Prolyl hydroxylation is a post-translational modification that affects the structure,
stability and function of proteins including collagen by catalysing hydroxylation of
proline to hydroxyproline through action of collagen prolyl hydroxylases3 (C-P3H) and 4
(C-P4H). Three C-P3Hs (nomenclature was amended according to approval by the HGNC
symbols and names at (http://www.genenames.org) and Entrez database at (http://www.ncbi.nlm.nih.gov/gene)
leucineproline-enriched proteoglycan (leprecan) 1 (Lepre1), leprecan-like 1
(Leprel1), leprecan-like 2 (Leprel2) and two paralogs
Cartilage-Related Protein (CRTAP) and leprecan-like 4 (Leprel4) are
found in humans. The C-P4Hs are tetrameric proteins comprising a variable
α subunit, encoded by the P4HA1, P4HA2 and
P4HA3 genes and a constant β subunit encoded by
We used RT–PCR, qPCR, pyrosequencing, methylation-specific PCR, western blotting
and immunohistochemistry to investigate expression and regulation of the C-P3H
and C-P4H genes in B lymphomas and normal bone marrow.
C-P3H and C-P4H are downregulated in lymphoma. Down-regulation is associated with
methylation in the CpG islands and is detected in almost all common types of B-cell
lymphoma, but the CpG islands are unmethylated or methylated at lower levels in DNA
isolated from normal bone marrow and lymphoblastoid cell lines. Methylation of multiple
C-P3H and C-P4H genes is present in some lymphomas, particularly
Methylation of C-P3H and C-P4H is common in B lymphomas and may have
utility in differentiating disease subtypes.
non-Hodgkin lymphoma; Prolyl hydroxylases; methylation; epigenetics
There are anecdotal data that lower levels of vitamin D may be associated with increased levels of toxicity in individuals receiving chemotherapy; we therefore wished to investigate this further.
From a cohort of over 11 000 individuals, we included those who had vitamin D levels (serum 1,25(OH)2D3) measured before and during chemotherapy. They were analysed for side effects correlating Chemotherapy Toxicity Criteria with vitamin D levels, normalising data for general markers of patient health including C-reactive protein and albumin.
A total of 241 (2% of the total cohort) individuals entered the toxicity analysis. We found no overall difference in toxicity effects experienced by patients depending on whether they were vitamin D depleted or had sufficient levels (P=0.78).
This pilot study suggests routine vitamin D measurement during treatment does not appear to be necessary in the management of chemotherapy-induced toxicity.
vitamin D; chemotherapy; toxicity; side effects
Current markers available for screening normal populations and for monitoring prostate cancer (PCa) treatment lack sensitivity and selectivity. Sphingosine-1-phosphate (S1P) is a circulating lipid second messenger involved in cell growth and migration, the immune response, angiogenesis, and malignant transformation.
Eighty-eight patients with localised, locally advanced, or metastatic PCa were recruited into this prospective single-centre study. Plasma S1P levels were measured and compared with age-matched controls with benign prostate hyperplasia (BPH) (n=110) or with young healthy males with the very small chance of having PCa foci (n=20).
Levels of circulating S1P were significantly higher in healthy subjects (10.36±0.69 pmol per mg protein, P<0.0001) and patients with BPH (9.39±0.75, P=0.0013) than in patients with PCa (6.89±0.58, ANOVA, P=0.0019). Circulating S1P levels were an early marker of PCa progression to hormonal unresponsiveness and correlated with prostate-specific antigen (PSA) levels and lymph node metastasis. During the course of the study, nine patients have died of PCa. Importantly, their circulating S1P levels were significantly lower (5.11±0.75) than in the surviving patients (7.02±0.22, n=79, P=0.0439). Our data suggest that the decrease in circulating S1P during PCa progression may stem from a highly significant downregulation of erythrocyte sphingosine kinase-1 (SphK1) activity (2.14±0.17 pmol per mg protein per minute in PCa patients vs 4.7±0.42 in healthy individuals, P<0.0001), which may be a potential mechanism of cancer-induced anaemia.
This current study has provided a potential mechanism for cancer-related anaemia and the first evidence that plasma S1P and erythrocyte SphK1 activity are the potential markers for the diagnosis, monitoring, and predicating for PCa mortality.
prostate cancer; molecular marker; diagnosis; prognosis; sphingosine-1-phosphate; anaemia
The aim of this study was to gain insight into breast cancer dormancy by examining different measures of minimal residual disease (MRD) over time in relation to known prognostic factors.
Sixty-four primary breast cancer patients on follow-up (a median of 8.3 years post surgery) who were disease free had sequential bone marrow aspirates and blood samples taken for the measurement of disseminated tumour cells (DTCs), circulating tumour cells (CTCs) by CellSearch and qPCR measurement of overlapping (96-bp and 291-bp) amplicons in circulating free DNA (cfDNA).
The presence of CTCs was correlated with the presence of DTCs measured by immunocytochemistry (P=0.01) but both were infrequently detected. Increasing cfDNA concentration correlated with ER, HER2 and triple-negative tumours and high tumour grade, and the 291-bp amplicon was inversely correlated with DTCs measured by CK19 qRT-PCR (P=0.047).
Our results show that breast cancer patients have evidence of MRD for many years after diagnosis despite there being no overt evidence of disease. The inverse relationship between bone marrow CK19 mRNA and the 291-bp amplicon in cfDNA suggests that an inverse relationship between a measure of cell viability in the bone marrow (DTCs) and cell death in the plasma occurs during the dormancy phase of breast cancer.
circulating tumour cells; disseminated tumour cells; circulating-free DNA; dormancy; breast cancer
The aromatase inhibitor (AI)-associated musculoskeletal syndrome (AIMSS) occurs in approximately 50% of AI-treated patients. Inflammatory mediators are associated with oestrogen signalling and may change with oestrogen depletion. We hypothesised that AIMSS may be associated with changes in circulating inflammatory markers.
Patients with breast cancer were enroled in a trial of adjuvant AI therapy. Changes in pain and function during therapy were assessed prospectively. We selected 30 cases with AIMSS and 22 controls without AIMSS, matched for demographics and prior therapy. Serum samples collected at baseline and during treatment were assayed for multiple inflammatory cytokines and lipid mediators using multiplex assays.
Before AI therapy, mean serum concentrations of 6 of 36 assayed factors were statistically significantly lower in cases than controls (all P<0.003). No statistically significant changes during AI therapy relative to pre-treatment were observed between cases and controls for any of the inflammatory markers tested.
AIMSS is probably not associated with a systemic inflammatory response. Pre-treatment cytokine levels may predict for development of AIMSS.
aromatase inhibitor; arthralgia; breast cancer; cytokine
The mitogen-activated protein kinase (MAPK) phosphatases or dual specificity phosphatases (DUSPs) are a family of proteins that catalyse the inactivation of MAPK in eukaryotic cells. Little is known of the expression, regulation or function of the DUSPs in human neoplasia.
We used RT–PCR and quantitative PCR (qPCR) to examine the expression of DUSP16 mRNA. The methylation in the DUSP16 CpG island was analysed using bisulphite sequencing and methylation-specific PCR. The activation of MAPK was determined using western blotting with phospho-specific antibodies for extra-cellular signal-related kinase (ERK), p38 and c-Jun N-terminal kinase (JNK). The proliferation of cell lines was assessed using the CellTiter 96 Aqueous One assay.
The expression of DUSP16, which inactivates MAPK, is subject to methylation-dependent transcriptional silencing in Burkitt's Lymphoma (BL) cell lines and in primary BL. The silencing is associated with aberrant methylation in the CpG island in the 5′ regulatory sequences of the gene blocking its constitutive expression. In contrast to BL, the CpG island of DUSP16 is unmethylated in other non-Hodgkin's lymphomas (NHLs) and epithelial malignancies. In BL cell lines, neither constitutive nor inducible ERK or p38 activity varied significantly with DUSP16 status. However, activation of JNK was increased in lines with DUSP16 methylation. Furthermore, methylation in the DUSP16 CpG island blocked transcriptional induction of DUSP16, thereby abrogating a normal physiological negative feedback loop that limits JNK activity, and conferred increased cellular sensitivity to agents, such as sorbitol and anthracycline chemotherapeutic agents that activate JNK.
DUSP16 is a new epigenetically regulated determinant of JNK activation in BL.
DUSP; Epigenetics; HIV; Burkitt's lymphoma
In addition to the direct targeting effects on HER2-positive cells, trastuzumab may have a therapeutic role modulating the activity of the cellular immune system in patients with breast cancer. To investigate this further, the balance of T-regulatory (Treg), Th17, natural killer (NK) and NK T (NKT) cells before, during and after trastuzumab therapy was investigated. Sequential frequencies of circulating Treg cells, Th17 cells, NK and NKT cells were measured in peripheral blood of breast cancer patients and normal controls throughout therapy. Individuals with breast cancer had significantly higher Treg frequencies of peripheral blood compared with healthy controls (9.2 or 8.6 vs 6%; P<0.05), and no significant differences in Treg frequencies were observed between HER2-positive and HER2-negative individuals. The number of Th17 cells was lowest in HER2-positive patients compared with both healthy controls and HER2-negative patients (0.31 vs 0.75% or 0.84%; P=0.01). There appeared to be an inverse relationship between Treg and Th17 frequencies in metastatic breast cancer (MBC) with Treg levels significantly reduced during treatment with trastuzumab (P=0.04), whereas Th17 frequencies were concomitantly increased (P=0.04). This study supports earlier data that Treg cells are present at higher frequencies in breast cancer patients compared with healthy individuals. For the first time, we show that HER2-positive individuals with breast carcinomas have reduced numbers of circulating Th17 cells, which appear, in turn to have an inverse relationship with Treg frequency in MBC. The change in balance of the Treg : Th17 ratio appears to characterise the cancer state, and furthermore, is disrupted by trastuzumab therapy.
trastuzumab; Treg; Th17; HER2; immunology
The purpose of this study was to determine whether primary breast cancer patients showed evidence of circulating tumour cells (CTCs) during follow-up as an alternative to monitoring disseminated bone marrow tumour cells (DTCs) by immunocytochemistry and reverse transcriptase (RT)–PCR for the detection of micrometastases. We planned to compare CTC and DTC frequency in low-risk and high-risk patients. We identified two cohorts of primary breast cancer patients who were at low (group II, T1N0, n=18) or high (group III, >3 nodes positive (with one exception, a patient with two positive nodes) n=33) risk of relapse who were being followed up after primary treatment. We tested each cohort for CTCs using the CellSearch system on 1–7 occasions and for DTCs by immunocytochemistry and RT–PCR on 1–2 occasions over a period of 2 years. We also examined patients with confirmed metastatic disease (group IV, n=12) and 21 control healthy volunteers for CTCs (group I). All group I samples were negative for CTCs. In contrast, 7 out of 18 (39%) group II primary patients and 23 out of 33 (70%) group III patients were positive for CTCs (P=0.042). If we count only samples with >1 cell as positive: 2 out of 18 (11%) group II patients were positive compared with 10 out of 33 (30%) in group III (P=0.174). In the case of DTCs, 1 out of 13 (8%) group II patients were positive compared with 19 out of 27 (70%) in group III (P<0.001). Only 10 out of 33 (30%) patients in group III showed no evidence of CTCs in all tests over the period of testing, compared with 11 out of 18 (61%) in group II (P=0.033). A significant proportion of poor prognosis primary breast cancer patients (group III) have evidence of CTCs on follow-up. Many also have evidence of DTCs, which are more often found in patients who were lymph node positive. As repeat sampling of peripheral blood is more acceptable to patients, the measurement of CTCs warrants further investigation because it enables blood samples to be taken more frequently, thus possibly enabling clinicians to have prior warning of impending overt metastatic disease.
micrometastasis; detection; disseminated; circulating; CellSearch
Background: Increasing numbers of patients with early cancer undergo routine staging using computerized tomography (CT). Those in whom indeterminate pulmonary nodules are visualized without the presence of other metastatic lesions represent a clinical dilemma regarding their management as early breast cancer or metastatic disease.
Patients and methods: Medical records of breast cancer patients who underwent thoracic CT scans between the years 2002 and 2008 were analyzed. Those with obvious metastatic disease were excluded. Patients were identified via the radiology database by searching for the terms: ‘suspicious lung metastases’ and ‘indeterminate nodules’.
Results: Out of 1578 new patients assessed from 2002 to 2008, we carried out 802 staging CT scans. Thirty-four cases (4.2%) with indeterminate pulmonary nodules were identified. We categorized cases by size and number of nodules. At a median follow-up of 18 months, there were no changes in lesion size in 86% of patients with a solitary nodule <1 cm and 89% with multiple subcentimeter nodules. In contrast, in 100% of cases with pulmonary nodules >1 cm, the nodules had progressed at follow-up (χ2, P = 0.004).
Conclusions: Breast cancer cases with subcentimeter indeterminate pulmonary lesions and no evidence of metastases elsewhere are unlikely to represent metastatic disease. Treatment with curative intent or entry into clinical trials should not be excluded.
breast cancer; CT; indeterminate; lung nodule; suspicious
This commentary provides a consensus view by two reviewers on a paper appearing in this issue that investigates the change in the quality of evidence published in clinical journals over a period of 25 years.
Atazanavir, an azadipeptide protease inhibitor (PI) with once daily dosing, a lack of insulin resistance, lipid increase, and gastrointestinal toxicities, is approved in combination with other antiretrovirals for the treatment of patients infected with HIV. Unboosted atazanavir is also used in highly active antiretroviral therapy (HAART) naive patients.
The study prospectively followed up an established cohort of patients who received atazanavir, and for whom one year of follow up data were available.
It was found that use of atazanavir in intent to treat and on treatment analyses, maintained and led to virological suppression and increases in CD4 count in both PI naive and experienced patients. Virological failure occurred in 7% of patients and the main toxicity was hyperbilirubinaemia, which led to treatment withdrawal in 2%. Its efficacy and safety profile was similar to that seen in previous randomised studies investigating its use.
These data should provide reassurance for clinicians wishing to introduce a new antiretroviral into an established cohort.
HIV; AIDS; compliance; atazanavir; lipid
There are data suggesting that inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase signalling may reverse resistance to fluoropyrimidine treatment. To investigate this further, the INFORM study was an open-label, non-comparative phase II study of gefitinib (Iressa, ZD1839; AstraZeneca, Wilmington, DE, USA) 250 mg daily in combination with 5-fluorouracil (5-FU administered as an intravenous 400 mg m−2 bolus injection followed by 2800 mg m−2 infusion over 46 h and folinic acid administered as a 350 mg infusion over 2 h) every 2 weeks for up to 12 cycles in 24 patients with metastatic colorectal cancer refractory to previous fluoropyrimidine treatment. There were no objective responses. The stable disease rate was 37.5% (95% CI: 18.80, 59.41), median progression-free survival measured 116 days and overall survival was 226 days. Quality of life was unchanged compared to baseline values, and the commonest toxicities were diarrhoea, rash and fatigue with 7 out of 24 (29%) patients having a grade 3 or 4 toxicity. Gefitinib does not sensitise patients with fluoropyrimidine refractory metastatic colorectal cancer to 5-FU chemotherapy.
refractory; colorectal cancer; gefitinib; 5-fluorouracil; phase II
Several commonly prescribed antibiotics are known to interact with warfarin, increasing its anticoagulant effect by different mechanisms. Retroperitoneal bleeding with consequent haematoma is recognised as a complication of over‐anticoagulation. Consequences, which are potentially fatal, include hypovolaemic shock and compression of retroperitoneal structures such as the ureter and inferior vena cava.
Antibiotic; complication; haematoma; retroperitoneal; warfarin
Background: The CD4 count is a dominant prognostic and predictive factor in HIV infection. This study assessed the utility of the total lymphocyte count (TLC) in place of the CD4 count to predict the development of AIDS defining opportunistic infections (ADOI).
Methods: The Chelsea and Westminster cohort was used to identify those people with a first episode of an ADOI. Corresponding CD4 and TLCs were recorded before diagnosis or at the time of first prescribing prophylaxis; patients without an AIDS defining opportunistic infection were defined as being at "risk" and receiver operating characteristic (ROC) curves were used to display the results of sensitivity and the false positive error rate of total lymphocyte and CD4 count groups.
Results: A significant linear correlation was seen between the log10 CD4 count and log10 TLC (Pearson's correlation coefficient = 0.70, p<0.001). The finer cut off value for TLC where false positive error rate is minimum and sensitivity maximum was 1500–2000 cells/mm3. Patients with TLC 1000–1500 cells/mm3 were estimated to be at 40% increased risk of developing an ADOI. The cut off value for CD4 counts measured 200 cells/mm3 above which the risk developing an ADOI decreased. Patients with a CD4 count of 150–200 cells/mm3 were at a 34% increased risk of developing an ADOI. The area under the ROC curve for TLC was 10% lower than that for CD4 count.
Conclusions: The TLC is minimally less reliable than the CD4 count as a predictor of ADOIs. In the absence of expensive equipment for CD4 measurement, the TLC is a useful test.
There is no consensus as to the management of untreated poor prognosis or relapsed/refractory germ cell tumours. We have studied an intensive cisplatin-based regimen that incorporates high-dose methotrexate (HD MTX) and actinomycin-D and etoposide every 14 days (GAMEC). Sixty-two patients were enrolled in a phase 2 study including 27 who were untreated (IGCCCG, poor prognosis) and 35 with progression despite conventional platinum based chemotherapy. The pharmacokinetics of the drugs were correlated with standard outcome measures. Twenty of the untreated patients were progression free following GAMEC and appropriate surgery, as were 18 individuals in the pretreated group. None of the established prognostic factors for therapy for pretreated patients could identify a poor-prognosis group. Five out of nine late relapses to prior chemotherapy were progression free following GAMEC and appropriate surgery. All patients had at least one episode of febrile neutropenia and there were five (8%) treatment-related deaths. PK values were not predictive of efficacy or toxicity, although the dose intensity in the pretreated group of patients, especially of HD MTX, was significantly correlated with progression-free survival (PFS). GAMEC is a novel intensive regimen for this group of patients producing encouraging responses, although with significant toxicity. For those in whom it fails, further therapy is still possible with durable responses being seen.
GAMEC; methotrexate; high dose; germ cell
Background: Previous studies have reached differing conclusions about the utility of anal cytology as a screening tool for anal intraepithelial neoplasia (AIN). There is a need also to establish whether HPV typing offers a useful adjunct to screening.
Methods: We analysed data from 99 consecutive homosexual/bisexual male patients (89 HIV-1 positive) who underwent high resolution anoscopy. Follow up visits for these patients were also included, giving a total of 160 anoscopic procedures. Comparison was made between results of anal cytology using the sampling method of Palefsky, and histological findings of biopsies taken from abnormal areas seen on high resolution anoscopic examination of the anal canal. Swabs taken concurrently with the cytology were analysed for the presence of human papillomavirus (HPV) DNA and compared with the cytological and histological findings.
Results: The sensitivity of the cytology was 83%, and the specificity 38% when compared with histology. At screening of 34 asymptomatic men, 83% had anal cytological dysplasia and 78% had AIN. There were no significant differences in the prevalence of hrHPV genotypes between different cytological or histological grades of abnormalities.
Conclusion: Anal cytology by the Palefsky method is simple to undertake, has a sensitivity and specificity comparable with cervical cytology, and can therefore be used as the basis of a pilot screening project in centres with large cohorts of HIV positive homosexual men who have a high risk of developing anal carcinoma. HPV genotyping is not a useful adjunct to cytological screening.
The majority of women requiring chemotherapy for gestational trophoblastic disease (GTN) are cured with their initial chemotherapy treatment. However, a small percentage either become refractory to treatment, or relapse after the completion of treatment. This study investigates the characteristics and outcome of these patients. Patients were identified from the Charing Cross Hospital GTD database. The outcome of these patients with relapsed disease was compared to those with refractory disease. Between 1980 and 2004, 1708 patients were treated with chemotherapy for GTN. Sixty (3.5%) patents relapsed following completion of initial therapy. The overall 5-year survival for patients with relapsed GTN was 93% (95% CI 86–100%). The overall survival for patients with low-risk and high-risk disease at presentation, who subsequently relapsed was 100% (n=35), and 84% (n=25) (95% CI: 66–96%: P<0.05), respectively. Eleven patients were identified who failed to enter remission and had refractory disease. These patients had a worse outcome compared to patients with relapsed disease (5-year survival 43% (95% CI:12–73% P<0.01)). The outcome of patients with relapsed GTN is good. However, patients with primary chemo-refractory disease do poorly and novel therapies are required for this group of patients.
gestational trophoblastic neoplasia; relapse; chemotherapy
Ocular involvement of systemic non-Hodgkin's lymphoma is rare. This report describes the unusual occurrence of a biopsy confirmed low grade lymphoma recurring in the conjunctiva, three years after initial diagnosis of systemic disease. The tumour was surgically resected and the patient remains disease free four years later. After the diagnosis of lymphoma, long term follow up is advised as recurrences in unusual sites can occur.
It is thought that the time interval between the antecedent pregnancy and diagnosis of gestational trophoblastic tumours (GTTs) may influence the outcome of these patients. In this study, we investigate the significance of this time interval. Multivariate analysis was used to investigate if the time interval was of prognostic significance from our cohort of 241 high-risk patients with GTT. Subsequent cutpoint analysis was used to determine an optimal cutpoint for the interval covariate. The outcome of these patients was plotted according to the Kaplan–Meier method. The time interval was of prognostic significance on multivariate analysis. A period of greater than 2.8 years after pregnancy was found to be of most significance. The 5-year overall survival was 62.0% (95% CI: 47–76%) for greater than 2.8 years vs 94% (95% CI: 91–97%) for less than 2.8 years (P<0.001). Multivariate analysis showed the presence of liver metastasis and the number of metastasis was also of prognostic importance. The interval between antecedent pregnancy and diagnosis in high-risk GTT is of prognostic significance. This gives some insight into the pathogenesis of the disease.
gestational trophoblastic disease; interval; survival
Background: Research is an increasingly important aspect of higher medical training for many doctors. Studies investigating sources of stress, isolation, and workplace bullying have not previously sought information in this setting.
Methods: An internet based questionnaire survey of doctors undertaking research (n = 259) was conducted to examine stressors and levels of job satisfaction in this potentially vulnerable group. In order to assess overall levels of satisfaction, we asked whether doctors would recommend their research post to a colleague.
Results: There was a statistically significant association between those who would not recommend their post to a colleague and those who had difficulties in arranging funding and in writing up (p<0.001). Further significant correlations were found between dissatisfaction with the post and lack of help, support, and advice from supervisors and colleagues, wanting to change supervisors, experience of the major categories of workplace bullying, and having an inadequate clinical commitment (p<0.001). When the significant variables were entered into a multivariate analysis, the results showed that dissatisfaction was associated with wanting to change supervisors and with a threat to professional status.
Conclusions: Stress and bullying are common in doctors undertaking research. These findings have important implications for medical training and for doctors choosing research projects. Setting up systems of support may have important benefits.
Antibiotic resistance profiles are useful in directing therapeutic strategies during bacterial infections. Patterns of antimicrobial resistance in Streptococcus pneumoniae and Pseudomonas aeruginosa associated pneumonia were investigated in an HIV-1 infected cohort during the era of highly active antiretroviral therapy. The median CD4 count at presentation was significantly lower for cases of P aeruginosa than for S pneumoniae. However, the number of antibiotic resistant cases of P aeruginosa decreased throughout the study period, while the incidence of S pneumoniae remained unchanged. In contrast to pneumococcal pneumonia, we show that antiretrovirals have protected from pneumonia due to antibiotic resistant P aeruginosa. These findings have implications for the treatment of individuals presenting with serious infections in which antibiotic therapy needs to be instituted before identification and sensitivities are known.