Table_1_Whole-exome sequencing of calcitonin-producing pancreatic neuroendocrine neoplasms indicates a unique molecular signature.xlsx

IntroductionCalcitonin-producing pancreatic neuroendocrine neoplasms (CT-pNENs) are an extremely rare clinical entity, with approximately 60 cases reported worldwide. While CT-pNENs can mimic the clinical and diagnostic features of medullary thyroid carcinoma, their molecular profile is poorly understood.MethodsWhole-exome sequencing (WES) was performed on tumor and corresponding serum samples of five patients with increased calcitonin serum levels and histologically validated calcitonin-positive CT-pNENs. cBioPortal analysis and DAVID gene enrichment analysis were performed to identify dysregulated candidate genes compared to control databases. Immunohistochemistry was used to detect the protein expression of MUC4 and MUC16 in CT-pNEN specimens.ResultsMutated genes known in the literature in pNENs like MEN1 (35% of cases), ATRX (18-20% of cases) and PIK3CA (1.4% of cases) were identified in cases of CT-pNENs. New somatic SNVs in ATP4A, HES4, and CAV3 have not been described in CT- pNENs, yet. Pathogenic germline mutations in FGFR4 and DPYD were found in three of five cases. Mutations of CALCA (calcitonin) and the corresponding receptor CALCAR were found in all five tumor samples, but none of them resulted in protein sequelae or clinical relevance. All five tumor cases showed single nucleotide variations (SNVs) in MUC4, and four cases showed SNVs in MUC16, both of which were membrane-bound mucins. Immunohistochemistry showed protein expression of MUC4 in two cases and MUC16 in one case, and the liver metastasis of a third case was double positive for MUC4 and MUC16. The homologous recombination deficiency (HRD) score of all tumors was low.DiscussionCT-pNENs have a unique molecular signature compared to other pNEN subtypes, specifically involving the FGFR4, DPYD, MUC4, MUC16 and the KRT family genes. However, a major limitation of our study was the relative small number of only five cases. Therefore, our WES data should be interpreted with caution and the mutation landscape in CT-pNENs needs to be verified by a larger number of patients. Further research is needed to explain differences in pathogenesis compared with other pNENs. In particular, multi-omics data such as RNASeq, methylation and whole genome sequencing could be informative.</p

Automated Image Analysis of Hodgkin Lymphoma

<p>Hodgkin lymphoma is an unusual type of lymphoma, arising from malignant B-cells. Morphological and immunohistochemical features<br>of malignant cells and their distribution differ from other cancer types. Based on systematic tissue image analysis, computer-aided exploration<br>can provide new insights into Hodgkin lymphoma pathology.</p> <p>Here, we report results from an image analysis of CD30 immunostained classical Hodgkin lymphoma (cHL) tissue section images. We have imple-<br>mented an automatic procedure to handle and explore image data in Aperio's SVS format. We use pre-processing approaches to separate the image<br>objects from the background, then select regions of interest and split the large images into tiles. Then, we use a CellProfiler pipeline to detect primary objects. Therefore, the images are split into their color stains using a color deconvolution approach. By setting a threshold in the CD30 stain image we identify CD30 positive cells and compute their shape descriptors. We label the cells based on size, elongation and compactness. We present results for a small set of nodular sclerosis, mixed type and non-lymphoma images.</p> <p> </p

DataSheet_1_Whole-exome sequencing of calcitonin-producing pancreatic neuroendocrine neoplasms indicates a unique molecular signature.docx

IntroductionCalcitonin-producing pancreatic neuroendocrine neoplasms (CT-pNENs) are an extremely rare clinical entity, with approximately 60 cases reported worldwide. While CT-pNENs can mimic the clinical and diagnostic features of medullary thyroid carcinoma, their molecular profile is poorly understood.MethodsWhole-exome sequencing (WES) was performed on tumor and corresponding serum samples of five patients with increased calcitonin serum levels and histologically validated calcitonin-positive CT-pNENs. cBioPortal analysis and DAVID gene enrichment analysis were performed to identify dysregulated candidate genes compared to control databases. Immunohistochemistry was used to detect the protein expression of MUC4 and MUC16 in CT-pNEN specimens.ResultsMutated genes known in the literature in pNENs like MEN1 (35% of cases), ATRX (18-20% of cases) and PIK3CA (1.4% of cases) were identified in cases of CT-pNENs. New somatic SNVs in ATP4A, HES4, and CAV3 have not been described in CT- pNENs, yet. Pathogenic germline mutations in FGFR4 and DPYD were found in three of five cases. Mutations of CALCA (calcitonin) and the corresponding receptor CALCAR were found in all five tumor samples, but none of them resulted in protein sequelae or clinical relevance. All five tumor cases showed single nucleotide variations (SNVs) in MUC4, and four cases showed SNVs in MUC16, both of which were membrane-bound mucins. Immunohistochemistry showed protein expression of MUC4 in two cases and MUC16 in one case, and the liver metastasis of a third case was double positive for MUC4 and MUC16. The homologous recombination deficiency (HRD) score of all tumors was low.DiscussionCT-pNENs have a unique molecular signature compared to other pNEN subtypes, specifically involving the FGFR4, DPYD, MUC4, MUC16 and the KRT family genes. However, a major limitation of our study was the relative small number of only five cases. Therefore, our WES data should be interpreted with caution and the mutation landscape in CT-pNENs needs to be verified by a larger number of patients. Further research is needed to explain differences in pathogenesis compared with other pNENs. In particular, multi-omics data such as RNASeq, methylation and whole genome sequencing could be informative.</p

Stem cell colonogenic potential of t(9;22) fusion proteins.

<p>(A) Experimental strategy for studying the influence of t(9;22) fusion proteins on the biology of murine HSCs. Sca1⁺/lin^- bone marrow (BM) cells were infected with the indicated retroviruses and maintained for 9 days in liquid culture supplemented with the indicated growth factors. 1 x 10⁴ cells were inoculated into lethally irradiated recipients that were sacrificed at day 12 after transplantation; (B) Number of colonies in the spleens (n = 3); the experiment was performed a total of three times with similar results. One representative experiment is given; (C) Gene expression profile induced by t(9;22) fusion proteins in spleen from the CFU-S12. Clustering was done by selecting genes with the highest SD and sorted according to the similarity in expression level; (D) <i>Seven representative cellular pathways known to be influenced by BCR/ABL related to cell cycle regulation</i>, <i>proliferation and apoptosis are presented here</i>. <i>The numbers indicate the number of differentially expressed genes between p185</i>^{<i>BCR/ABL</i>}<i>and p96</i>^{<i>ABL/BCR</i>} + p185^{<i>BCR/ABL</i>} (p96+p185)-positive cells from the total number of the genes (related to each of the pathways); (E) Total RNA was isolated from spleens from the CFU-S12. The expression levels of Tp53, Gadd45α, and Cdkn1a were analyzed using q-RT-PCR. The Ct values were normalized to that of Gapdh and results are represented as 2^-ΔΔCt. The mean of three independent experiments each done in triplicates is given ± SD; (F) PH and BV were lentivirally transduced with shRNA against p96^{<i>ABL/BCR</i>} (siR961 and siR962) and a control shRNA (NTC). The expression of GADD45α was detected by q-RT-PCR. The Ct values were normalized to that of GAPDH and results are represented as 2^-ΔΔCt.</p

The leukemogenic potential of t(9;22) fusion proteins.

<p>(A) Schematic representation of the experimental procedure. Sca1⁺ bone marrow cells were infected with the indicated retroviruses and inoculated into sub-lethally irradiated mice. Empty vector-transduced cells were used as control; (B) Kaplan Maier curves present the probability of survival upon primary induction of leukemia and re-transplantation of leukemic cells in order to induce secondary (II) leukemia; (C) May-Grünwald-Giemsa staining of cytospins from BM and spleens of p185^{<i>BCR/ABL</i>} and p96+p185-positive leukemia of one representative mouse in each group; (D) Expression of differentiation specific surface markers (Mac1: monocytes- macrophage, Gr1: granulocytes and B220: mature B-cells) of one representative mouse in p185^{<i>BCR/ABL</i>} and <i>p96</i>^{<i>ABL/BCR</i>} + p185^{<i>BCR/ABL</i>} (p96+p185) groups. (E) Co-expression of differentiation specific surface markers (Mac1/Gr-1—myeloid leukemia; B220/CD19: B-cell leukemia) of one representative mouse in p185^{<i>BCR/ABL</i>} and <i>p96</i>^{<i>ABL/BCR</i>} + p185^{<i>BCR/ABL</i>} (p96+p185) groups and secondary transplanted sup.</p

Targeting p96^{<i>ABL/BCR</i>} in Ph⁺ ALL cells.

<p>(A) SupB15 and K562 cells were lentivirally transduced with shRNA against p96^{<i>ABL/BCR</i>} (siR961 and siR962) and a control shRNA (NTC). The expression of p96^{<i>ABL/BCR</i>} and/or BCR was detected by immunoblotting using anti-BCR antibody. Tubulin was used as loading control. Proliferation was measured using XTT-assay after 3 days. One representative experiment in triplicates ± SD of at least three yielding similar results is given; (B) The effect of targeting p96^{<i>ABL/BCR</i>} by shRNA in SupB15 on STAT5 and ERK1/2 pathway was detected using the indicated antibodies; (C) Down-regulation of p96^{<i>ABL/BCR</i>} in Ph⁺ ALL PD-LTCs by shRNA. Ph⁺ ALL PD-LTCs—PH: fully TKI-responsive; BV: TKI-resistant; as controls were used: HP (Ph^- ALL patient) and VG: t(12;9)-TEL/ABL-positive ALL. The effect of shRNAs on the expression of p96^{<i>ABL/BCR</i>} was tested by immunoblotting using the indicated antibodies and by q-RT-PCR for PH and BV. The Ct values were normalized to that of GAPDH and results are represented as 2^-ΔΔCt. Proliferation was measured by XTT-assay. The mean of at least experiments is given ± SD.</p

Effect of the anti-CD30 drug conjugate Brentuximab Vedotin to L-428 cells.

<p>Cells from the cHL line L-428 were sorted according to FSC in subpopulations of small and big cells (each 20% of total live cell population). Total live gates were sorted as control (bulk). Thereafter, subpopulations were seeded at a density of 1 x 10⁵ cells per ml in triplicates and the indicated amount of Brentuximab Vedotin was added. After 48 h cell numbers were determined by FACS. * p = 0.0117, unpaired t-test.</p

Western blot analysis of the NCF1 protein.

<p>NCF1 protein expression in 8 non-cHL lymphoma cell lines (LM1, Karpas 299 (CD30⁺), DEV (CD30⁺), DG-75, Ca 46, Karpas 422, Daudi, Granta 519) and 6 cHL cell lines (L540, UHO1, L1236, KMH2, HDLM2, L428 - all CD30⁺). AU – arbitrary units after normalization to actin signal strength. Each bar presents the mean result of 6 independent Western blots and is exemplified by a blot presented below. cHL cell lines show significantly lower (p<0.005) expression of the NCF1 protein as compared to the control cell lines.</p

Microarray expression analysis of NADPH oxidase encoding genes CYBA, CYBB, NCF1, NCF2 and NCF4.

<p>Relative expression of the five genes in 4 cHL cell lines and 20 normal B-cell samples. CB - centroblasts, CC - centrocytes, N- naive B-cells, M - memory B-cells. The p value is given only for genes showing significant changes in expression between cHL and controls. Based on published Genechip data [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084928#B13" target="_blank">13</a>].</p

Unsupervised hierarchical clustering of gene expression profiles obtained from microdissected small and giant HRS cells of the cHL cell lines L-428 and L-1236 shows a strong heterogeneity between the two cell lines.

<p>Small HRS cells of both cell lines show more strongly expressed probesets (red color) than giant HRS cells. 958 differentially expressed probesets between the four samples representing a standard deviation of > 1 were considered for the analysis.</p