Tumor infiltration levels of CD3, Foxp3 (+) lymphocytes and CD68 macrophages at diagnosis predict 5-year disease-specific survival in patients with oropharynx squamous cell carcinoma

Head and neck cancer (HNC) is the sixth most common cancer worldwide. Oropharyngeal (OP) cancers are of special interest because of possible underlying HPV infection which is tied to prognosis. Influxes of inflammatory cells into tumors may vary with prognoses. We wanted to study whether the number of tumor-infiltrating lymphocytes (TIL) and tumor-associated macrophages (TAM) in tumors correlated to HPV status and predicted 5-year disease-specific survival (DSS). Formalin-fixed paraffin-embedded (FFPE) biopsies cut sections from 170 patients treated for OP cancer were stained by immunohistochemistry and evaluated for the number of CD68 (+) TAMs, CD3 (+), and Foxp3 (+) (T regulatory) TILs. From FFPE slides HPV by PCR and p16 by immunohistochemistry were established. From FFPE Hematoxylin-Eosin slides, levels of tumor nuclear polymorphism, tumor invasion, desmoplasia, and inflammation were determined as previously published. Levels of TIL CD3 (+) and TIL Foxp3 (+) were increased among the HPV (+) compared to the HPV (−) patients. High levels of TIL Foxp3 (+) and CD68 (+) macrophages predicted better 5-year DSS. TIL Foxp3 (+) levels predicted independent of age, gender, TNM stage, and HPV infection as well as level of stromal desmoplasia, tumor invasion, and nuclear polymorphism, but more pronounced among tumor HPV (+) than HPV (−) patients.publishedVersio

Impact of KRAS and BRAF mutations on treatment efficacy and survival in high-grade gastroenteropancreatic neuroendocrine neoplasms

High-grade gastroenteropancreatic neuroendocrine neoplasms (HG GEP-NEN) typically disseminate early. Treatment of metastatic disease has limited benefit and prognosis is generally discouraging. Data on the clinical impact of mutations in HG GEP-NEN are scarce. There is an unmet need for reliable biomarkers to predict treatment outcome and prognosis in metastatic HG GEP-NEN. Patients with metastatic HG GEP-NEN diagnosed at three centres were selected for KRAS-, BRAF mutation and microsatellite instability (MSI) analyses. Results were linked to treatment outcome and overall survival. After pathological re-evaluation, 83 patients met inclusion criteria: 77 (93%) GEP neuroendocrine carcinomas (NEC) and six (7%) GEP neuroendocrine tumours (NET) G3. NEC harboured higher frequency of mutations than NET G3. Colon NEC harboured a particular high frequency of BRAF mutations (63%). Immediate disease progression on first-line chemotherapy was significantly higher for NEC with BRAF mutation (73%) versus wild-type (27%) (p = .016) and for colonic primary (65%) versus other NEC (28%) (p = .011). Colon NEC had a significant shorter PFS compared to other primary sites, a finding independent of BRAF status. Immediate disease progression was particularly frequent for BRAF mutated colon NEC (OR 10.2, p = .007). Surprisingly, BRAF mutation did not influence overall survival. KRAS mutation was associatedwith inferior overall survival for the wholeNEC population (HR 2.02, p = .015), but not for those given first-line chemotherapy. All long-term survivors (>24 m) were double wild-type. Three NEC cases (4.8%) were MSI. Colon NEC with BRAF mutation predicted immediate disease progression on first-line chemotherapy, but did not affect PFS or OS. Benefit of first-line platinum/etoposide treatment seems limited for colon NEC, especially for BRAF mutated cases. KRAS mutations did not influence treatment efficacy nor survival for patients receiving first-line chemotherapy. Both frequency and clinical impact of KRAS/BRAF mutations in digestive NEC differ from prior results on digestive adenocarcinoma.publishedVersio

Homologous Recombination Deficiency Across Subtypes of Primary Breast Cancer

Homologous recombination; Primary breast cancerRecombinació homòloga; Càncer de mama primariRecombinación homóloga; Cáncer de mama primarioPURPOSE Homologous recombination deficiency (HRD) is highly prevalent in triple-negative breast cancer (TNBC) and associated with response to PARP inhibition (PARPi). Here, we studied the prevalence of HRD in non-TNBC to assess the potential for PARPi in a wider group of patients with breast cancer. METHODS HRD status was established using targeted gene panel sequencing (360 genes) and BRCA1 methylation analysis of pretreatment biopsies from 201 patients with primary breast cancer in the phase II PETREMAC trial (ClinicalTrials.gov identifier: NCT02624973). HRD was defined as mutations in BRCA1, BRCA2, BRIP1, BARD1, or PALB2 and/or promoter methylation of BRCA1 (strict definition; HRD-S). In secondary analyses, a wider definition (HRD-W) was used, examining mutations in 20 additional genes. Furthermore, tumor BRCAness (multiplex ligation-dependent probe amplification), PAM50 subtyping, RAD51 nuclear foci to test functional HRD, tumor-infiltrating lymphocyte (TIL), and PD-L1 analyses were performed. RESULTS HRD-S was present in 5% of non-TNBC cases (n = 9 of 169), contrasting 47% of the TNBC tumors (n = 15 of 32). HRD-W was observed in 23% of non-TNBC (n = 39 of 169) and 59% of TNBC cases (n = 19 of 32). Of 58 non-TNBC and 30 TNBC biopsies examined for RAD51 foci, 4 of 4 (100%) non-TNBC and 13 of 14 (93%) TNBC cases classified as HRD-S had RAD51 low scores. In contrast, 4 of 17 (24%) non-TNBC and 15 of 19 (79%) TNBC biopsies classified as HRD-W exhibited RAD51 low scores. Of nine non-TNBC tumors with HRD-S status, only one had a basal-like PAM50 signature. There was a high concordance between HRD-S and either BRCAness, high TIL density, or high PD-L1 expression (each P < .001). CONCLUSION The prevalence of HRD in non-TNBC suggests that therapy targeting HRD should be evaluated in a wider breast cancer patient population. Strict HRD criteria should be implemented to increase diagnostic precision with respect to functional HRD.Supported by unrestricted grants from The K.G. Jebsen Foundation [SKGJ-MED-020 to H.P.E., S.K., P.E.L.], Helse Vest [912008 to P.E.L.], The Norwegian Research Council [273354 to P.E.L.] and The Norwegian Cancer Society [190281-2017 to S.K., 190275-2017 to P.E.L.]. Research funding was provided by Grieg Foundation (to H.P.E. and T.A.), Helse Vest [912252; Clinical researcher fellowship to H.P.E.], Generalitat de Catalunya [PERIS, SLT017/20/000081 to A.H.R.], “la Caixa” Foundation and European Institute of Innovation and Technology/Horizon 2020 [LCF/TR/CC19/52470003 to A.L.G.], Asociación Española Contra el Cáncer [AECC, INVES20095LLOP to A.L.G.], Generalitat de Catalunya (PERIS Fellowship SLT002/16/00477 to A.L.G.) ERA PerMed [2019-215 to V.S.] and Miguel Servet fellowship (CPII19/00033 to V.S.). Additional funding and study medication was provided by Illumina [grant number 9529854], Pfizer [WI206347] and AstraZeneca [ESR-14-10077] to H.P.E., S.K., P.E.L

A Comparison of p53 Isoform Profiles and Apoptosis Induced by Camptothecin or a Herbal Khat Extract (Catha Edulis (Vahl) Forssk. ex Endl.) in Leukemic Cell Lines: Exploring Cellular Responses in Therapy Development

Khat (Catha edulis (Vahl) Forssk. ex Endl.) is habitually used as a natural stimulant by millions of people, but is associated with adverse effects on gastrointestinal, cardiovascular and central neural systems. At the cellular level khat toxicity involves p53 induction and cell cycle arrest, decreased mitochondrial function and activation of receptor- and mitochondria-mediated cell death pathways. In this study we have examined an extract of khat for induction of p53 post-translational modifications (PTMs) and the functional role of p53 in khat-mediated cell death. Khat was shown to induce phosphorylation and acetylation of p53 in both the khat-sensitive MOLM-13 and the khat-resistant MV-4-11 cell line, but accumulation of the full-length p53 isoform was only observed in the khat sensitive cell line. Small molecule inhibitors of p38 MAP kinase sensitized MV-4-11 cells for khat-treatment without concomitant stabilization of p53. Experiments using a p53 knock-down cell line and murine p53 knock-out bone marrow cells indicated that p53 was redundant in khat-mediated cell death in vitro. We suggest that analysis of isoform patterns and p53 PTMs are useful for elucidation of biological effects of complex plant extracts, and that p53 protein analysis is particularly useful in the search for new chemical probes and experimental cancer therapeutics.publishedVersio

Spontaneous Splenic Artery Rupture as the First Symptom of Systemic Amyloidosis

Spontaneous splenic rupture is a life-threatening condition leading to a rapidly progressing hypovolemic shock due to intra-abdominal blood loss, with a mortality rate of about 10%. Spontaneous splenic rupture can be caused by widely different disorders including acute and chronic infections, neoplastic disorders, and inflammatory noninfectious disorders. In this case report, we present a 67-year-old male patient with hemorrhagic shock caused by an acute bleeding from the splenic artery. The patient was massively transfused with blood products and fluids and underwent laparotomy for hemostatic control and clinical stabilization. Multiorgan involvement by amyloid light-chain amyloidosis (AL-amyloidosis) caused by plasma cell dyscrasia, specifically with infiltration of the spleen artery, was found to be the underlying cause of his life-threatening bleeding. Based on this case, we discuss the features of serious spleen bleeding, massive transfusion therapy in the intensive care setting, and AL-amyloidosis pathophysiology and treatment.publishedVersio

Tumor HPV status, level of regulatory T cells and macrophage infiltration predict up to 20-year non-disease-specific survival in oropharynx squamous cell carcinoma patients

Oropharynx squamous cell carcinoma (OPSCC) is of special interest because human papilloma virus (HPV) and/or smoking cause this disease. Influxes of inflammatory cells into such tumors are known to vary with prognoses. Aims: To study whether the density of tumor-infiltrating T lymphocytes and tumor-infiltrating macrophages predicted general 20-year overall survival (OS), as well as OS with only disease-specific survival (DSS) patients included. Methods: Biopsies from patients treated for OPSCC (n = 180) were stained by immunohistochemistry and the tumor cell macrophage (CD68), pan T lymphocytes (CD3), and regulatory T lymphocytes (Foxp3) densities were determined. The HE-determined percentage of matured tumor cells and the rate of invasion were calculated, and stromal desmoplasia were performed. Tumor HPV presence was studied by PCR. Twenty-year OS and five-year DSS patients were determined. Results: Tumor HPV status strongly predicted survival. High tumor infiltration of CD3, Foxp3 and CD68-positive cells predicted better twenty-year OS, with and without HPV stratification. Foxp3 and CD68 levels predicted OS, and 20-year among DSS patients, primarily among HPV(+) patients. Tumor HE-derived variables did not predict such survival. Conclusions: Tumor HPV status, level of Foxp3 tumor-infiltrating lymphocytes and CD68 tumor-infiltrating macrophages predicted up to 20-year OS of both all patients and disease-specific survived patients.publishedVersio

Development of a high dimensional imaging mass cytometry panel to investigate spatial organization of tissue microenvironment in formalin-fixed archival clinical tissues

To decipher the interactions between various components of the tumor microenvironment (TME) and tumor cells in a preserved spatial context, a multiparametric approach is essential. In this pursuit, imaging mass cytometry (IMC) emerges as a valuable tool, capable of concurrently analyzing up to 40 parameters at subcellular resolution. In this study, a set of antibodies was selected to spatially resolve multiple cell types and TME elements, including a comprehensive panel targeted at dissecting the heterogeneity of cancer-associated fibroblasts (CAF), a pivotal TME component. This antibody panel was standardized and optimized using formalin-fixed paraffin-embedded tissue (FFPE) samples from different organs/lesions known to express the markers of interest. The final composition of the antibody panel was determined based on the performance of conjugated antibodies in both immunohistochemistry (IHC) and IMC. Tissue images were segmented employing the Steinbock framework. Unsupervised clustering of single-cell data was carried out using a bioinformatics pipeline developed in R program. This paper provides a detailed description of the staining procedure and analysis workflow. Subsequently, the panel underwent validation on clinical FFPE samples from head and neck squamous cell carcinoma (HNSCC). The panel and bioinformatics pipeline established here proved to be robust in characterizing different TME components of HNSCC while maintaining a high degree of spatial detail. The platform we describe shows promise for understanding the clinical implications of TMA heterogeneity in large patient cohorts with FFPE tissues available in diagnostic biobanks worldwide.publishedVersio

Granulocyte macrophage-colony stimulating factor and keratinocyte growth factor control of early stages of differentiation of oral epithelium

Oral epithelial differentiation is known to be directed by underlying fibroblasts, but the responsible factor(s) have not been identified. We aimed here to identify fibroblast-derived factors responsible for oral epithelial differentiation. Primary normal human oral keratinocytes and fibroblasts were isolated from healthy volunteers after informed consent (n = 5) and 3D-organotypic (3D-OT) cultures were constructed. Various growth factors were added at a range of 0.1-100 ng/ml. 3D-OTs were harvested after ten days and assessed histologically, by immunohistochemistry and the TUNEL method. Epithelium developed in 3D-OT without fibroblasts showed an undifferentiated phenotype. Addition of granulocyte macrophage-colony stimulating factor (GM-CSF) induced expression of cytokeratin 13 in suprabasal cell layers. Admixture of GM-CSF and keratinocyte growth factor (KGF) induced, in addition, polarization of epidermal growth factor (EGF) receptor and β1-integrin to basal cell layer and collagen IV deposition. Terminal differentiation with polarization of TUNEL-positive cells to superficial layers occurred only in the presence of fibroblasts in collagen gels either in direct contact or at distance from normal oral keratinocytes. Taken together, these results show that major aspects of oral epithelial differentiation are regulated by the synergic combination of GM-CSF and KGF. However, the terminal stage seems to be controlled by other yet unidentified fibroblast-derived diffusible factor(s).publishedVersio

Camptothecin and khat (Catha edulis Forsk.) induced distinct cell death phenotypes involving modulation of c-FLIPL, Mcl-1, procaspase-8 and mitochondrial function in acute myeloid leukemia cell lines

<p>Abstract</p> <p>Background</p> <p>An organic extract of the recreational herb khat (<it>Catha edulis </it>Forsk.) triggers cell death in various leukemia cell lines <it>in vitro</it>. The chemotherapeutics camptothecin, a plant alkaloid topoisomerase I inhibitor, was tested side-by-side with khat in a panel of acute myeloid leukemia cell lines to elucidate mechanisms of toxicity.</p> <p>Results</p> <p>Khat had a profound effect on MOLM-13 cells inducing mitochondrial damage, chromatin margination and morphological features of autophagy. The effects of khat on mitochondrial ultrastructure in MOLM-13 correlated with strongly impaired routine respiration, an effect neither found in the khat-resistant MV-4-11 cells nor in camptothecin treated cells. Enforced expression of anti-apoptotic Bcl-2 protein provided protection against camptothecin-induced cell death and partly against khat toxicity. Khat-induced cell death in MOLM-13 cells included reduced levels of anti-apoptotic Mcl-1 protein, while both khat and camptothecin induced c-FLIP_Lcleavage and procaspase-8 activation.</p> <p>Conclusion</p> <p>Khat activated a distinct cell death pathway in sensitive leukemic cells as compared to camptothecin, involving mitochondrial damage and morphological features of autophagy. This suggests that khat should be further explored in the search for novel experimental therapeutics.</p