Heinz Sill

Complex Tumor Genomes Inferred from Single Circulating Tumor Cells by Array-CGH and Next-Generation Sequencing

Circulating tumor cells (CTC) released into blood from primary cancers and metastases reflect the current status of tumor genotypes, which are prone to changes. Here, we conducted the first comprehensive genomic profiling of CTCs using array-comparative genomic hybridization (CGH) and next-generation sequencing. We used the U.S. Food and Drug Administration-cleared CellSearch system, which detected CTCs in 21 of 37 patients (range, 1-202/7.5 mL sample) with stage IV colorectal carcinoma. In total, we were able to isolate 37 intact CTCs from six patients and identified in those multiple colorectal cancer-associated copy number changes, many of which were also present in the respective primary tumor. We then used massive parallel sequencing of a panel of 68 colorectal cancer-associated genes to compare the mutation spectrum in the primary tumors, metastases, and the corresponding CTCs from two of these patients. Mutations in known driver genes [e.g., adenomatous polyposis coli (APC), KRAS, or PIK3CA] found in the primary tumor and metastasis were also detected in corresponding CTCs. However, we also observed mutations exclusively in CTCs. To address whether these mutations were derived from a small subclone in the primary tumor or represented new variants of metastatic cells, we conducted additional deep sequencing of the primary tumor and metastasis and applied a customized statistical algorithm for analysis. We found that most mutations initially found only in CTCs were also present at subclonal level in the primary tumors and metastases from the same patient. This study paves the way to use CTCs as a liquid biopsy in patients with cancer, providing more effective options to monitor tumor genomes that are prone to change during progression, treatment, and relapse.

BAALC, the human member of a novel mammalian neuroectoderm gene lineage, is implicated in hematopoiesis and acute leukemia

The molecular basis of human leukemia is heterogeneous. Cytogenetic findings are increasingly associated with molecular abnormalities, some of which are being understood at the functional level. Specific therapies can be developed based on such knowledge. To search for new genes in the acute leukemias, we performed a representational difference analysis. We describe a human gene in chromosome 8q22.3, BAALC (brain and acute leukemia, cytoplasmic), that is highly conserved among mammals but evidently absent from lower organisms. We characterized BAALC on the genomic level and investigated its expression pattern in human and mouse, as well as its complex splicing behavior. In vitro studies of the protein showing its subcellular localization suggest a function in the cytoskeleton network. Two isoforms are specifically expressed in neuroectoderm-derived tissues, but not in tumors or cancer cell lines of nonneural tissue origin. We show that blasts from a subset of patients with acute leukemia greatly overexpress eight different BAALC transcripts, resulting in five protein isoforms. Among patients with acute myeloid leukemia, those overexpressing BAALC show distinctly poor prognosis, pointing to a key role of the BAALC products in leukemia. Our data suggest that BAALC is a gene implicated in both neuroectodermal and hematopoietic cell functions.

Establishment of tumor‐specific copy number alterations from plasma DNA of patients with cancer

With the increasing number of available predictive biomarkers, clinical management of cancer is becoming increasingly reliant on the accurate serial monitoring of tumor genotypes. We tested whether tumor‐specific copy number changes can be inferred from the peripheral blood of patients with cancer. To this end, we determined the plasma DNA size distribution and the fraction of mutated plasma DNA fragments with deep sequencing and an ultrasensitive mutation‐detection method, i.e., the Beads, Emulsion, Amplification, and Magnetics (BEAMing) assay. When analyzing the plasma DNA of 32 patients with Stage IV colorectal carcinoma, we found that a subset of the patients (34.4%) had a biphasic size distribution of plasma DNA fragments that was associated with increased circulating tumor cell numbers and elevated concentration of mutated plasma DNA fragments. In these cases, we were able to establish genome‐wide tumor‐specific copy number alterations directly from plasma DNA. Thus, we could analyze the current copy number status of the tumor genome, which was in some cases many years after diagnosis of the primary tumor. An unexpected finding was that not all patients with progressive metastatic disease appear to release tumor DNA into the circulation in measurable quantities. When we analyzed plasma DNA from 35 patients with metastatic breast cancer, we made similar observations suggesting that our approach may be applicable to a variety of tumor entities. This is the first description of such a biphasic distribution in a surprisingly high proportion of cancer patients which may have important implications for tumor diagnosis and monitoring.

Signaling Through RAS-RAF-MEK-ERK: from Basics to Bedside

Aberrant signaling caused by mutations in the RAS-RAF-MEK-ERK pathway and its upstream activators critically contributes to human tumor development. Strategies, which aim at inhibiting hyperactive signaling molecules, appear conceptually straight forward, but their translation into clinical practice has been hampered by many setbacks. Understanding structure, function and regulation of this intracellular pathway as well as its crosstalk with other signaling activities in the cell will be essential to ensure reasonable usage of new therapeutic possibilities. This review provides an understanding of this signaling cascade as revealed by genetic and biochemical approaches and discusses the existing or arising possibilities to interfere with unphysiological activation in cancer. Signaling aberrations and signal transduction therapies will be discussed exemplary for two types of hematological neoplasia, acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS). In the future understanding the role of tumor stem cells, both as a source of tumor recurrence and tumor heterogeneity, the signals controlling their fate as well as epigenetic changes in cancer will be the next critical steps to further advance the applicability of these novel therapeutic strategies.

Two transforming C-RAF germ-line mutations identified in patients with therapy-related acute myeloid leukemia.

Mutations leading to activation of the RAF-mitogen-activated protein kinase/extracellular signal-regulated (ERK) kinase (MEK)-ERK pathway are key events in the pathogenesis of human malignancies. In a screen of 82 acute myeloid leukemia (AML) samples, 45 (55%) showed activated ERK and thus were further analyzed for mutations in B-RAF and C-RAF. Two C-RAF germ-line mutations, S427G and I448V, were identified in patients with therapy-related AML in the absence of alterations in RAS and FLT3. Both exchanges were located within the kinase domain of C-RAF. In vitro and in vivo kinase assays revealed significantly increased activity for (S427G)C-RAF but not for (I448V)C-RAF. The involvement of the S427G C-RAF mutation in constitutive activation of ERK was further confirmed through demonstration of activating phosphorylations on C-RAF, MEK, and ERK in neoplastic cells, but not in nonneoplastic cells. Transformation and survival assays showed oncogenic and antiapoptotic properties for both mutations. Screening healthy individuals revealed a <1/400 frequency of these mutations and, in the case of I448V, inheritance was observed over three generations with another mutation carrier suffering from cancer. Taken together, these data are the first to relate C-RAF mutations to human malignancies. As both mutations are of germ-line origin, they might constitute a novel tumor-predisposing factor.

Common alterations in gene expression and increased proliferation in recurrent acute myeloid leukemia.

Recurrent disease following high-dose chemotherapy is a major problem in patients with acute myeloid leukemia (AML). To identify its characteristics, we performed expression profiling in blasts from untreated AML and relapse, using a specific cDNA microarray comprising 4128 genes generated by cDNA subtraction supplemented with cancer-associated genes. Expression analysis of 18 AML bone marrow specimens showed that recurrent AML is commonly associated with the mRNA expression changes in a set of 58 genes. Increased cellular proliferation was indicated by the overexpression of the transferrin receptor, proliferating cell nuclear antigen, and G1 cyclins. An immunohistochemical study for Ki-67-positive blasts in 18 paired bone marrow biopsy samples confirmed a highly significant (P<0.0001) increase in the proliferation fraction at relapse. In addition, we found enhanced activation of the RAF/MEK/ERK cascade as mRNAs of MKP-1, c-jun, c-fos, and egr-1 were significantly increased at relapse. Immunohistochemistry and immunoblotting analyses for biphosphorylated ERK1/2 protein provide additional evidence for enhanced activation of the RAF/MEK/ERK pathway. The degree of increase is significantly correlated with the increased proliferation. Furthermore, the genes identified provide a rationale for further studies on predictive diagnosis and therapeutic intervention.

Therapy-related myeloid neoplasms: pathobiology and clinical characteristics

Therapy‐related myeloid neoplasms (t‐MNs) are serious long‐term consequences of cytotoxic treatments for an antecedent disorder. t‐MNs are observed after ionizing radiation as well as conventional chemotherapy including alkylating agents, topoisomerase‐II‐inhibitors and antimetabolites. In addition, adjuvant use of recombinant human granulocyte‐colony stimulating factor may also increase the risk of t‐MNs. There is clinical and biological overlap between t‐MNs and high‐risk de novo myelodysplastic syndromes and acute myeloid leukaemia suggesting similar mechanisms of leukaemogenesis. Human studies and animal models point to a prominent role of genetic susceptibilty in the pathogenesis of t‐MNs. Common genetic variants have been identified that modulate t‐MN risk, and t‐MNs have been observed in some cancer predisposition syndromes. In either case, establishing a leukaemic phenotype requires acquisition of somatic mutations – most likely induced by the cytotoxic treatment. Knowledge of the specific nature of the initiating exposure has allowed the identification of crucial pathogenetic mechanisms and for these to be modelled in vitro and in vivo. Prognosis of patients with t‐MNs is dismal and at present, the only curative approach for the majority of these individuals is haematopoietic stem cell transplantation, which is characterized by high transplant‐related mortality rates. Novel transplantation strategies using reduced intensity conditioning regimens as well as novel drugs – demethylating agents and targeted therapies – await clinical testing and may improve outcome. Ultimately, individual assessment of genetic risk factors may translate into tailored therapies and establish a strategy for reducing t‐MN incidences without jeopardizing therapeutic success rates for the primary disorders.

Posaconazole plasma concentrations and invasive mould infections in patients with haematological malignancies

Posaconazole (PCZ) is a triazole antifungal agent that has broad activity against pathogenic fungi and is increasingly used for prophylaxis and treatment of invasive mould infections (IMIs). PCZ is only available as an oral formulation, with varying absorption from the gastrointestinal tract. However, reports correlating PCZ plasma concentrations (PPCs) with breakthrough IMIs are rare. In this study, PPCs were analysed in a prospective, observational, single-centre study and the correlation of PPCs with breakthrough IMIs in patients with haematological malignancies was evaluated. Risk factors associated with low PPCs were further evaluated. A total of 109 PPCs were measured in 34 cases receiving PCZ prophylaxis (n=31) or treatment (n=3). Levels below the target of 0.5 μg/mL were detected in 24 (71%) of the 34 cases; in 15 (63%) of these 24 cases concentrations were found to be <0.20 μg/mL. Three patients receiving PCZ prophylaxis met the criteria of breakthrough infection. Notably, prior to development of IMI, PPCs were below the target in all three individuals. Associated risk factors for insufficient PPCs varied from previous reports. In conclusion, these data demonstrate that therapeutic drug monitoring of PCZ is mandatory in all patients with haematological malignancies as low PPCs are common and may be associated with development of IMIs.

RT-PCR and FISH analysis of acute myeloid leukemia with t(8;16)(p11;p13) and chimeric MOZ and CBP transcripts: breakpoint cluster region and clinical implications.

The translocation t(8;16)(p11;p13) is associated with acute myeloid leukemia displaying monocytic differentiation (AML FAB M4/5) and fuses the MOZ (also named MYST3) gene (8p11) with the CBP (also named CREBBP) gene (16p13). Detection of the chimeric RNA fusions has proven difficult; only three studies have described successful amplification of the chimeric MOZ-CBP and CBP-MOZ fusions by reverse transcriptase-polymerase chain reaction (RT-PCR). We analyzed four cases of AML M4/5 with t(8;16)(p11;p13) by RT-PCR and fluorescence in situ hybridization (FISH) and characterized the reciprocal RNA fusions from three cases. We cloned both genomic translocation breakpoints from one case by long-range PCR and successfully applied RT-PCR to monitor minimal residual disease (MRD) between clinical complete remission and relapse. In three cases, the genomic breakpoints occurred in MOZ intron 16 and CBP intron 2. In one case, no fusion transcript was detected. The available data suggest clustering of t(8;16)(p11;p13) breakpoints in these introns leading to reciprocal in-frame MOZ exon 16/CBP exon 3 and in-frame CBP exon 2/MOZ exon 17 chimeric transcripts in the majority of cases. The described RT-PCR strategy may be valuable both for the routine detection of the t(8;16)(p11;p13) as well as for monitoring of MRD in this prognostically unfavorable patient group.

Adoptive transfer of vitiligo after allogeneic bone marrow transplantation for non-Hodgkin's lymphoma

Vitiligo developed in a 50-year-old man 9 months after allogeneic transplantation from his HLA-identical sister who had had this disease for several years. Our findings suggest adoptive transfer of vitiligo by haematopoietic stem cell transplantation, and lend support to the autoimmune nature of this disease.