High-dose chemotherapy and autologous stem cell transplantation for primary central nervous system lymphoma: a multi-centre retrospective analysis from the United Kingdom.

The prognosis of patients with primary central nervous system lymphoma (PCNSL) has improved in recent years. This has partly been achieved by remission induction protocols incorporating high-dose methotrexate (HD-MTX) and rituximab. Given the high rates of relapse, consolidation therapy is usually considered in first response. Whole brain radiotherapy may prolong PFS but appears to confer no long-term survival advantage and is associated with significant neurocognitive dysfunction. Attempts to improve efficacy and reduce neurotoxicity of consolidation therapy have included thiotepa-based high-dose chemotherapy and autologous stem cell transplant (HDC-ASCT). This multi-centre, retrospective study reports the outcome of 70 patients undergoing HDC-ASCT for PCNSL in the United Kingdom. The median age at diagnosis was 56 years and all patients received HD-MTX-containing induction regimens. All patients underwent HDC-ASCT in first response. The rate of complete response increased from 50% before HDC-ASCT to 77% following HDC-ASCT. Treatment-related mortality was 6%. At a median follow-up of 12 months from HDC-ASCT, the estimated 1- and 2-year PFS rates were 71.5% and overall survival 86.4% and 83.3%, respectively. These data are comparable to published studies of HDC-ASCT for PCNSL, supporting its feasibility and efficacy.

Phase II study of copanlisib, a PI3K inhibitor, in relapsed or refractory, indolent or aggressive lymphoma.

BACKGROUND: Copanlisib is a pan-class I phosphatidylinositol 3-kinase inhibitor with predominant activity against the α- and δ-isoforms. PATIENTS AND METHODS: This phase II study evaluated the response rate of copanlisib administered intravenously on days 1, 8, and 15 of a 28-day cycle, in patients with indolent or aggressive malignant lymphoma. Archival tumor tissues were used for immunohistochemistry, gene-expression profiling, and mutation analysis. RESULTS: Thirty-three patients with indolent lymphoma and 51 with aggressive lymphoma received copanlisib. Follicular lymphoma (48.5%) and peripheral T-cell lymphoma (33.3%) were the most common histologic subtypes. Most patients (78.6%) had received prior rituximab and 54.8% were rituximab-refractory. Median duration of treatment was 23 and 8 weeks in the indolent and aggressive cohorts, respectively (overall range 2-138). Eighty patients were evaluated for efficacy. The objective response rate was 43.7% (14/32) in the indolent cohort and 27.1% (13/48) in the aggressive cohort; median progression-free survival was 294 days (range 0-874) and 70 days (range 0-897), respectively; median duration of response was 390 days (range 0-825) and 166 days (range 0-786), respectively. Common adverse events included hyperglycemia (57.1%; grade ≥3, 23.8%), hypertension (54.8%; grade ≥3, 40.5%), and diarrhea (40.5%; grade ≥3, 4.8%), all generally manageable. Neutropenia occurred in 28.6% of patients (grade 4, 11.9%). Molecular analyses showed enhanced antitumor activity in tumors with upregulated phosphatidylinositol 3-kinase pathway gene expression. CONCLUSION: Intravenous copanlisib demonstrated promising efficacy and manageable toxicity in heavily pretreated patients with various subtypes of indolent and aggressive malignant lymphoma. Subtype-specific studies of copanlisib in patients with follicular, peripheral T-cell, and mantle cell lymphomas are ongoing. This trial is registered with ClinicalTrials.gov number NCT01660451 (Part A).

A study to investigate dose escalation of doxorubicin in ABVD chemotherapy for Hodgkin lymphoma incorporating biomarkers of response and toxicity.

BACKGROUND: Myelotoxicity during initial cycles of chemotherapy for Hodgkin lymphoma is associated with better outcome, supporting the concept of individualised dosing based on pharmacodynamic end points to optimise results. This study was performed to identify the maximum tolerated dose (MTD) of doxorubicin within cycles 1-3 ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine). Circulating biomarkers of response (nucleosomal DNA, nDNA) and epithelial toxicity (Cytokeratin 18, CK18) were also measured. METHODS: Dose escalation of doxorubicin in cycles 1-3 ABVD supported by pegfilgrastim was performed on a six-patient cohort basis (35, 45 and 55 mg m(-2)) with doxorubicin reduced to 25 mg m(-2) or omitted in cycles 4-6 to maintain cumulative exposure of 103-130% standard ABVD. BVD was given at standard doses throughout. Six additional subjects were recruited at the MTD. RESULTS: Twenty-four subjects were recruited. Dose-limiting toxicities (DLTs) of grade 3 neuropathy, pneumonitis, palmar-plantar erythema and neutropenic infection were observed at 55 mg m(-2), so 45 mg m(-2) was declared the MTD. In patients who subsequently experienced DLT at any time, large increases in CK18 were seen on day 3 of cycle 1 ABVD. CONCLUSION: Escalated ABVD incorporating doxorubicin at 45 mg m(-2) in cycles 1-3 can be delivered safely with pegfilgrastim support. Circulating cell death biomarkers may assist in the development of future individualised dosing strategies.

Symptoms do not predict colorectal cancer in an FOB screened population.

BACKGROUND AND AIMS: The Scottish Bowel Screening Programme aims to detect cancer in asymptomatic individuals. We aimed to measure the prevalence of lower gastrointestinal symptoms in faecal occult blood (FOB) screen-positive patients, to correlate the symptoms with neoplasia and to compare the predictive value of FOB screening with urgent symptomatic referrals in Ayrshire and Arran. METHODS: Data were collected prospectively on FOB screen-positive patients undergoing colonoscopy. Patients completed a symptom questionnaire. Positive predictive values (PPVs) for detecting neoplasia were calculated and a chi-square test was performed to determine any influence of symptoms in diagnosing neoplasia. Symptomatic patients undergoing colonoscopy via a general practice fast-track system were compared. RESULTS: A total of 378 FOB screen-positive patients were included. In all, 198 (52%) had colorectal symptoms. Overall, 32 were diagnosed with colorectal cancer and 93 had polyps . FOB positivity and symptoms gave a PPV of 34% for neoplasia. FOB positivity without symptoms gave a PPV of 32% for neoplasia. Urgent referral of symptomatic patients had a lower PPV of 21% for neoplasia (p < 0.001). CONCLUSION: Half the FOB screen-positive patients had bowel symptoms. Symptoms in these patients had no correlation with an increased rate of neoplasia. The PPV for neoplasia is superior in symptomatic and asymptomatic screen-positive patients when compared to conventional urgent symptom-based referral.

Assessment of circulating biomarkers for potential pharmacodynamic utility in patients with lymphoma.

PURPOSE: Treatment efficacy and toxicity are difficult to predict in lymphoma patients. In this study, the utility of circulating biomarkers in predicting and/or monitoring treatment efficacy/toxicity were investigated. PATIENTS AND METHODS: Circulating biomarkers of cell death (nucleosomal DNA (nDNA) and cytokeratin 18 (CK18)), and circulating FLT3 ligand, a potential biomarker of myelosuppression, were assessed before and serially after standard chemotherapy in 49 patients with Hodgkin and non-Hodgkin lymphoma. Cytokeratin 18 is not expressed in lymphoma cells so is a potential biomarker of epithelial toxicity in this setting. Tumour response was assessed before and after completion of chemotherapy by 2D and 3D computed tomography radiological response. RESULTS: Baseline nDNA level was significantly higher in all lymphoma subtypes compared with 61 healthy controls and was prognostic for progression-free survival in diffuse large B-cell lymphoma (DLBCL). Decreases in nDNA levels were observed in the first week after chemotherapy; in FL, early falls in nDNA predicted for long remission following therapy. In DLBCL, elevations in nDNA occurred in cases with progressive disease. Circulating CK18 increased within 48 h of chemotherapy and was significantly higher in patients experiencing epithelial toxicity graded >3 by Common Terminology for Classification of Adverse Events criteria. FLT3 ligand was elevated within 3-8 days of chemotherapy initiation and predicted those patients who subsequently developed neutropenic sepsis. CONCLUSION: These data suggest circulating biomarkers contribute useful information regarding tumour response and toxicity in patients receiving standard chemotherapy and have potential utility in the development of individualised treatment approaches in lymphoma. These biomarkers are now being tested within multicentre phase III trials to progress their qualification.

The UK national breast cancer screening programme for survivors of Hodgkin lymphoma detects breast cancer at an early stage.

BACKGROUND: Supradiaphragmatic radiotherapy (SRT) to treat Hodgkin's lymphoma (HL) at a young age increases the risk of breast cancer (BC). A national notification risk assessment and screening programme (NRASP) for women who were treated with SRT before the age of 36 years was instituted in the United Kingdom in 2003. In this study, we report the implementation and screening results from the largest English Cancer Network. METHODS: A total of 417 eligible women were identified through cancer registry/hospital databases and from follow-up (FU) clinics. Screening results were collated retrospectively, and registry searches were used to capture BC cases. RESULTS: Of the 417 women invited for clinical review, 243 (58%) attended. Of these 417 women, 23 (5.5%) have been diagnosed with BC, a standardised incidence ratio of 2.9 compared with the age-matched general population. Of five invasive BCs diagnosed within the NRASP, none involved axillary lymph nodes compared with 7 of 13 (54%) diagnosed outside the programme (P<0.10). The mean latency for BC cases was 19.5+/-8.35 years and the mean FU duration for those unaffected by BC was 14.6+/-9.11 years (P<0.01), suggesting that those unaffected by BC remain at high risk. Recall and negative biopsy rates were acceptable (10.5 and 0.8%, respectively). CONCLUSIONS: The NRASP appears to detect BC at an early stage with acceptable biopsy rates, although numbers are small. Determination of NRASP results on a national basis is required for the accurate evaluation of screening efficacy in women previously treated with SRT.

Contribution of variant alleles of ABCB11 to susceptibility to intrahepatic cholestasis of pregnancy.

BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) has a complex aetiology with a significant genetic component. ABCB11 encodes the bile salt export pump (BSEP); mutations cause a spectrum of cholestatic disease, and are implicated in the aetiology of ICP. METHODS: ABCB11 variation in ICP was investigated by screening for five mutant alleles (E297G, D482G, N591S, D676Y and G855R) and the V444A polymorphism (c.1331T>C, rs2287622) in two ICP cohorts (n = 333 UK, n = 158 continental Europe), and controls (n = 261) for V444A. PCR primers were used to amplify and sequence patient and control DNA. The molecular basis for the observed phenotypes was investigated in silico by analysing the equivalent residues in the structure of the homologous bacterial transporter Sav1866. RESULTS: E297G was observed four times and D482G once. N591S was present in two patients; D676Y and G855R were not observed. The V444A polymorphism was associated with ICP (allelic analysis for C vs T: OR 1.7 (95% CI 1.4 to 2.1, p<0.001)). In addition, CC homozygotes were more likely to have ICP than TT homozygotes: OR 2.8 (95% CI 1.7 to 4.4 p<0.0001). Structural analyses suggest that E297G and D482G destabilize the protein fold of BSEP. The molecular basis of V444A and N591S was not apparent from the Sav1866 structure. CONCLUSIONS: Heterozygosity for the common ABCB11 mutations accounts for 1% of European ICP cases; these two mutants probably reduce the folding efficiency of BSEP. N591S is a recurrent mutation; however, the mechanism may be independent of protein stability or function. The V444A polymorphism is a significant risk factor for ICP in this population.

Imaging the effects of castration on bone turnover and hormone-independent prostate cancer colonization of bone.

INTRODUCTION: Tumor populations may selectively colonize bone that is being actively remodeled. In prostate cancer patients, androgen deprivation directly inhibits tumor growth initially, whilst induced bone loss may facilitate tumor colonization of bone by androgen-insensitive cells. We have tested this hypothesis using a xenograft model of early growth of prostate cancer in bone. METHODS: PC3 cells transfected with Green fluorescent protein (GFP) were injected into castrated and non-castrated athymic mice via intrabial and intracardiac routes. In vivo tumor growth was monitored daily and animals sacrificed 6-9 days following initial GFP-based detection of tumors. Tumor bearing and contra-lateral non-tumor bearing tibias were analyzed extensively by micro-CT and histology/immunohistochemistry for the presence of tumor cells and the effects of tumor and/or castration on bone cells and bone structure evaluated. RESULTS: GFP-positive tumors in bone were visible from 12 days post-injection following intratibial injection, allowing tumors <1 mm diameter to be monitored in live animals. Castration did not affect tumor frequency, tumor volume, or time to initial appearance of tumors injected via intratibial or intracardiac routes. Castration decreased trabecular bone volume in all mice. Significant tumor-induced suppression of numbers of osteoblasts, coupled with increased numbers of activated osteoclasts, was evident in both intact animals and castrated animals. CONCLUSIONS: In vivo GFP imaging allows the detection of early tumor growth at intra-osseous sites. Castration induces bone loss, but PC3-GFP cells are also capable of inducing bone remodeling in intact animals at early time points, independently of pre-existing castration-induced alterations to bone.

Acquisition of biologically relevant gene expression data by Affymetrix microarray analysis of archival formalin-fixed paraffin-embedded tumours.

Robust protocols for microarray gene expression profiling of archival formalin-fixed paraffin-embedded tissue (FFPET) are needed to facilitate research when availability of fresh-frozen tissue is limited. Recent reports attest to the feasibility of this approach, but the clinical value of these data is poorly understood. We employed state-of-the-art RNA extraction and Affymetrix microarray technology to examine 34 archival FFPET primary extremity soft tissue sarcomas. Nineteen arrays met stringent QC criteria and were used to model prognostic signatures for metastatic recurrence. Arrays from two paired frozen and FFPET samples were compared: although FFPET sensitivity was low ( approximately 50%), high specificity (95%) and positive predictive value (92%) suggest that transcript detection is reliable. Good agreement between arrays and real time (RT)-PCR was confirmed, especially for abundant transcripts, and RT-PCR validated the regulation pattern for 19 of 24 candidate genes (overall R(2)=0.4662). RT-PCR and immunohistochemistry on independent cases validated prognostic significance for several genes including RECQL4, FRRS1, CFH and MET - whose combined expression carried greater prognostic value than tumour grade - and cmet and TRKB proteins. These molecules warrant further evaluation in larger series. Reliable clinically relevant data can be obtained from archival FFPET, but protocol amendments are needed to improve the sensitivity and broad application of this approach.

Response evaluation in gastrointestinal stromal tumours treated with imatinib: misdiagnosis of disease progression on CT due to cystic change in liver metastases.

Imatinib is a highly effective treatment for patients with metastatic gastrointestinal stromal tumours (GIST). In most instances, response to imatinib treatment is assessed with CT. We present two cases where CT demonstrated the appearance of new low density liver lesions after 8-12 weeks of imatinib treatment. While this finding is consistent with progressive disease due to new lesions appearing at a previously uninvolved site, we hypothesise that the appearance of new liver lesions is in fact due to cystic change within previously occult, solid metastases. These untreated solid metastases were not visible on conventional portal phase CT due to their small size and vascular nature. Our hypothesis is supported by the observation that extrahepatic sites of disease had reduced in size over the same period of imatinib treatment and by the subsequent disease outcomes of these two cases. One patient, who continued imatinib because of significant symptomatic improvement despite the CT findings, remained stable on the same dose of imatinib for 18 months. The other patient, whose disease progressed when imatinib was withdrawn, had a dramatic response to treatment when imatinib was restarted at the same dose 2 years later. It is important that radiologists and oncologists who are involved in the management of GIST recognize that the appearance of new, low-density liver lesions on CT may represent a response to treatment. This finding must be correlated with symptomatic response and with tumour sites outside the liver before erroneously withdrawing effective imatinib treatment.

A phase II trial with RFS2000 (rubitecan) in patients with advanced non-small cell lung cancer.

Rubitecan (RFS2000, 9 nitrocamptothecin,) is a new oral topoisomerase I inhibitor. We report a phase II, single-arm, open-label study of RFS2000 as first line treatment for non-small cell lung cancer (NSCLC). Seventeen treatment-naïve patients with stage IIIB (9/17) and IV (8/17) NSCLC (11 male and 6 female) were treated, the median age was 62 years (range 52-86), and the majority of patients (14/17) were of performance status 1. RFS2000 was given orally, daily for 5 days, repeated every week. The starting dose was 1.5 mg/m(2)/day, and dose adjustment was permitted based upon toxicity. Fifteen patients had a dose escalation to 1.75 mg/m(2)/day and 7 had a second dose escalation to the protocol maximum level of 2.0 mg/m(2)/day. RFS2000 was tolerated well. Almost all adverse events were grade 1 and 2. The most frequently encountered adverse events were diarrhoea, nausea, anorexia, and lethargy. Neutropenia and thrombocytopenia were not observed in any patient. There were no responders to RFS2000 treatment, 10 patients had stable disease as their best response, whilst five had tumour progression. Two patients were not assessable for tumour response. The median survival time was 257 days (95% CI = 222-352). RFS2000 appears to be inactive at dose levels of 1.5-2.0 mg/m(2)/day in advanced NSCLC patients. Since only mild toxicity and no myelosuppression were encountered, these dose level are too low for this treatment-naïve patient population with NSCLC. Further studies at an increased dose would be required to identify whether this agent has merit in the treatment of NSCLC.

Genetic study of the CD36 gene in a French diabetic population.

OBJECTIVES: CD36 is a multifunctional membrane receptor widely expressed in different tissues which binds and internalizes oxidized low-density lipoprotein. In rodents, CD36 gene variations modulate glucose homeostasis and contribute to metabolic syndrome associated with type 2 diabetes but the effects in human are unknown. METHODS: We screened the entire coding sequence of the CD36 gene in 272 individuals and we genotyped both rare and frequent variants in 454 T2D subjects and 221 controls. RESULTS: We detected five mutations, P191P and N247S were only found each in one family and did not segregate with diabetes, the three others (A/C-178 in the promoter, A/G-10 in intron 3 and (GGGTTGAGA) insertion in intron 13) being equally frequent in diabetic subjects and in controls. However, adiponectin levels, a marker for insulin sensitivity, were significantly associated with the -178 A/C promoter variant allele (p=0.003, p corrected for multiple testing=0.036), possibly reflecting association with insulin-resistance in the French population. CONCLUSION: Thus, the -178 A/C SNP promoter mutation in the CD36 gene represents a putative genetic marker for insulin-resistance in the French population, although it does not appear to contribute to the genetic risk for T2D.

Detailed characterization of cysteine-less P-glycoprotein reveals subtle pharmacological differences in function from wild-type protein.

1. Subtle alterations in the coupling of drug binding to nucleotide hydrolysis were observed following mutation of all seven endogenous cysteine residues to serines in the human multidrug resistance transporter, P-glycoprotein. Wild-type (wt) and the mutant (cys-less) forms of P-gp were expressed in Trichoplusia ni (High Five) cells and purified by metal affinity chromatography in order to undertake functional studies. 2. No significant differences were observed in substrate ([(3)H]-azidopine) binding to wt or cys-less P-gp. Furthermore, neither the transported substrate vinblastine, nor the modulator nicardipine, differed in their respective potencies to displace [(3)H]-azidopine from the wt or cys-less P-gp. These results suggest that respective binding sites for these drugs were unaffected by the introduced cysteine to serine substitutions. 3. The Michaelis-Menten characteristics of basal ATP hydrolysis of the two isoforms of P-gp were identical. The maximal ATPase activity in the presence of vinblastine was marginally reduced whilst the K(m) was unchanged in cys-less P-gp compared to control. However, cys-less P-gp displayed lower overall maximal ATPase activity (62%), a decreased K(m) and a lower degree of stimulation (76%) in the presence of the modulator nicardipine. 4. Therefore, the serine to cysteine mutations in P-gp may suggest that vinblastine and nicardipine transduce their effects on ATP hydrolysis through distinct conformational pathways. The wt and cys-less P-gp isoforms display similarity in their fundamental kinetic properties thereby validating the use of cys-less P-gp as a template for future cysteine-directed structure/function analysis.

Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle.

P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an approximately 10 A resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding affinity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P(i). Biochemical data suggest that these rearrangements may involve rotation of transmembrane alpha-helices. A mechanism for transport is suggested.

Heterozygous MDR3 missense mutation associated with intrahepatic cholestasis of pregnancy: evidence for a defect in protein trafficking.

Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy with serious consequences for the mother and fetus. Two pedigrees have been reported with ICP in the mothers of children with a subtype of autosomal recessive progressive familial intrahepatic cholestasis (PFIC) with raised serum gamma-glutamyl transpeptidase (gamma-GT). Affected children have homozygous mutations in the MDR3 gene (also called ABCB4 ), and heterozygous mothers have ICP. More frequently, however, ICP occurs in women with no known family history of PFIC and the genetic basis of this disorder is unknown. We investigated eight women with ICP and raised serum gamma-GT, but with no known family history of PFIC. DNA sequence analysis revealed a C to A transversion in codon 546 in exon 14 of MDR3 in one patient, which results in the missense substitution of the wild-type alanine with an aspartic acid. We performed functional studies of this mutation introduced into MDR1, a closely related homologue of MDR3. Fluorescence activated cell sorting (FACS) and western analysis indicated that this missense mutation causes disruption of protein trafficking with a subsequent lack of functional protein at the cell surface. The demonstration of a heterozygous missense mutation in the MDR3 gene in a patient with ICP with no known family history of PFIC, analysed by functional studies, is a novel finding. This shows that MDR3 mutations are responsible for the additional phenotype of ICP in a subgroup of women with raised gamma-GT.

Cysteine-scanning mutagenesis provides no evidence for the extracellular accessibility of the nucleotide-binding domains of the multidrug resistance transporter P-glycoprotein.

Multidrug resistance of cancer cells is, at least in part, conferred by overexpression of P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) superfamily of active transporters. P-gp actively extrudes chemotherapeutic drugs from cells, thus reducing their efficacy. As a typical ABC transporter, P-gp has four domains: two transmembrane domains, which form a pathway through the membrane through which substrates are transported, and two hydrophilic nucleotide-binding domains (NBDs), located on the cytoplasmic side of the membrane, which couple the energy of ATP hydrolysis to substrate translocation. It has been proposed that the NBDs of ABC transporters, including the histidine permease of Salmonella typhimurium and the cystic fibrosis transmembrane conductance regulator, are accessible from the extracellular surface of the cell, spanning the membrane directly or potentially contributing to the transmembrane pore. Such organization would have significant implications for the transport mechanism. We determined to establish whether the NBDs of P-gp are exposed extracellularly and which amino acids are accessible, using cysteine-scanning mutagenesis and limited proteolysis. In contrast to other transporters, the data provided no evidence that the P-gp NBDs are exposed to the cell surface. The implications for the structure and mechanism of P-gp and other ABC transporters are discussed.

Tonsillar metastasis from malignant pulmonary carcinoid tumour.

Tumour metastases to the tonsil are rare and are usually due to spread from malignant melanoma and carcinomas of the breast, lung, kidney or stomach. We describe the clinical and histological findings of a tonsillar metastasis from a malignant pulmonary carcinoid tumour, an occurrence not previously reported.