripheral T-cell lymphomas. pSTAT5 expression was detected frequently in follicular lymphomas, diffuse large B-cell lymphomas and AILTs. 9p24 gains correlated with increased proportions of tumor cells expressing pJAK2 (P=0.002) and pSTAT3 (P=0.001). In follicular lymphomas, concomitant expression of pJAK2 and pSTAT5 was linked to better prognosis, whereas expression of pSTAT3 in nongerminal center-like diffuse large B-cell lymphomas could identify a patient group with an inferior outcome. Our findings stress that despite the rarity of activating JAK2 mutations in lymphomas, JAK2 is recurrently targeted by numerical, and rarely by structural, genetic aberrations in distinct lymphoma subtypes and that JAK2-STAT pathway may play a role in lymphomagenesis.

Exome sequencing of fetal anomaly syndromes: novel phenotype-genotype discoveries.

The monogenic etiology of most severe fetal anomaly syndromes is poorly understood. Our objective was to use exome sequencing (ES) to increase our knowledge on causal variants and novel candidate genes associated with specific fetal phenotypes. We employed ES in a cohort of 19 families with one or more fetuses presenting with a distinctive anomaly pattern and/or phenotype recurrence at increased risk for lethal outcomes. Candidate variants were identified in 12 families (63%); in 6 of them a definite diagnosis was achieved including known or novel variants in recognized disease genes (MKS1, OTX2, FGFR2, and RYR1) and variants in novel disease genes describing new fetal phenotypes (CENPF, KIF14). We identified variants likely causal after clinical and functional review (SMAD3, KIF4A, and PIGW) and propose novel candidate genes (PTK7, DNHD1, and TTC28) for early human developmental disease supported by functional and cross-species phenotyping evidence. We describe rare and novel fetal anomaly syndromes and highlight the diagnostic utility of ES, but also its contribution to discovery. The diagnostic yield of the future application of prenatal ES will depend on our ability to increase our knowledge on the specific phenotype-genotype correlations during fetal development.

Human Papillomavirus (HPV) Detection in Cytologic Specimens: Similarities and Differences of Available Methodology.

Accumulating evidence regarding the causative role of human papillomavirus (HPV) in a wide range of malignant and nonmalignant diseases highlights the importance of HPV testing. This study describes and discusses the efficacy and characteristics of 4 well-established and commercially available tests. Here, 181 cytologic specimens from cervical smears were analyzed using the HPV SIGN PQ (Diatech) and the Linear Array (Roche) method. Discrepant results were further studied with the Real Time High-Risk HPV (Abbott) method and the INNO-LiPA (Fujirebio) method. Of 181 cytologic specimens, 61 (34%) showed discrepant results. High-risk HPV was not detected in 9 cases by HPV SIGN PQ, in 16 cases by Linear Array, in 10 cases by Real Time High-Risk HPV, and in 6 cases by INNO-LiPA, respectively. Lack of DNA detection or problems in interpreting the result were seen in 9 cases with HPV SIGN PQ, 8 cases with Linear Array, 3 cases with Real Time High-Risk HPV, and 3 cases with INNO-LiPA, respectively. This study indicates that the choice of HPV detection method has a substantial influence on the HPV risk classification of tested PAP smears and clinical follow-up decisions.

In Europe expression of EBNA2 is associated with poor survival in EBV-positive diffuse large B-cell lymphoma of the elderly.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) of the elderly occurs by definition in patients above the age of 50 years without any known underlying immunodeficiency. We investigated the incidence and clinical relevance of this subtype in Europe with special attention to the EBV-latency type. Among the 598 DLBCL, 15 EBV-positive lymphomas fulfilling the criteria of EBV-positive DLBCL of the elderly were identified (2.5%). Patients with EBV-positive DLBCL expressing EBNA2 showed a significantly poorer overall survival than patients with EBNA2-negative EBV-positive DLBCL (p = 0.0156). The incidence of EBV-positive DLBCL of the elderly in Europe is much lower than in Asian countries (2.5% of all cases of DLBCL). Interestingly, the likelihood of EBV positivity did not increase with age in patient above 50 years. Among EBV-positive DLBCL of the elderly a subgroup with EBV-latency type III expressing EBNA2 can be identified, which shows a poor outcome.

The PD-1/PD-L1 pathway: biological background and clinical relevance of an emerging treatment target in immunotherapy.

INTRODUCTION: The co-inhibitory receptor programmed death 1 (PD-1) and its ligands are key regulators in a wide spectrum of immune responses and play a critical role in autoimmunity and self-tolerance as well as in cancer immunology. Emerging evidence suggests that cancer cells might use the PD-1/PD-ligand (PD-L) pathway to escape anti-tumor immunity. Based on this evidence, early phase human clinical trials targeting the PD-1/PD-L pathway are currently underway for multiple human cancers. AREAS COVERED: The role of the PD-1/PD-L pathway in autoimmune disease, viral infections as well as in malignant neoplasms is discussed and an overview of the existing therapeutics as well as the results of clinical trials targeting this pathway in cancer is given. EXPERT OPINION: The PD-1/PD-L pathway represents an important mechanism of immune evasion for malignant neoplasms. Early clinical trials indicate effectiveness of PD-1/PD-L pathway blockade in several solid cancers. However, greater insight into the exact mechanisms by which tumors are able to evade anti-tumor immunity is needed to increase clinical effectiveness, for example by combination blockade of diverse co-inhibitory receptors.