Alterations in glutamine metabolism have been proposed to play critical roles in cancer cell growth and survival. Mitochondrial glutaminase is a key enzyme in the conversion of glutamine to glutamate and is highly expressed in colorectal cancer (CRC). CB-839 is a potent and selective inhibitor of glutaminase, and synergistic activity in vitro and in vivo have been seen with CB-839 in combination with chemotherapy. We also demonstrated synergy between CB-839 and anti-EGFR monoclonal antibody therapy in multiple human CRC cell lines and that the combination could overcome anti-EGFR resistance. Given this preclinical data, we conducted a phase I/II study evaluating CB-839 with panitumumab. Our phase I results were presented earlier; here we present phase II. Study enrolled adult patients with metastatic, RAS wildtype CRC who achieved at least stable disease from prior anti-EGFR therapy. Patients were treated at the recommended phase II dose from phase I: 6 mg/kg panitumumab biweekly and 800 mg/kg daily CB-839 800 mg/kg in 28-day cycles. Imaging was assessed every eight weeks and the primary endpoint was response rate (RR). Enrollment followed a Simon’s two-stage design that required at least one response in the first 10 patients to continue. A sample size of 29 patients would provide 80% power to reject the null hypothesis of low efficacy (RR£5%) with a true RR of 20%. Correlative studies included two research PET scans (baseline and after one cycle) using 18F-FSPG, which provides a measurement of glutamine metabolism, and baseline tumor biopsies for RNA-seq. Seventeen patients were enrolled [median age, 52; male (71%); white (77%)]. All had received at least three prior lines of therapy for metastatic disease. Two responses were observed at the interim analysis and the study continued. Unfortunately, the development of CB-839 was discontinued and the study closed. Of the 17 patients enrolled, 15 were evaluable for efficacy. The RR was 13.3% (2/15) and the disease control rate was 46.7% (7/15). The median progression-free survival (PFS) and overall survival (OS) was 2.23 months and 8.87 months, respectively. At six months, the PFS and OS rates were 20.1% and 50.1%, respectively, while the OS rate at 12 months was 28.7%. The most frequently reported toxicities were: rash (70.6%); hypomagnesemia and nausea (35.3%); elevated alanine aminotransferase (29.4%); fatigue, paronychia, and vomiting (23.5%). Most were Grade 1 or 2. Eight patients had two research PET scans; preliminary analysis demonstrated a positive correlation (R2=0.37) with change in 18F-FSPG uptake and lesion diameter, suggesting lesions that are responding to treatment have less tracer accumulation. RNA-seq analysis is ongoing and will be presented. Panitumumab and CB-389 was very tolerable with some efficacy in heavily pre-treated patients. Early analysis of PET imaging data suggests a potential relationship between 18F-FSPG uptake and response, but additional studies are needed. It is anticipated that RNA-seq will identify genes that are related to glutamine avidity and represent a key determinate of response to inhibitors of glutamine metabolism.

The mammalian target of rapamycin (mTOR) signaling has been implicated in esophageal cancer progression. Preclinical studies have shown synergy between everolimus, an mTOR inhibitor, radiation, and platinum agents. Preclinical data suggested that Aurora Kinase A (AURKA) plays an important role in the development of everolimus resistance and that this is mediated through upregulation of c-MYC. We postulated that patients with persistent disease at time of surgery after neoadjuvant therapy would have higher levels of both AURKA and c-MYC. We conducted a multi-institutional phase IB trial to determine the recommended phase II dose of concurrent everolimus with carboplatin and radiation in patients with localized esophageal cancer. Patients with Stage II/III esophageal cancer and ECOG performance status 0-1 were enrolled. Following two cycles of induction Capecitabine/Oxaliplatin (XELOX), patients were restaged and those without evidence of disease progression, received radiation to a total dose of 50.4 Gy in 28 fractions with concurrent weekly carboplatin (AUC=2), with escalating doses of everolimus. A standard 3+3 dose escalation design was used. The recommended phase II dose of everolimus with concurrent weekly carboplatin and radiation was determined to be 2.5 mg QOD. Paraffin-embedded tissue blocks from the surgical specimen were used for immunohistochemical analysis for AURKA and c-MYC and levels of these markers were correlated with pathologic response. Nineteen patients were enrolled. There were two screen failures (thrombocytopenia, metastases) and four patients were removed from study due to poor tolerance of XELOX (n = 2) or disease progression (n = 2). Thirteen patients completed concurrent chemoradiation therapy and were deemed resectable. All patients had adenocarcinoma, median age was 58 and 85% were males. All patients achieved an R0 resection with a pathologic response rate of 40% and a pathologic complete response rate of 23%. The 2-year PFS and OS were 50% and 49.6%, respectively. Only 6 of the 13 patients had tissue available for biomarker analysis. All 6 patients had viable disease on final pathologic assessment suggesting resistance to everolimus. Five patients with high levels of AURKA also had high levels of c-MYC expression (83%). The one patient with low level of AURKA also had low levels of c-MYC expression (16%). Biomarker studies from this Phase IB trial suggest that AURKA is associated with c-MYC dependent resistance to mTOR inhibition. Targeting AURKA may be a novel therapeutic strategy that can be used in everolimus-resistant tumors that show overexpression of AURKA.

The persistence of minimal residual disease (MRD) post-treatment may provide an early indicator of impending relapse in acute myeloid leukaemia (AML) (Raanani & Ben-Bassat, 2004). The absence of an appropriate molecular signature in 40–60% of AML patients who have normal cytogenetics associated with intermediate risk has limited the analysis in this group. Through advances in mutation analysis a list of recurring gene mutations as potential MRD targets are now being compiled. Many AML cells have more than one recurring mutation, indicating that there is a multistep pathogenesis of disease. Mutation events in AML have been detected by analysis of the blast cells; therefore, chronology of mutational events within the stem cell and/or early progenitor populations has not been established. Many of these mutations result in the insertion or deletion of a small number of additional nucleotides that should allow for the design of a specific primer against the unique sequence generated by the mutation (Pabst et al, 2001). Found in approximately 20% of normal karyotype AML, the presence of CEBPA mutation segregates with favourable risk among this otherwise intermediate risk group. The CEBPA gene encodes the granulocytic differentiation factor CCAAT Enhancer Binding Protein-alpha and plays a crucial role in granulocytic differentiation of haematopoietic stem cells and has antiproliferative activity in various cell types. Mutation in CEBPA was investigated as a potential MRD marker because of its prevalence in normal karyotype AML, the frequency of insertion and deletion mutations enabling a straightforward design of mutation-specific primers and concordance between presentation and relapse (Tiesmeier et al, 2003). Four patients with normal karyotype AML FAB type M1 were selected for study based on the availability of diagnostic (patients 1, 3 and 4) or relapse (patient 2) bone marrow (BM) and on the presence of CEBPA mutations identified during previous screening (Snaddon et al, 2003). Samples were collected and analysed in accordance with the requirements of the local ethics committee. Five real-time quantitative polymerase chain reaction (RQ-PCR) assays were developed that selectively amplified mutated CEBPA DNA from four patients. Figure 1A and B demonstrates the RQ-PCR strategy used in this study. Three patients (1, 2 and 3) had an insertion mutation in the region of the gene that codes for the C-terminal DNA-binding/basic leucine zipper domain of the protein, the fourth contained mutations corresponding to both the N- and C-terminal regions of the C/EBP-α protein (4a and 4b respectively). Mutations in 1, 2, 4a, and 4b were all tandem duplications ranging from 3 to 57 base pairs (bp) in length while patient 3 had a 3-bp insertion. Mutation-specific forward primers were designed by making use of the unique sequences generated by the insertion mutations along with the introduction of single nucleotide changes in the primers to prevent annealing to the wild-type (WT) sequence. Patient-specific primer sequences are given in Fig 1C. Real-time PCR analysis was performed on 25 μl reaction mixture in duplicate using Taqman chemistry on the ABI PRISM 7700 Sequence Detector (Applied Biosystems, Warrington, UK) with default settings. β-2-Microglobulin was used as the endogenous reference gene to assess total cell number per well. (A) Real-time quantitative polymerase chain reaction (RQ-PCR) approach for the specific detection of C-terminal CEBPA mutations. All patient sequences are shown (P1, P2, P3, P4b). Forward and reverse primers are highlighted in yellow and purple respectively, and the Taqman probe is highlighted in green. A patient-specific forward primer was designed to preferentially anneal to a unique sequence generated by insertion mutation (red text and underlined). Base changes (lower case and blue highlighted) were introduced to the primer to prevent annealing and extension from wild-type (WT) sequence. The mean amplicon length was 158 bp (range 150–164) for patient assays 1, 2, 3 and 4b. Probe 1, 2, 3, 4b 5′-6-FAM-CTGGAGCTGACCAGTGACAATGACCGCCTGCGCAAGC-3′-Tamra (Genbank accession no. Y11525 bp 1095–1125). (B) RQ-PCR approach for the specific detection of N-terminal CEBPA mutation, patient sequence 4a (P4a) Probe 4a 5′-6-FAM-CCGCCTTCAACGACGAGTTCCTGGCCGACC-3′-Tamra (Genbank accession no. Y11525 bp 361–391). Amplicon length was 78 bp for patient assay 4a. (C) Characteristics of the CEBPA RQ-PCR assays. The specificity of each RQ-PCR assay was tested by amplifying 750 ng (equivalent to 125 000 cells) of each patient's diagnostic BM DNA, wt DNA, patient diagnostic BM DNA serially diluted in wt DNA and no template controls, in duplicate. An assay was deemed to be specific if no amplification was observed with wt DNA. To test the sensitivity of each assay a standard curve was generated by five serial dilutions of diagnostic DNA in water (ranging from 125 000 to 4 cells per well). In all amplification plots the same threshold was maintained to allow comparison between experiments. The correlation coefficient of the standard curves was at least 0·95, indicating that the assays had a high probability of precise quantification. The mean Ct for DNA equivalent to 125 000 cells was 24·6 (range 24–27). The mean slope of the dilution curves was −3·2 (range −3·5 to −2·8), indicating good amplification efficiencies (the perfect theoretical slope is −3·3). As shown in Fig 1C, the maximum reproducible sensitivity (defined as the dilution for which duplicates were both positive) for all the patient mutation assays was 10−4 (8·0 × 10−5; 10 in 125 000) and therefore were of the required order of sensitivity, >10−4, for an MRD monitoring assay. Insufficient material was available for retrospective MRD monitoring in these patients. Instead we have taken advantage of the assay's ability to quantify mutated cells for the determination of presence or absence of mutation within the leukaemic stem cell (LSC) population. The LSC generally accounts for approximately 1% of leukaemic cells (Hope et al, 2004). As with the normal haematopoietic stem cell (HSC) compartment, LSCs are not functionally homogeneous and comprise of distinct hierarchically arranged LSC classes (Hope et al, 2004). AML peripheral blood samples, with viable cells ranging from 2 to 27 × 106, from the four patients were flow sorted into three distinct hierarchal compartments. The most primitive stem cells, characterised by potential for long-term repopulation of multiple haematopoietic lineages, the common myeloid progenitors (CMPs) and their downstream progeny, the granulocyte/macrophage progenitors (GMPs). These populations were sorted based on the lack of lineage markers in all cases; the stem cells expressed CD34 cell surface marker, but not CD38 (Lin−/CD34+/CD38−). The CMPs and GMPs expressed CD34, CD38 and CD123 and were distinguished by presence (GMP) or absence (CMP) of CD45RA. DNA isolated from each compartment was tested with the RQ-PCR assays for mutation. In the stem cell compartment (Lin−/CD34+/CD38−), CEBPA mutation was detected in all patients, ranging from 15% to 65% mutation, with the exception of patient 1 where insufficient cells were collected for testing. The downstream populations, CMPs and GMPs, tested positive for the mutations in all cases. These assays, therefore, showed that the CEBPA mutations were present within the stem cell compartment in all evaluated patients. In conclusion we have developed five mutation-specific assays for four patients and have demonstrated these assays to be both sensitive and specific. As about 60% of CEBPA mutations in AML reported in the literature (Fröhling et al, 2004) are insertions or deletions this makes them an attractive RQ-PCR target for MRD monitoring. Indeed a survey of the sequences shows clustering of mutations and indicates that Probe 1,2,3,4b described herein could be applied to about 40% of these. This is the first study describing specific RQ-PCR for CEBPA mutations. Since the presence of CEBPA mutation among normal karyotype AML distinguishes a group with favourable prognosis, it is of potential therapeutic significance to stratify these patients and monitor them over the course of their disease. Its application and correlation with clinical outcome may now be evaluated, alongside other approaches, such as multiparametric flow cytometry and WT1-expression changes, for its ability to quantify MRD in AML informatively. Applying the same principles mutations in other genes also have potential as appropriate targets that may increase the number of AML patients with normal karyotype amenable to MRD monitoring. This study was funded by CRUK and Newman Foundation.

Background: Proteasome inhibition has been shown to be effective against a variety of tumours. In multiple myeloma the response rates to bortezomib (B) in relapsed patients is 46% (APEX study ≥ MR), but are likely to be up to 75% when combined with melphalan (M). Mechanisms underlying this effect are yet to be fully determined.

A subset of patients (pts.) with follicular lymphoma (FL) will transform to a more aggressive histological sub-type, most typically diffuse large B-cell lymphoma (DLBCL). In general response to therapy is poor and survival short. Paired analysis of samples pre- and post transformation suggest that the molecular mechanisms underlying transformation (Tx) are heterogeneous. In order to independently validate recurring changes in gene/protein expression at transformation (GC phenotype of TxDLBCL, Davies et al., 2002; loss of follicular dendritic Cell (FDC) markers, Shiozawa et al., 2003) a Tx-tissue microarray (Tx-TMA) was created comprising serial samples from 35 pts. (median age 54yrs (22–81) at the time of transformation). In these pts. transformation occurred a median of 3.1years from diagnosis (range 0–15.4) and for each pt. ‘set' at least 1 pre-Tx FL sample (1–3; n=56), and 1 (1–4; n=44) post transformation sample were represented on the array. To ensure that the Tx-TMA cores accurately represented the corresponding full tissue sections a panel of routine immunohistochemical (IHC) diagnostic markers (n=9) were scored. The concordance between Tx-TMA and full sections (n=10) was >90%. The Tx-TMA was then used to investigate the phenotype of transformed DLBCL, according to the germinal centre (GC)/non-GC like model of de novo DLBCL. Using CD10, BCL6 and MUM1 expression to discriminate between the two subclasses of DLBCL the methodology was first validated on a de novo DLBCL TMA (n=31; 20/31 (65%) non-GC, 11/31 (35%) GC phenotype; 5-yr survival for non-GC pts. 51% and for GC pts., 73%). IHC confirmed the results of gene expression profiling indicating that in 31/35 (89%) pts. transformed DLBCL was of GC phenotype (28/35 (80%) CD10+ and 3/35 (9%) CD10-, BCL6+, MUM1-). Of the remainder, 4/35 were CD10-, BCL6+, of which 3/4 were MUM1+ (3/35 (9%) non-GC phenotype; 1/4 MUM1 was not assessable). Similarly the Tx-TMA confirmed loss of FDC markers (CD21 and CD23) on transformation. Samples from 28 pts were evaluable for CD21 and CD23 IHC expression. In 71% (20/28) of pts. the FDC meshwork was lost or became more sparse on transformation (CD21 loss 15/28 (54%); CD23 loss 17/28 (61%)). The most discriminating changes in gene expression on transformation are now being assessed by IHC. Aurora kinase B (ARKB) is an attractive therapeutic target given that disruption of ARK function results in the induction of apoptosis in RL, a t(14:18) positive DLBCL cell line (Harrington et al. 2004). The observed elevation in ARKB transcription on transformation was confirmed by IHC in this series. The Tx-TMA showed ARKB expression increased on Tx in 13/33 (40%) pts., potentially defining a subset of pts. who might be considered for ARKB directed therapies. Expression of ARKB was low throughout in 18/33 (55%) pts and decreased in 2/33 (6%) pts.; difference in ARKB expression was not significantly associated with survival. These preliminary studies suggest that the availability of TMA of serial biopsies from pts. with transformed FL will provide a powerful means of assessing the relevance of gene expression, both within the tumour and the microenvironment while facilitating the selection of patients most likely to benefit from directed therapeutic approaches.

Follicular lymphoma (FL) is characterised by the presence of the t(14;18)(q32,q21) and represents similar to 25% of new cases, of non-Hodgkin's lymphoma. While the t(14,18) is a welf-documenterl rearrangement, the role of secondary cytogenetic abnormalities in the development and progression of these tumours remains unclear. Comparative genomic hybridisation was used to characterise changes in DNA copy number in tumour DNA from patients with this malignancy. The, mean numbers of deletion and amplification events found in each of the 45 samples studied were 1.8 and 23, respectively. Regions of recurrent (> 10% tumour samples) gain involved chromosomes 2p 13-16 (16%), 7 (20%), 12 (16%), 13q21-33 (18%), 18 (27%), and X (36%) and frequent losses localised to 6q (29%) and 17p (20%). Amplification of chromosome 13 represents a novel finding in FL The minimal amplified region was refined to a 6.8-Mb interval of 13q32-33 between the BAC clones 88K16 and 44H20 by fluorescence in situ hybridisation studies using metaphase chromosomes derived from tumour material. There are a number of reports in the literature suggesting that amplification of chromosome 13 also occurs in other human cancers. The, location of the putative oncogene on 13q described here In follicular and transformed lymphoma may also be important in the evolution of many other malignancies. (C) 2001 Wiley-Liss, Inc.

An unusual case of leukaemia in a patient with Hodgkin's disease is described. The leukaemic blast cell population was typified by the presence of a substantial proportion of binucleate and multinucleate cells, many of which had the morphological features of Sternberg-Reed cells. The circulating and bone marrow blast cells were shown by immunophenotyping to be of myeloid origin.