A genetically defined signature of responsiveness to erlotinib in early-stage pancreatic cancer patients: Results from the CONKO-005 trial.

BACKGROUND: high recurrence rates of up to 75% within 2 years in pancreatic ductal adenocarcinoma (PDAC) patients resected for cure indicate a high medical need for clinical prediction tools and patient specific treatment approaches. Addition of the EGFR inhibitor erlotinib to adjuvant chemotherapy failed to improve outcome but its efficacy in some patients warrants predictors of responsiveness. PATIENTS AND METHODS: we analysed tumour samples from 293 R0-resected patients from the randomized, multicentre phase III CONKO-005 trial (gemcitabine ± erlotinib) with targeted sequencing, copy number, and RNA expression analyses. FINDINGS: a total of 1086 mutations and 4157 copy-number aberrations (CNAs) with a mean of 17.9 /tumour were identified. Main pathways affected by genetic aberrations were the MAPK-pathway (99%), cell cycle control (92%), TGFß signalling (77%), chromatin remodelling (71%), and the PI3K/AKT pathway (65%). Based on genetic signatures extracted with non-negative matrix factorization we could define five patient clusters, which differed in mutation patterns, gene expression profiles, and survival. In multivariable Cox regression analysis, SMAD4 aberrations were identified as a negative prognostic marker in the gemcitabine arm, an effect that was counteracted when treated with erlotinib (DFS: HR=1.59, p = 0.016, and OS: HR = 1.67, p = 0.014). Integration of differential gene expression analysis established SMAD4 alterations with low MAPK9 expression (n = 91) as a predictive biomarker for longer DFS (HR=0.49; test for interaction, p = 0.02) and OS (HR = 0.32; test for interaction, p = 0.001). INTERPRETATION: this study identified five biologically distinct patient clusters with different actionable lesions and unravelled a previously unappreciated association of SMAD4 alteration status with erlotinib effectiveness. Confirmatory studies and mechanistic experiments are warranted to challenge the hypothesis that SMAD4 status might guide addition of erlotinib treatment in early-stage PDAC patients.

Trabectedin Reduces Skeletal Prostate Cancer Tumor Size in Association with Effects on M2 Macrophages and Efferocytosis.

Macrophages play a dual role in regulating tumor progression. They can either reduce tumor growth by secreting antitumorigenic factors or promote tumor progression by secreting a variety of soluble factors. The purpose of this study was to define the monocyte/macrophage population prevalent in skeletal tumors, explore a mechanism employed in supporting prostate cancer (PCa) skeletal metastasis, and examine a novel therapeutic target. Phagocytic CD68+ cells were found to correlate with Gleason score in human PCa samples, and M2-like macrophages (F4/80+CD206+) were identified in PCa bone resident tumors in mice. Induced M2-like macrophages in vitro were more proficient at phagocytosis (efferocytosis) of apoptotic tumor cells than M1-like macrophages. Moreover, soluble factors released from efferocytic versus nonefferocytic macrophages increased PC-3 prostate cancer cell numbers in vitro. Trabectedin exposure reduced M2-like (F4/80+CD206+) macrophages in vivo. Trabectedin administration after PC-3 cell intracardiac inoculation reduced skeletal metastatic tumor growth. Preventative pretreatment with trabectedin 7 days prior to PC-3 cell injection resulted in reduced M2-like macrophages in the marrow and reduced skeletal tumor size. Together, these findings suggest that M2-like monocytes and macrophages promote PCa skeletal metastasis and that trabectedin represents a candidate therapeutic target.

Frequent somatic mutations in epigenetic regulators in newly diagnosed chronic myeloid leukemia.

Although tyrosine kinase inhibitors (TKIs) have significantly improved the prognosis of chronic myeloid leukemia (CML), the ability of TKIs to eradicate CML remains uncertain and patients must continue TKI therapy for indefinite periods. In this study, we performed whole-exome sequencing to identify somatic mutations in 24 patients with newly diagnosed chronic phase CML who were registered in the JALSG CML212 study. We identified 191 somatic mutations other than the BCR-ABL1 fusion gene (median 8, range 1-17). Age, hemoglobin concentration and white blood cell counts were correlated with the number of mutations. Patients with mutations ⩾6 showed higher rate of achieving major molecular response than those<6 (P=0.0381). Mutations in epigenetic regulator, ASXL1, TET2, TET3, KDM1A and MSH6 were found in 25% of patients. TET2 or TET3, AKT1 and RUNX1 were mutated in one patient each. ASXL1 was mutated within exon 12 in three cases. Mutated genes were significantly enriched with cell signaling and cell division pathways. Furthermore, DNA copy number analysis showed that 2 of 24 patients had uniparental disomy of chromosome 1p or 3q, which disappeared major molecular response was achieved. These mutations may play significant roles in CML pathogenesis in addition to the strong driver mutation BCR-ABL1.

The Role of Osteoclasts in Early Dissemination of Prostate Cancer Tumor Cells.

Prostate cancer (PCa) is one of the most common neoplasms that metastasize to bone. The aim of this study was to determine if osteoclasts play a role in the seeding of disseminated tumor cells to the bone marrow by mobilizing hematopoietic stem cells (HSCs) out of their marrow niche. Human PC-3Luc cells were introduced into male SCID mice by intracardiac (i.c.) injection after mice were treated with the antiresorptive agent Zoledronic Acid (bisphosphonate (BP)) and/or AMD3100, which mobilizes HSCs out of the marrow. Short term homing of PC-3 was assessed at 24 hours by QPCR for human Alu and luciferase and HSC number was determined by FACS. Mice also received pre and/or post treatments of BP by intraperiteneal (i.p.) injections, in addition to PC-3 luc by intratibial (i.t.) injections. TRAP assays were used to determine the osteoclast (OC) number in both studies. AMD3100 enhanced the release of HSCs from the bone marrow, while BP increased the retention of HSCs. PCa entry into bone was facilitated in AMD3100, BP, and AMD3100+BP treatments. Before PCa injection, the number of TRAP+ OC was increased in mice treated with AMD3100, while treatment with BP resulted in relatively lower TRAP+ OCs. TRAP+ OCs were not detected in the AMD3100 + BP treatment. After PCa injection, however, the number of TRAP+ OCs was dramatically increased, but did not differ significantly amongst the treatment groups. The pre and post BP treatments in the Nude mice decreased the size of PCa lesions in the tibia compared to the control. The results indicate that OC activation is not necessary for PCa metastasis to bone at the earliest stages. These findings are critical in proving that OCs' contribution to metastasis occur during the growth phase of the tumor rather than at the initiation phase.

Reduced TET2 function leads to T-cell lymphoma with follicular helper T-cell-like features in mice.

TET2 (Ten Eleven Translocation 2) is a dioxygenase that converts methylcytosine (mC) to hydroxymethylcytosine (hmC). TET2 loss-of-function mutations are highly frequent in subtypes of T-cell lymphoma that harbor follicular helper T (Tfh)-cell-like features, such as angioimmunoblastic T-cell lymphoma (30-83%) or peripheral T-cell lymphoma, not otherwise specified (10-49%), as well as myeloid malignancies. Here, we show that middle-aged Tet2 knockdown (Tet2(gt/gt)) mice exhibit Tfh-like cell overproduction in the spleen compared with control mice. The Tet2 knockdown mice eventually develop T-cell lymphoma with Tfh-like features after a long latency (median 67 weeks). Transcriptome analysis revealed that these lymphoma cells had Tfh-like gene expression patterns when compared with splenic CD4-positive cells of wild-type mice. The lymphoma cells showed lower hmC densities around the transcription start site (TSS) and higher mC densities at the regions of the TSS, gene body and CpG islands. These epigenetic changes, seen in Tet2 insufficiency-triggered lymphoma, possibly contributed to predated outgrowth of Tfh-like cells and subsequent lymphomagenesis. The mouse model described here suggests that TET2 mutations play a major role in the development of T-cell lymphoma with Tfh-like features in humans.

Haploinsufficiency of Sf3b1 leads to compromised stem cell function but not to myelodysplasia.

SF3B1 is a core component of the mRNA splicing machinery and frequently mutated in myeloid neoplasms with myelodysplasia, particularly in those characterized by the presence of increased ring sideroblasts. Deregulated RNA splicing is implicated in the pathogenesis of SF3B1-mutated neoplasms, but the exact mechanism by which the SF3B1 mutation is associated with myelodysplasia and the increased ring sideroblasts formation is still unknown. We investigated the functional role of SF3B1 in normal hematopoiesis utilizing Sf3b1 heterozygous-deficient mice. Sf3b1(+/-) mice had a significantly reduced number of hematopoietic stem cells (CD34(-)cKit(+)ScaI(+)Lin(-) cells or CD34(-)KSL cells) compared with Sf3b1(+/+) mice, but hematopoiesis was grossly normal in Sf3b1(+/-) mice. When transplanted competitively with Sf3b1(+/+) bone marrow cells, Sf3b1(+/-) stem cells showed compromised reconstitution capacity in lethally irradiated mice. There was no increase in the number of ring sideroblasts or evidence of myeloid dysplasia in Sf3b1(+/-) mice. These data suggest that SF3B1 plays an important role in the regulation of hematopoietic stem cells, whereas SF3B1 haploinsufficiency itself is not associated with the myelodysplastic syndrome phenotype with ring sideroblasts.

Landscape of genetic lesions in 944 patients with myelodysplastic syndromes.

High-throughput DNA sequencing significantly contributed to diagnosis and prognostication in patients with myelodysplastic syndromes (MDS). We determined the biological and prognostic significance of genetic aberrations in MDS. In total, 944 patients with various MDS subtypes were screened for known/putative mutations/deletions in 104 genes using targeted deep sequencing and array-based genomic hybridization. In total, 845/944 patients (89.5%) harbored at least one mutation (median, 3 per patient; range, 0-12). Forty-seven genes were significantly mutated with TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and RUNX1 mutated in >10% of cases. Many mutations were associated with higher risk groups and/or blast elevation. Survival was investigated in 875 patients. By univariate analysis, 25/48 genes (resulting from 47 genes tested significantly plus PRPF8) affected survival (P<0.05). The status of 14 genes combined with conventional factors revealed a novel prognostic model ('Model-1') separating patients into four risk groups ('low', 'intermediate', 'high', 'very high risk') with 3-year survival of 95.2, 69.3, 32.8, and 5.3% (P<0.001). Subsequently, a 'gene-only model' ('Model-2') was constructed based on 14 genes also yielding four significant risk groups (P<0.001). Both models were reproducible in the validation cohort (n=175 patients; P<0.001 each). Thus, large-scale genetic and molecular profiling of multiple target genes is invaluable for subclassification and prognostication in MDS patients.

Effects of erythropoietin on the bone microenvironment.

It has been well established that blood and bone share a unique, regulatory relationship with one another, though the specifics of this relationship still remain unanswered. Erythropoietin (Epo) is known primarily for its role as a hematopoietic hormone. However, after the discovery of Epo receptor outside the hematopoietic tissues, Epo has been avidly studied for its possible nonhematopoietic effects. It has been proposed that Epo interacts with bone both directly, by activating bone marrow stromal cells, and indirectly, through signaling pathways on hematopoietic stem cells. Yet, the role of Epo in regulating skeletal maintenance and regeneration remains controversial. Here, we review the current state of knowledge pertaining to the effects of Epo on the skeleton.

The bone marrow niche: habitat to hematopoietic and mesenchymal stem cells, and unwitting host to molecular parasites.

In post-fetal life, hematopoiesis occurs in unique microenvironments or 'niches' in the marrow. Niches facilitate the maintenance of hematopoietic stem cells (HSCs) as unipotent, while supporting lineage commitment of the expanding blood populations. As the physical locale that regulates HSC function, the niche function is vitally important to the survival of the organism. This places considerable selective pressure on HSCs, as only those that are able to engage the niche in the appropriate context are likely to be maintained as stem cells. Since niches are central regulators of stem cell function, it is not surprising that molecular parasites like neoplasms are likely to seek out opportunities to harvest resources from the niche environment. As such, the niche may unwittingly participate in tumorigenesis as a leukemic or neoplastic niche. The niche may also promote metastasis or chemo-resistance of hematogenous neoplasms or solid tumors. This review focuses on what is known about the physical structures of the niche, how the niche participates in hematopoiesis and neoplastic growth and what molecules are involved. Further understanding of the interactions between stem cells and the niche may be useful for developing therapeutic strategies.

Granulocytic sarcoma of the thymus in a nonleukaemic patient.

We report a case of granulocytic sarcoma arising from the thymus in a 17-year-old nonleukaemic patient. The patient presented with an anterior mediastinal tumour and underwent surgical resection. Histological examination showed a diffuse infiltrate of immature round cells in the thymus. Tumour cells were diffusely peroxidase positive, but naphthol AS-D chloroacetate esterase negative. Immunohistochemical staining revealed expression of CD34 and terminal deoxynucleotidyl transferase (TdT), but not of CD13 and CD33. Ultrastructurally, electron-dense or medium-density granules were present in the cytoplasm. Four months after successful autogenic bone marrow transplantation, pleural and pericardial fluid contained tumour cells with azurophilic granules, which expressed CD13 and CD33, but not CD34 and TdT. The patient died of the disease 18 months after clinical manifestation, but still without developing leukaemia. The granulocytic sarcoma in the present case may have originated from myeloid precursors in the thymus and remained within the extramedullary site despite the differentiation into a more committed myeloid lineage at the relapse.

Neuroendocrine differentiation in thymic epithelial tumors with special reference to thymic carcinoma and atypical thymoma.

To determine the neuroendocrine (NE) features of thymic epithelial tumor, immunohistochemistry and electron microscopy studies were performed on eight NE tumors (thymic carcinoids) and 26 non-NE tumors (nine thymic carcinomas, five atypical thymomas, and 12 thymomas other than lymphocytic thymoma). Immunohistochemical studies were performed with antibodies against general markers for NE cells (synaptophysin, alpha subunit of a guanine nucleotide-binding protein, Go, and small-cell lung carcinoma cluster 1 antigen), and a broad panel of antibodies for hormonal substances. Thymic carcinoid showed synchronous diffuse immunoreactivity for the three NE markers and contained cells that were positive for a variety of hormonal products: human chorionic gonadotropin (hCG) alpha-subunit (eight of eight), hCG beta-subunit (three of eight), adrenocorticotropic hormone (ACTH) (three of eight), calcitonin (two of eight), calcitonin gene-related peptide (two of eight), and serotonin (one of eight). Conversely, although positivity for NE markers was neither synchronous nor diffuse in non-NE tumors, seven of nine thymic carcinomas, three of five atypical thymomas (focal or dispersed distribution), and none of the five thymomas were positive for at least two of these NE markers. A small number of neoplastic cells were positive for hCGalpha-subunit or ACTH in three thymic carcinomas and one atypical thymoma. Ultrastructurally, dense core granules (DCG) were much more frequent in thymic carcinoid, but several DCG-like granules were identified in 12 of 13 non-NE tumors with or without immunoexpression of NE markers. The presence of focal or dispersed NE cells in thymic carcinoma and atypical thymoma may reflect multidirectional differentiation within the tumor, which, like cytological atypia, epithelial CD5 expression, and lack of immature T cell infiltration, may be another feature of this group at thymic tumors.

Fibrillary inclusions in neoplastic and fetal acinar cells of the pancreas.

We report a case of pancreatic acinar cell carcinoma which contained a large number of pleomorphic inclusions with fibrillary internal structures and mature zymogen granules. To clarify the significance of fibrillary inclusions in the differentiation of acinar cells of the pancreas, we further investigated fetal pancreases (gestational weeks 16, 17, 19, 20 and 28). We found two types of inclusions: type A, corresponding to fibrillary inclusion of neoplastic acinar cells, was observed only in a 19-week fetus; type B showed a homogeneous density similar to that of zymogen granules. Type B was observed in all the fetuses after the 17th gestational week. Although the type A inclusion might be generated through a different mechanism than the type B inclusion, the appearance of a large number of fibrillary inclusions in neoplastic acinar cells may represent a transient form of zymogen granule.

Production of recombinant human monoclonal antibody using ras-amplified BHK-21 cells in a protein-free medium.

A ras oncogene-amplified recombinant BHK-21 cell line (ras-rBHK-IgG) has been established, and was shown to hyperproduce the recombinant IgG chimeric human monoclonal antibody (hMAb) AE6F4, which recognizes lung cancer cells. We found that the ras-rBHK-IgG cell could be easily cultured in a protein-free ERDF medium supplemented with iron(III) nitrate, hydroxyethyliminodiacetic acid, and non-protein synthetic attachment factor as well as in a serum-free ERDF medium supplemented with insulin, transferrin, ethanolamine, and sodium selenite. The productivity of recombinant hMAb from the cells cultured in dishes at high cell densities was higher in protein-free medium than in serum-containing medium. True high density culture of the ras-rBHK-IgG cells was done in protein-free medium using the Tecnomouse, which is a novel hollow fiber bioreactor system. After culture for 30 days in protein-free culture, a total amount of about 14 mg of the recombinant hMAb AE6F4 was obtained, and was shown to be reactive against lung cancer cells in tissues.

Immune cell migration through the arterial wall in the murine lung during a pulmonary inflammatory response.

Perivascular accumulation of leukocytes in the lung was induced by intratracheal administration of sheep erythrocyte antigen to primed mice. The route of migration of intravascular leukocytes to the perivascular space in the lung, in particular from arteries, and the structure of lymphatic vessels among the aggregated leukocytes were examined by transmission electron microscopy. Leukocytes--lymphocytes, granulocytes, monocytes and macrophages--were demonstrated to adhere to the endothelial surface and to migrate between endothelial cells to reach the internal elastic lamina of arteries. Becoming conspicuously constricted, the leukocytes penetrate through this elastic lamina. They further migrate through the smooth muscle layer to the interstitium, passing through the external elastic laminar region. At 2 days after antigen administration, dilated lymphatic vessels containing large numbers of leukocytes in the lumen and bearing endothelial gaps open to the interstitium began to be seen. The lymphatic walls were more convoluted and richer in pinocytotic vesicles than those prior to antigen challenge. This study confirms the light microscopic findings by Curtis et al. (1990) that arteries, besides veins, venules and capillaries, may represent a major route of inflammatory cell entry into the lung parenchyme in an acute and vigorous immune response. In addition, lymphatic vessels were suggested to be newly formed for the transport of fluid and immune cells from the sites of inflammation in the lung.

Atypical endocrine granules in atypical endocrine tumor (AET) of the lung. An immunoelectron microscopic study.

Highly dense granules are a hallmark for recognizing atypical endocrine tumor (AET) of the lung. We report a case of AET with many atypical neurosecretory-type granules: moderately dense granules (mean size 373.7 nm) and "target" granules with a central dense core (425.1 nm), both apparently larger than the highly dense granules (223.3 nm). Immunoelectron microscopical studies demonstrated that all three types of granule were positive for gastrin-releasing peptide (GRP), human chorionic gonadotropin alpha-subunit (hCG alpha), calcitonin or serotonin. Although the size profiles of positive granules were similar for calcitonin and hCG alpha, they were different from those of GRP or serotonin granules. The presence of atypical granules and the different size profiles of hormonal products in AET indicate that caution is required in ultrastructural evaluation of granules in lung carcinomas.

Human chorionic gonadotropin alpha-subunit in endocrine cells of fibrotic and neoplastic lung. Its mode of localization and the size profile of granules.

To investigate the nature of various endocrine cells immunoreactive for human chorionic gonadotropin (hCG alpha) in the lung, immunoelectron microscopic study was performed on fibrotic adult lungs and endocrine neoplasms of the lung. The mode of localization of hCG alpha and the size profile of hCG alpha granules were different among endocrine cells under various proliferative conditions. The population of hCG alpha granules in the grouped type of endocrine cells was more variable with a shift to smaller size (mean area: 1.395 x 10(-2) microns 2, mean maximum diameter: 149.8 nm), than that in solitary ones (1.493 x 10(-2) microns 2, 155.4 nm). Tumorlet endocrine cells had larger hCG alpha granules (1.800 x 10(-2) microns 2, 171.3 nm) without change of SD of size parameters. In carcinoid tumors, the size profile of hCG alpha granules was considerably different from that in the three types described above. Moreover, hCG alpha granules were significantly smaller in size in carcinoid tumors without lymph node metastasis (2.295 x 10(-2) microns 2, 189.8 nm) than those in malignant carcinoid tumors with metastasis (3.368 x 10(-2) microns 2, 230.5 nm). The population of hCG alpha granules in atypical endocrine tumor was the parallel shift to a larger scale (6.251 x 10(-2) microns 2, 307.5 nm) from that of malignant carcinoids and the distribution pattern was different from that in benign carcinoids. In small cell carcinoma of the lung, hCG alpha immunoreaction was preferentially present in perinuclear space and dilated rough endoplasmic reticulum. The mode of localization of hCG alpha and the size profile of hCG alpha granules, representing specific features of intracellular processing of hCG alpha, may be closely related with some qualitative changes in the neoplastic process of pulmonary endocrine cells.

Human chorionic gonadotropin in the thymus. An immunocytochemical study on discordant expression of subunits.

To demonstrate discordant expression of human chorionic gonadotropin (hCG) subunits in the human thymus and thymic tumors, immunocytochemical studies were performed with immunoelectron microscopy. In the fetus, hCG beta-positive cells were identified in excess of hCG alpha-positive cells (hCG alpha: 1.38 +/- 0.18/mm2, hCG beta: 4.50 +/- 1.13/mm2). They were usually different populations in serial sections. There were 0.18 +/- 0.03/mm2 synchronously positive cells using the double immunostaining method, comprising approximately 3% of the total positive cells. The immunoreactive material for both subunits was present at the rough endoplasmic reticulum (RER), perinuclear space, and vesicular structures with protruding microvilli in the lumens. hCG alpha was expressed preferentially in the endocrine tumors of the thymus and non-germ-cell tumor, with rare positivity for beta-subunits of glycoprotein hormones. The hCG alpha-immunoreactive material also was present in the granules as well as in RER of the neoplastic cells. In four teratomas of the thymus, hCG alpha-positivity only was present in endocrinelike cells. The subunit profile of hCG was unbalanced in the thymus and isolated hCG alpha-expression in the fetal thymus may represent the endocrine cell with a primitive storage mechanism. The discordant expression of hCG subunits may be common in nontrophoblastic tissues.

Human chorionic gonadotropin alpha-subunit in rectal carcinoids. Its mode of presence and the change of granule morphology.

To investigate the nature of endocrine cells immunoreactive for human chorionic gonadotropin alpha-subunit (hCG alpha), rectal carcinoid tumors were studied with immunohistochemistry and immunoelectron microscopy. There were two types of rectal carcinoids: Type A (n = 5) was diffusely argyrophilic and immunoreactive for serotonin with many hCG alpha-positive cells (16.7%-91.1%). Type B (n = 5) was dispersedly argyrophilic and contained, at most, 5% positive cells for pancreatic polypeptide (PP) with hCG alpha cells in 1.4% to 9.7%. By double immunostaining, 55.0% to 89.7% of hCG alpha cells were synchronously immunoreactive for serotonin in Type A and 3.2% to 11.8% of hCG alpha cells showed PP-positivity in Type B. HCG alpha-positive granules had a constant relationship between perimeter (P) and area (A), log10 A approximately D log10 P, in each case (n = 5). The inverse correlation was found between the value of D and the frequency of hCG alpha in the tumor or in the neoplastic cells (P less than 0.05). HCG alpha may represent the quantitative difference of rectal carcinoids and its expression may have some relation with granule morphology in neoplastic endocrine cells of the rectum.