Transcriptomic analysis of hepatocellular carcinoma reveals molecular features of disease progression and tumor immune biology.

Hepatocellular carcinoma (HCC) develops in the context of chronic inflammatory liver disease and has an extremely poor prognosis. An immunosuppressive tumor microenvironment may contribute to therapeutic failure in metastatic HCC. Here, we identified unique molecular signatures pertaining to HCC disease progression and tumor immunity by analyzing genome-wide RNA-Seq data derived from HCC patient tumors and non-tumor cirrhotic tissues. Unsupervised clustering of gene expression data revealed a gradual suppression of local tumor immunity that coincided with disease progression, indicating an increasingly immunosuppressive tumor environment during HCC disease advancement. IHC examination of the spatial distribution of CD8+ T cells in tumors revealed distinct intra- and peri-tumoral subsets. Differential gene expression analysis revealed an 85-gene signature that was significantly upregulated in the peri-tumoral CD8+ T cell-excluded tumors. Notably, this signature was highly enriched with components of underlying extracellular matrix, fibrosis, and epithelial-mesenchymal transition (EMT). Further analysis condensed this signature to a core set of 23 genes that are associated with CD8+ T cell localization, and were prospectively validated in an independent cohort of HCC specimens. These findings suggest a potential association between elevated fibrosis, possibly modulated by TGF-ß, PDGFR, SHH or Notch pathway, and the T cell-excluded immune phenotype. Indeed, targeting fibrosis using a TGF-ß neutralizing antibody in the STAM™ model of murine HCC, we found that ameliorating the fibrotic environment could facilitate redistribution of CD8+ lymphocytes into tumors. Our results provide a strong rationale for utilizing immunotherapies in HCC earlier during treatment, potentially in combination with anti-fibrotic therapies.

Randomized, phase II, placebo-controlled trial of onartuzumab and/or bevacizumab in combination with weekly paclitaxel in patients with metastatic triple-negative breast cancer.

BACKGROUND: Increased hepatocyte growth factor/MET signaling is associated with an aggressive phenotype and poor prognosis in triple-negative breast cancer (TNBC). We evaluated the benefit of adding onartuzumab, a monoclonal anti-MET antibody, to paclitaxel with/without bevacizumab in patients with TNBC. PATIENTS AND METHODS: Women with metastatic TNBC were randomized to receive onartuzumab plus placebo plus weekly paclitaxel (OP; n = 60) or onartuzumab plus bevacizumab plus paclitaxel (OBP; n = 63) or placebo plus bevacizumab plus paclitaxel (BP; n = 62). The primary end point was progression-free survival (PFS); additional end points included overall survival (OS), objective response rate (ORR), and safety. This trial was hypothesis generating and did not have power to detect minimum clinically meaningful differences between treatment arms. RESULTS: There was no improvement in PFS with the addition of onartuzumab to BP [hazard ratio (HR), 1.08; 95% confidence interval (CI) 0.69-1.70]; the risk of a PFS event was higher with OP than with BP (HR, 1.74; 95% CI 1.13-2.68). Most patients had MET-negative tumors (88%); PAM50 subtype analysis showed basal-like tumors in 68% of samples. ORR was higher in the bevacizumab arms (OBP: 42.2%; 95% CI 28.6-57.1; BP: 54.7%; 95% CI 41.0-68.4) compared with OP (27.5%; 95% CI 15.9-40.6). Median OS was shorter with OBP (HR, 1.36; 95% CI 0.75-2.46) and OP (HR, 1.92; 95% CI 1.03-3.59), than with BP. Peripheral edema was more frequent in the onartuzumab arms (OBP, 51.8%; OP, 58.6%) versus BP (17.7%). CONCLUSION: This study did not show a clinical benefit of the addition of onartuzumab to paclitaxel with/without bevacizumab in patients with predominantly MET-negative TNBC. CLINICALTRIALSGOV: NCT01186991.

Anti-CD22 and anti-CD79B antibody drug conjugates are active in different molecular diffuse large B-cell lymphoma subtypes.

Antibody drug conjugates (ADCs), in which cytotoxic drugs are linked to antibodies targeting antigens on tumor cells, represent promising novel agents for the treatment of malignant lymphomas. Pinatuzumab vedotin is an anti-CD22 ADC and polatuzumab vedotin an anti-CD79B ADC that are both linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE). In the present study, we analyzed the activity of these agents in different molecular subtypes of diffuse large B-cell lymphoma (DLBCL) both in vitro and in early clinical trials. Both anti-CD22-MMAE and anti-CD79B-MMAE were highly active and induced cell death in the vast majority of activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL cell lines. Similarly, both agents induced cytotoxicity in models with and without mutations in the signaling molecule CD79B. In line with these observations, relapsed and refractory DLBCL patients of both subtypes responded to these agents. Importantly, a strong correlation between CD22 and CD79B expression in vitro and in vivo was not detectable, indicating that patients should not be excluded from anti-CD22-MMAE or anti-CD79B-MMAE treatment because of low target expression. In summary, these studies suggest that pinatuzumab vedotin and polatuzumab vedotin are active agents for the treatment of patients with different subtypes of DLBCL.

Structural and functional analysis of the interaction between the agonistic monoclonal antibody Apomab and the proapoptotic receptor DR5.

Activation of the proapoptotic receptor death receptor5 (DR5) in various cancer cells triggers programmed cell death through the extrinsic pathway. We have generated a fully human monoclonal antibody (Apomab) that induces tumor cell apoptosis through DR5 and investigated the structural features of its interaction with DR5. Biochemical studies showed that Apomab binds DR5 tightly and selectively. X-ray crystallographic analysis of the complex between the Apomab Fab fragment and the DR5 ectodomain revealed an interaction epitope that partially overlaps with both regions of the Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand binding site. Apomab induced DR5 clustering at the cell surface and stimulated a death-inducing signaling complex containing the adaptor molecule Fas-associated death domain and the apoptosis-initiating protease caspase-8. Fc crosslinking further augmented Apomab's proapoptotic activity. In vitro, Apomab triggered apoptosis in cancer cells, while sparing normal hepatocytes even upon anti-Fc crosslinking. In vivo, Apomab exerted potent antitumor activity as a single agent or in combination with chemotherapy in xenograft models, including those based on colorectal, non-small cell lung and pancreatic cancer cell lines. These results provide structural and functional insight into the interaction of Apomab with DR5 and support further investigation of this antibody for cancer therapy.

Achaete-scute like 2 (ascl2) is a target of Wnt signalling and is upregulated in intestinal neoplasia.

Achaete-scute like (ASCL)2 is a basic helix-loop-helix transcription factor essential for the maintenance of proliferating trophoblasts during placental development. Using oligonucleotide microarrays we identified ascl2 as a gene significantly upregulated in colorectal adenocarcinomas (n=36 cancers, n=16 normals; 15-fold, P<0.0001). This finding was confirmed by quantitative reverse transcriptase (RT)-PCR on large intestinal cancers (n=29 cancers, n=16 normals; 10-fold, P<0.0001). In situ hybridization for ascl2 demonstrated expression at the base of small and large intestinal crypts (n=304), but in no other normal tissues excepting placenta. By in situ hybridization, 52-71% of colorectal adenomas (n=187), 50-73% of large (n=327) and 33-64% of small intestinal adenocarcinomas (n=124) were positive for ascl2 expression. Upregulation of murine ascl2 was also observed using oligonucleotide microarrays, quantitative RT-PCR and in situ hybridization on apcmin/+ and apc1638N/+ smad4-/+ tumours. Tumour cell lines stably transfected with LEF1(DN) or APC2, or transiently transfected with short-interfering RNA (siRNA) against beta-catenin showed a significant downregulation of ascl2. Colocalization of ascl2 with nuclear beta-catenin was observed in 73 small intestinal adenocarcinomas (P=0.0008) and apcmin/+ tumours. Preliminary in vitro data suggest ascl2 may promote progression through the G2/M cell cycle checkpoint. In summary, ascl2 is a putative regulator of proliferation that is overexpressed in intestinal neoplasia.

Repressor roles for TCF-4 and Sfrp1 in Wnt signaling in breast cancer.

Mutations in Wnt pathway genes are rare in human breast cancer, yet activation of the pathway is evident from the misolocalization of beta-catenin. We searched for relationships in the expression of Wnt pathway genes and found that both secreted frizzled related protein 1 (Sfrp1) and TCF-4 transcripts were all highly downregulated in a common subset of breast cancers relative to normal breast tissue. Sfrp1 has been previously characterized as a Wnt inhibitor, and we found that interfering with its expression in the human mammary epithelial cell line MCF10A activated Wnt signaling. Reduction of TCF-4 levels in breast cancer was surprising as it is a transcription factor that is responsive to Wnt signaling. Therefore, we investigated a possible inhibitory role for TCF-4 in human breast cells as well as further characterizing Sfrp1. We identified CD24 as a Wnt target in MCF10A cells and used its expression a marker of Wnt signaling. Interfering with either Sfrp1 or TCF-4 in this cell line enhanced CD24 expression. Furthermore, removal of TCF/LEF binding sites in a CD24-luciferase reporter resulted in elevated reporter gene expression. Our results indicate that both Sfrp1 and TCF-4 repress Wnt signaling in breast tissue and their downregulation contributes to the activation of Wnt signaling.

Expression of vascular endothelial growth factor, hypoxia inducible factor 1alpha, and carbonic anhydrase IX in human tumours.

AIMS: To measure vascular endothelial growth factor (VEGF-A) mRNA in a large, diverse cohort of tumours and to investigate whether VEGF-A expression is associated with markers of hypoxia, including hypoxia inducible factor 1alpha (HIF-1alpha) and carbonic anhydrase IX (CA9). METHODS: The expression of VEGF-A and CA9 was assessed in 5067 fresh frozen human tissue samples and 238 cell lines by DNA microarray analysis. In addition, tissue microarrays were constructed from 388 malignancies to investigate the expression of VEGF-A and HIF-1alpha by in situ hybridisation and immunohistochemistry, respectively. RESULTS: VEGF-A was significantly upregulated in primary malignancies of the breast, cervix, colon and rectum, oesophagus, head and neck, kidney, ovary, skin, urinary system, and white blood cells by DNA microarray analysis. However, VEGF-A expression only correlated with CA9 expression in renal tissues. In the tissue microarrays, HIF-1alpha positive cores showed a significant increase in VEGF-A expression in lung, ovary, soft tissue, and thyroid malignancies. CONCLUSIONS: The expression of VEGF-A is upregulated in a large proportion of human malignancies, and may be associated with markers of hypoxia. VEGF-A expression can be induced in the absence of hypoxia and hypoxia does not always provoke VEGF-A upregulation in tumours.

Dynamics of notch expression during murine prostate development and tumorigenesis.

Notch signaling has been widely demonstrated to be responsible for cell fate determination during normal development and implicated in human T-cell leukemia and mouse mammary carcinomas. Here we show that Notch signaling may be involved in prostatic development and cancer cell growth. In situ hybridization and reverse transcription-PCR analyses revealed that Notch1 was expressed in prostate epithelial cells during normal development and in prostate cancer cells. Characterization of Notch1-green fluorescent protein transgenic mice, in which the expression of reporter green fluorescent protein is under the control of the Notch1 promoter, indicated that Notch1-expressing cells were associated with the basal epithelial cell population in the prostate. Examination of the transgenic adenocarcinoma of the mouse prostate showed that expression of Notch1 was elevated in malignant prostatic epithelial cells of primary and metastatic tumors. Expression of Notch ligands, however, was low or undetectable in cultured prostate cancer cells or in malignant prostatic epithelial cells in transgenic adenocarcinoma of the mouse prostate. Furthermore, overexpression of a constitutively active form of Notch1 inhibited the proliferation of various prostate cancer cells, including DU145, LNCaP, and PC3 cells. Taken together, our data indicate for the first time that Notch signaling may play a role in murine prostatic development and tumorigenesis.

Isotype-dependent inhibition of tumor growth in vivo by monoclonal antibodies to death receptor 4.

To explore an approach for death receptor targeting in cancer, we developed murine mAbs to human death receptor 4 (DR4). The mAb 4H6 (IgG1) competed with Apo2L/TNF-related apoptosis-inducing ligand (DR4's ligand) for binding to DR4, whereas mAb 4G7 (IgG2a) did not. In vitro, both mAbs showed minimal intrinsic apoptosis-inducing activity, but each triggered potent apoptosis upon cross-linking. In a colon tumor nude mouse model in vivo, mAb 4H6 treatment without addition of exogenous linkers induced apoptosis in tumor cells and caused complete tumor regression, whereas mAb 4G7 partially inhibited tumor growth. An IgG2a isotype switch variant of mAb 4H6 was much less effective in vivo than the parent IgG1-4H6, despite similar binding affinities to DR4. The same conclusion was obtained by comparing other IgG1 and IgG2 mAbs to DR4 for their anti-tumor activities in vivo. Thus, the isotype of anti-DR4 mAb may be more important than DR4 binding affinity for tumor elimination in vivo. Anti-DR4 mAbs of the IgG1 isotype may provide a useful tool for investigating the therapeutic potential of death receptor targeting in cancer.

Selective expression of murine prostate stem cell antigen in fetal and adult tissues and the transgenic adenocarcinoma of the mouse prostate model of prostate carcinogenesis.

Prostate stem cell antigen (PSCA) is a GPI-anchored membrane protein whose expression is reportedly up-regulated in a majority of human prostate cancers, including advanced stages and metastases. In this study, we investigate the expression pattern of the murine orthologue of PSCA by in situ hybridization in fetal and adult mouse tissues. Murine PSCA is expressed during fetal development in the urogenital sinus, skin, and gastrointestinal tract. The expression in these tissues is restricted to the most superficial cell layer. In the adult mouse, expression is highest in the mucosal lining of the urinary tract. In the normal adult prostate, expression of PSCA is detected exclusively in the secretory epithelium. Examination of PSCA during carcinogenesis of the murine prostate in the transgenic adenocarcinoma of the mouse prostate model showed a markedly increased expression in areas of neoplasia. The transgenic adenocarcinoma of the mouse prostate model may represent a valuable model for the study of PSCA as a potential target for immunotherapy of prostate cancer, despite potential differences in the pattern of expression between mice and humans.

PTEN 2, a Golgi-associated testis-specific homologue of the PTEN tumor suppressor lipid phosphatase.

The tumor suppressor PTEN is a phosphatidylinositol phospholipid phosphatase, which indirectly down-regulates the activity of the protein kinase B/Akt survival kinases. Examination of sequence data bases revealed the existence of a highly conserved homologue of PTEN. This homologue, termed PTEN 2, contained an extended amino-terminal domain having four potential transmembrane motifs, a lipid phosphatase domain, and a potential lipid-binding C2 domain. Transcript analysis demonstrated that PTEN 2 is expressed only in testis and specifically in secondary spermatocytes. In contrast to PTEN, PTEN 2 was localized to the Golgi apparatus via the amino-terminal membrane-spanning regions. Molecular modeling suggested that PTEN 2 is a phospholipid phosphatase with potential specificity for the phosphate at the 3 position of inositol phosphates. Enzymatic analysis of PTEN 2 revealed substrate specificity that is similar to PTEN, with a preference for the dephosphorylation of the phosphatidylinositol 3,5-phosphate phospholipid, a known mediator of vesicular trafficking. Together, these data suggest that PTEN 2 is a Golgi-localized, testis-specific phospholipid phosphatase, which may contribute to the terminal stages of spermatocyte differentiation.

Overexpression of HER2/neu in solid tumours: an immunohistochemical survey.

AIMS: Using a standardized immunohistochemical assay we have evaluated 575 primary neoplasms of different histogenesis to determine the incidence of HER2 overexpression in some of the most common categories of human solid neoplasms. This study addresses the variable incidence of HER2 overexpression previously published for some tumour types. METHODS AND RESULTS: The immunohistochemical staining was performed on paraffin sections of surgical specimens and a well-defined scoring system based upon numbers of HER2 receptors expressed on the cell surface was applied. Overexpression of HER2 as defined as a HER2 score of equal or greater than 2 was seen in breast cancer (22%), pulmonary adenocarcinoma (28%), colorectal adenocarcinomas (17%), pulmonary squamous (11%) and gastric adenocarcinomas (11%). As expected, the proportion of cases with a HER2 score of 3 was highest in breast cancer. Contrary to published results prostate and pancreas adenocarcinomas showed a very low incidence of HER2 overexpression. CONCLUSIONS: Overexpression of HER2 is detected immunohistochemically in a proportion of epithelial neoplasms of diverse histogenesis in addition to ductal breast cancer. The standardized format of the assay will allow comparative analyses of studies performed at different institutions.

Safety and antitumor activity of recombinant soluble Apo2 ligand.

TNF and Fas ligand induce apoptosis in tumor cells; however, their severe toxicity toward normal tissues hampers their application to cancer therapy. Apo2 ligand (Apo2L, or TRAIL) is a related molecule that triggers tumor cell apoptosis. Apo2L mRNA is expressed in many tissues, suggesting that the ligand may be nontoxic to normal cells. To investigate Apo2L's therapeutic potential, we generated in bacteria a potently active soluble version of the native human protein. Several normal cell types were resistant in vitro to apoptosis induction by Apo2L. Repeated intravenous injections of Apo2L in nonhuman primates did not cause detectable toxicity to tissues and organs examined. Apo2L exerted cytostatic or cytotoxic effects in vitro on 32 of 39 cell lines from colon, lung, breast, kidney, brain, and skin cancer. Treatment of athymic mice with Apo2L shortly after tumor xenograft injection markedly reduced tumor incidence. Apo2L treatment of mice bearing solid tumors induced tumor cell apoptosis, suppressed tumor progression, and improved survival. Apo2L cooperated synergistically with the chemotherapeutic drugs 5-fluorouracil or CPT-11, causing substantial tumor regression or complete tumor ablation. Thus, Apo2L may have potent anticancer activity without significant toxicity toward normal tissues.

Chromosome 18 breakpoint in t(11;18)(q21;q21) translocation associated with MALT lymphoma is proximal to BCL2 and distal to DCC.

The t(11;18)(q21;q21) translocation has recently been identified as a recurring chromosomal abnormality in a subset of extranodal marginal zone B-cell lymphoma, a low-grade lymphoma of mucosa-associated lymphoid tissue (MALT). Neither the 11q21 nor the 18q21 breakpoints have been characterized by molecular genetic analysis. As a prelude to isolation of the gene(s) involved in this translocation, we have mapped the 18q21 breakpoint region by fluorescence in situ hybridization (FISH) of YAC and PAC clones. We mapped 37 YACs assigned to a 29-cM region within the chromosomal band 18q21. Using nine of these YACs in single- and/or dual-color FISH to analyze three cases of MALT lymphomas with the t(11;18)(q21;q21) translocation, we localized the breakpoints within a 1.6-Mb nonchimeric YAC (938E1). This YAC is useful for the detection of the translocation in metaphase and in interphase cells. A nonchimeric YAC contig of an 8-cM region around the breakpoint comprising nine YACs and a PAC contig of YAC 938E1 were constructed, which enabled the refinement of the breakpoint region in the proximal region of the YAC within a <820-kb segment. This breakpoint is proximal to the BCL2 locus and distal to DCC and DPC4 loci in chromosomal band 18q21.

New entities, issues, and controversies in the classification of malignant lymphoma.

The classification of malignant lymphoma has, for decades, provided a fertile area for controversy, discussion, and change. Such debate is necessary in order to appropriately assimilate new knowledge regarding this group of neoplasms. In this review, we provide a brief account of the evolution of classification schemes for lymphoma, and emphasize the recently proposed Revised European-American Lymphoma (REAL) classification as a synthesis of current knowledge of clinical, morphologic, immunologic, and molecular data. Specific entities that are recently described or for which there are current controversies are discussed. The necessity of communication between clinicians and pathologists in the workup of a patient with malignant lymphoma is emphasized.

An interleukin 12-related cytokine is up-regulated in ulcerative colitis but not in Crohn's disease.

BACKGROUND & AIMS: Interleukin 12 (IL-12) is a heterodimeric, macrophage-derived cytokine that is elevated in Crohn's disease (CD). Epstein-Barr virus-induced gene 3 (EBI3) is a recently characterized human glycoprotein that is homologous to the 40-kilodalton chain of IL-12 and forms a heterodimer with the 35-kilodalton chain of IL-12. We investigated the expression of EBI3 in colonic mucosa of normal control subjects, patients with ulcerative colitis (UC), and patients with CD. METHODS: Colonic tissue was analyzed for messenger RNA (mRNA) expression by quantitative polymerase chain reaction and for protein expression by immunohistology and Western blotting. RESULTS: EBI3 mRNA was present in intestinal biopsy specimens from healthy subjects and patients with CD but was elevated only in active UC. EBI3 levels in UC specimens correlated with histological scores of activity and T-cell infiltration. EBI3-positive cells that had a shape consistent with that of macrophages were identified in the lamina propria, and protein was detected by Western blotting. CONCLUSIONS: EBI3 is a novel IL-12-related cytokine that is expressed by macrophage-like cells in normal intestine and CD and has enhanced expression in active UC but not in active CD.

Morphologic bone marrow changes in patients with posttransplantation lymphoproliferative disorders.

The clinical workup of patients with posttransplantation lymphoproliferative disorders (PTLPDs) frequently involves bone marrow biopsies. However, little is known about the morphologic bone marrow changes in patients with PTLPD. To define the spectrum of morphologic bone marrow changes in such patients, we evaluated the bone marrow biopsy samples of 26 transplant patients with proven extramedullary PTLPD and of 20 transplant patients without PTLPD. Morphologic changes were present in 14 of 26 patients with PTLPD (54%) and consisted of aggregates of lymphoid and plasma cells with variable histologic and cytologic features. Cells expressing Epstein-Barr virus-encoded small transcripts (EBER) were seen in 9 of 13 bone marrow biopsy samples with morphologic changes and in none of the biopsy samples without morphologic changes. Bone marrow changes were significantly more frequent in patients with PTLPD who were younger than 18 years of age (76%) compared with those who were older than 18 years of age (11%). The difference in mortality rates between the patient groups with and without bone marrow changes was statistically not significant, possibly because of the small sample size. The finding that children with PTLPD have an increased incidence of bone marrow changes supports the notion that Epstein-Barr virus-associated PTLPD involves different pathogenetic mechanisms in pediatric patients than in adults.

Antigenic cancer cells grow progressively in immune hosts without evidence for T cell exhaustion or systemic anergy.

One enigma in tumor immunology is why animals bearing malignant grafts can reject normal grafts that express the same nonself-antigen. An explanation for this phenomenon could be that different T cell clones react to the normal graft and the malignant cells, respectively, and only the tumor-reactive clonotypes may be affected by the growing tumor. To test this hypothesis, we used a T cell receptor transgenic mouse in which essentially all CD8(+) T cells are specific for a closely related set of self-peptides presented on the MHC class I molecule Ld. We find that the tumor expressed Ld in the T cell receptor transgenic mice but grew, while the Ld-positive skin was rejected. Thus, despite an abundance of antigen-specific T cells, the malignant tissue grew while normal tissue expressing the same epitopes was rejected. Therefore, systemic T cell exhaustion or anergy was not responsible for the growth of the antigenic cancer cells. Expression of costimulatory molecules on the tumor cells after transfection and preimmunization by full-thickness skin grafts was required for rejection of a subsequent tumor challenge, but there was no detectable effect of active immunization once the tumor was established. Thus, the failure of established tumors to attract and activate tumor-specific T cells at the tumor site may be a major obstacle for preventive or therapeutic vaccination against antigenic cancer.