Test, test, test for COVID-19 antibodies: the importance of sensitivity, specificity and predictive powers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody tests of varying specificity and sensitivity are now available. For informing individuals whether they have had coronavirus disease 2019 (COVID-19), they need to be very accurate. For measuring population prevalence of past infection, the numbers of false positives and negatives need to be roughly equal. With a series of worked examples for a notional population of 100,000 people, we show that even test systems with a high specificity can yield a large number of false positive results, especially where the population prevalence is low. For example, at a true population prevalence of 5%, using a test with 99% sensitivity and specificity, 16% of positive results will be false and thus 950 people will be incorrectly informed they have had the infection. Further confirmatory testing may be needed. Giving false reassurance on which personal or societal decisions might be based could be harmful for individuals, undermine public confidence and foster further outbreaks.

Gene editing in the context of an increasingly complex genome.

The reporting of the first draft of the human genome in 2000 brought with it much hope for the future in what was felt as a paradigm shift toward improved health outcomes. Indeed, we have now mapped the majority of variation across human populations with landmark projects such as 1000 Genomes; in cancer, we have catalogued mutations across the primary carcinomas; whilst, for other diseases, we have identified the genetic variants with strongest association. Despite this, we are still awaiting the genetic revolution in healthcare to materialise and translate itself into the health benefits for which we had hoped. A major problem we face relates to our underestimation of the complexity of the genome, and that of biological mechanisms, generally. Fixation on DNA sequence alone and a 'rigid' mode of thinking about the genome has meant that the folding and structure of the DNA molecule -and how these relate to regulation- have been underappreciated. Projects like ENCODE have additionally taught us that regulation at the level of RNA is just as important as that at the spatiotemporal level of chromatin.In this review, we chart the course of the major advances in the biomedical sciences in the era pre- and post the release of the first draft sequence of the human genome, taking a focus on technology and how its development has influenced these. We additionally focus on gene editing via CRISPR/Cas9 as a key technique, in particular its use in the context of complex biological mechanisms. Our aim is to shift the mode of thinking about the genome to that which encompasses a greater appreciation of the folding of the DNA molecule, DNA- RNA/protein interactions, and how these regulate expression and elaborate disease mechanisms.Through the composition of our work, we recognise that technological improvement is conducive to a greater understanding of biological processes and life within the cell. We believe we now have the technology at our disposal that permits a better understanding of disease mechanisms, achievable through integrative data analyses. Finally, only with greater understanding of disease mechanisms can techniques such as gene editing be faithfully conducted.

Patient-derived xenografts effectively capture responses to oncology therapy in a heterogeneous cohort of patients with solid tumors.

BACKGROUND: While patient-derived xenografts (PDXs) offer a powerful modality for translational cancer research, a precise evaluation of how accurately patient responses correlate with matching PDXs in a large, heterogeneous population is needed for assessing the utility of this platform for preclinical drug-testing and personalized patient cancer treatment. PATIENTS AND METHODS: Tumors obtained from surgical or biopsy procedures from 237 cancer patients with a variety of solid tumors were implanted into immunodeficient mice and whole-exome sequencing was carried out. For 92 patients, responses to anticancer therapies were compared with that of their corresponding PDX models. RESULTS: We compared whole-exome sequencing of 237 PDX models with equivalent information in The Cancer Genome Atlas database, demonstrating that tumorgrafts faithfully conserve genetic patterns of the primary tumors. We next screened PDXs established for 92 patients with various solid cancers against the same 129 treatments that were administered clinically and correlated patient outcomes with the responses in corresponding models. Our analysis demonstrates that PDXs accurately replicate patients' clinical outcomes, even as patients undergo several additional cycles of therapy over time, indicating the capacity of these models to correctly guide an oncologist to treatments that are most likely to be of clinical benefit. CONCLUSIONS: Integration of PDX models as a preclinical platform for assessment of drug efficacy may allow a higher success-rate in critical end points of clinical benefit.

Angiosarcoma and Dialysis-related Arteriovenous Fistulae: A Comprehensive Review.

OBJECTIVE/BACKGROUND: To conduct a comprehensive review of cases, presentation, diagnosis, and management of angiosarcoma in arteriovenous fistulae (AVF) created for haemodialysis. METHODS: Two authors independently conducted systematic searches and extraction of articles from the Embase, AMED, Health Management Information Consortium, and MEDLINE databases in keeping with the inclusion/exclusion criteria and Preferred Reporting Items for Systematic Reviews and Meta-Analyses standards. RESULTS: Twenty-two unique patient cases were identified; 20 of the cases were men and mean ± SD age of presentation was 54.9 ± 13.6 years. Nineteen cases were post-transplant and 18 were on antirejection agents. The most common presenting symptom was pain, with or without a mass. The initial diagnosis was most often thrombosis/infection of the AVF and the diagnostic interval to a correct diagnosis of angiosarcoma was between 2 and 40 weeks. Mean ± SD time to presentation of symptoms from fistula formation was 118.9 ± 57.5 months, while from transplant it was 96.9 ± 70.0 months. Amputation was the most common treatment modality and mean ± SD survival was 8.8 ± 3.7 months. CONCLUSION: Angiosarcoma should be suspected in previously quiescent AVF that presents with pain. The presence of a rapidly enlarging mass or bleeding/bruising should be taken as alarm indicators and warrant urgent investigation in accordance with local cancer guidelines. Any surgical procedure should involve histological samples as a matter of course.

Initiation of adjuvant chemotherapy within 8 weeks of elective colorectal resection improves overall survival regardless of reoperation.

AIM: Reoperation after elective colorectal resection may delay the start of adjuvant chemotherapy (AC). The study investigated the dual impact of a reoperation and AC delay on overall survival (OS). METHOD: The Hospital Episode Statistics database was analysed between 1997 and 2012. Patients were divided into colon and rectal cancer cohorts and data were analysed based on whether there was delay in receiving AC beyond 8 weeks and whether a patient suffered reoperation within 30 days. Multivariate regression analysis was undertaken to investigate the relationship between delay in giving AC and reoperation and their combined effect on OS. RESULTS: Logistic regression showed reoperation, amongst other things, to be an independent predictor of AC delay, in both colon and rectal cancer (colon, odds ratio 2.31, P < 0.001; rectal, odds ratio 2.19, P < 0.001). There was no significant difference in OS between patients who had no AC delay but suffered a reoperation and patients who had no AC delay and no reoperation. Patients who had AC delay but no reoperation, however, had significantly worse OS compared to those who had no AC delay and no reoperation [colon, hazard ratio (HR) 1.16, P < 0.001; rectal, HR 1.17, P < 0.001]. Individuals who had both AC delay and a reoperation also had worse OS compared with patients who had neither (colon, HR 1.33, P = 0.037; rectal, HR 1.38, P < 0.001). CONCLUSION: Delayed receipt of AC beyond 8 weeks after surgery is associated with significantly reduced OS regardless of reoperation status in both colon and rectal cancer patients.

Genetic variants of kinase suppressors of Ras (KSR1) to predict survival in patients with ERα-positive advanced breast cancer.

In patients with breast cancer (BC), deregulation of estrogen receptor (ERα) activity may account for most resistance to endocrine therapies. Our previous study used a whole-human kinome siRNA screen to identify functional actors in ERα modulation and showed the implication of proteins kinase suppressors of ras (KSR1). From those findings we evaluated the clinical impact of KSR1 variants in patients with ERα+ BC treated with TAM. DNA was obtained from 222 patients with advanced ERα+ BC treated with TAM who had undergone surgery from 1981 to 2003. We selected three potentially functional relevant KSR1 polymorphisms; two within the 3'UTR (rs224190, rs1075952) and one in the coding exon 7 (rs2293180). The primary end points were overall survival (OS) and disease-free survival (DFS). After a 6.4-year median follow-up, patients carrying the rs2241906 TT genotype showed shorter DFS (2.1 vs 7.1 years, P=0.005) and OS (2.6 vs 8.4 years P=0.002) than those with the TC or TT genotypes. Those associations remained significant in the multivariable analysis adjusting age, lymph node status, LMTK3 and IGFR variants and HER2 status. The polymorphisms rs2241906 and rs1075952 were in linkage disequilibrium. No association was shown between rs2293180 and survival. Among the actors of ERα signaling, KSR1 rs2241906 variants may predict survival in patients with advanced ERα+ BC treated with adjuvant TAM.

1000 Port-A-Cath ® placements by subclavian vein approach: single surgeon experience.

INTRODUCTION: Totally implantable venous access ports are widely used for the administration of chemotherapy in patients with cancer. Although there are several approaches to implantation, here we describe Port-A-Cath(®) (PAC) placement by percutaneous puncture of the subclavian vein with ultrasonographic guidance. PATIENTS AND METHODS: Data on our vascular access service were collected prospectively from June 2004. This service included port-a-caths and Hickman lines. Once 1000 consecutive port-a-caths(®) had been reached the study was closed and data analysed for the port-a-caths(®) alone. The left subclavian vein was the preferred site for venous access, with the right subclavian and jugular veins being the alternative choices if the initial approach failed. Patients were followed up in the short-term, and all the procedures were carried out by a single surgeon at each one of two institutions. RESULTS: Venous access by PAC was established in 100 % of the 1,000 cases. Of the 952 patients where the left subclavian vein was chosen for the first attempt of puncture, the success rate of PAC placement was 95 % (n = 904). Pneumothorax occurred in 12 patients (1.2 %), and a wound haematoma occurred in 4 (0.4 %) out of the total 1,000 patients. No infections were recorded during the immediate post-operative period but only in the long-term post-operative use with 8 patients requiring removal of the PAC due to infection following administration of chemotherapy. CONCLUSION: This is a very large series of PAC placement with an ultrasound-guided approach for left subclavian vein and X-ray confirmation, performed by a single surgeon, demonstrating both the safety and effectiveness of the procedure.

Prognostic performance of inflammation-based prognostic indices in primary operable non-small cell lung cancer.

BACKGROUND: At least 30% of patients with primary resectable non-small cell lung cancer (NSCLC) will experience a relapse in their disease within 5 years following definitive treatment. Clinicopathological predictors have proved to be suboptimal in identifying high-risk patients. We aimed to establish whether inflammation-based scores offer an improved prognostic ability in terms of estimating overall (OS) and recurrence-free survival (RFS) in a cohort of operable, early-stage NSCLC patients. METHODS: Clinicopathological, demographic and treatment data were collected prospectively for 220 patients operated for primary NSCLC at the Hammersmith Hospital from 2004 to 2011. Pretreatment modified Glasgow Prognostic Score (mGPS), neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) were tested together with established prognostic factors in uni- and multivariate Cox regression analyses of OS and RFS. RESULTS: Half of the patients were male, with a median age of 65. A total of 57% were classified as stage I with adenocarcinoma being the most prevalent subtype (60%). Univariate analyses of survival revealed stage (P<0.001), grade (P=0.02), lymphovascular (LVI, P=0.001), visceral pleural invasion (VPI, P=0.003), mGPS (P=0.02) and NLR (P=0.04) as predictors of OS, with stage (P<0.001), VPI (P=0.02) and NLR (P=0.002) being confirmed as independent prognostic factors on multivariate analyses. Patients with more advanced stage (P<0.001) and LVI (P=0.008) had significantly shorter RFS. CONCLUSIONS: An elevated NLR identifies operable NSCLC patients with a poor prognostic outlook and an OS difference of almost 2 years compared to those with a normal score at diagnosis. Our study validates the clinical utility of the NLR in early-stage NSCLC.

Spontaneous antibodies against Engrailed-2 (EN2) protein in patients with prostate cancer.

We reported the expression of the homeodomain-containing transcription factor Engrailed-2 (EN2) in prostate cancer and showed that the presence of EN2 protein in the urine was highly predictive of prostate cancer. This study aimed to determine whether patients with prostate cancer have EN2 autoantibodies, what the prevalence of these antibodies is and whether they are associated with disease stage. The spontaneous immunoglobulin (Ig)G immune response against EN2 and for comparison the tumour antigen New York Esophageal Squamous Cell Carcinoma 1 (NY-ESO-1), were tested by enzyme-linked immunosorbent assay (ELISA) in three different cohorts of prostate cancer patients as well as a group of men genetically predisposed to prostate cancer. Thirty-two of 353 (9·1%) of the SUN cohort representing all stages of prostate cancer demonstrated EN2 IgG responses, 12 of 107 patients (11·2%) in the advanced prostate cancer patients showed responses, while only four of 121 patients (3·3%) with castrate-resistant prostate cancer showed EN2 autoantibodies. No significant responses were found in the predisposed group. Anti-EN2 IgG responses were significantly higher in patients with prostate cancer compared to healthy control males and similarly prevalent to anti-NY-ESO-1 responses. While EN2 autoantibodies are not a useful diagnostic or monitoring tool, EN2 immunogenicity provides the rationale to pursue studies using EN2 as an immunotherapeutic target.

Insulin-like growth factor receptor polymorphism defines clinical outcome in estrogen receptor-positive breast cancer patients treated with tamoxifen.

Compelling evidence points to a key role for insulin-like growth factor 1 (IGF1) signaling in breast cancer development and progression. In addition, IGF1 receptor (IGF1R) expression has been correlated and functionally linked with estrogen receptor (ER) signaling. Recent translational studies support a cross talk between IGF1R and ERα at different levels and data suggest enhanced IGF1R signaling as a causative mechanism of tamoxifen (TAM) resistance. We tested whether functional germline variations in the IGF pathway are associated with clinical outcome in ER-positive primary invasive breast cancer patients, who were treated with surgery and adjuvant TAM. Tissue samples of 222 patients with ER+ primary invasive breast cancer, who had undergone surgery at Charing Cross Hospital, London, UK between 1981 and 2003, were analyzed. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue samples and six functional IGF1 pathway polymorphisms were analyzed using direct DNA sequencing and PCR-restriction fragment length polymorphism. In multivariable analysis, patients with primary invasive breast cancer carrying IGF1R_rs2016347 G allele had a significantly increased risk of early tumor progression (hazard ratio (HR) 2.01; adjusted P=0.004) and death (HR 1.84; adjusted P=0.023) compared with patients carrying G/T or T/T, independent of established clinicopathological determinants. This association remained significant after adjusting for multiple testing. In addition, we were able to demonstrate that IRS1_rs1801123 and IGFBP3_rs2854744 were significantly associated with lymph node involvement and tumor size, respectively. We provide the first evidence for IGF1R_rs2016347 as an independent prognostic marker for ER+ breast cancer patients treated with TAM and support a rational for combined treatment strategies.

SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1.

BACKGROUND: We have previously identified kinase suppressor of ras-1 (KSR1) as a potential regulatory gene in breast cancer. KSR1, originally described as a novel protein kinase, has a role in activation of mitogen-activated protein kinases. Emerging evidence has shown that KSR1 may have dual functions as an active kinase as well as a scaffold facilitating multiprotein complex assembly. Although efforts have been made to study the role of KSR1 in certain tumour types, its involvement in breast cancer remains unknown. METHODS: A quantitative mass spectrometry analysis using stable isotope labelling of amino acids in cell culture (SILAC) was implemented to identify KSR1-regulated phosphoproteins in breast cancer. In vitro luciferase assays, co-immunoprecipitation as well as western blotting experiments were performed to further study the function of KSR1 in breast cancer. RESULTS: Of significance, proteomic analysis reveals that KSR1 overexpression decreases deleted in breast cancer-1 (DBC1) phosphorylation. Furthermore, we show that KSR1 decreases the transcriptional activity of p53 by reducing the phosphorylation of DBC1, which leads to a reduced interaction of DBC1 with sirtuin-1 (SIRT1); this in turn enables SIRT1 to deacetylate p53. CONCLUSION: Our findings integrate KSR1 into a network involving DBC1 and SIRT1, which results in the regulation of p53 acetylation and its transcriptional activity.

'Being there' for women with metastatic breast cancer: a pan-European patient survey.

BACKGROUND: Understanding their experiences of diagnosis is integral to improving the quality of care for women living with advanced/metastatic breast cancer. METHODS: A survey, initiated in March 2011, was conducted in two stages. First, the views of 47 breast cancer-related patient groups in eight European countries were sought on standards of breast cancer care and unmet needs of patients. Findings were used to develop a patient-centric survey to capture personal experiences of advanced breast cancer to determine insights into the 'trade-off' between extending overall survival and side effects associated with its treatment. The second online survey was open to women with locally advanced or metastatic breast cancer, or their carers, and responders were recruited through local patient groups. Data were collected via anonymous local language questionnaires. RESULTS: The online stage II survey received a total of 230 responses from 17 European countries: 94% of respondents had locally advanced or metastatic breast cancer and 6% were adult carers. Although the overall experience of care was generally good/excellent (77%), gaps were still perceived in terms of treatment choice and information provision. Treatment choice for patients was felt to be lacking by 32% of responders. In addition, 68% of those who responded would have liked more information about future medical treatments and research, with 57% wishing to receive this information from their oncologist. Two-thirds (66%) of women with advanced breast cancer, or their carers, believed life-extending treatment to be important so that they can spend more time with family and friends, and 67% said that the treatment was worthwhile, despite potential associated side effects. CONCLUSION: These findings show a continuing need to provide women with advanced breast cancer with better information and emphasise the importance that these patients often place on prolonging survival.

An open-label study of lapatinib in women with HER-2-negative early breast cancer: the lapatinib pre-surgical study (LPS study).

BACKGROUND: This phase II, open-label, multicentre study aimed to evaluate changes in cell proliferation and biomarkers, as well as efficacy of lapatinib in treatment-naïve patients with HER-2-negative primary breast cancer. PATIENTS AND METHODS: Patients received 1500 mg lapatinib for 28-42 days before surgery with repeat biopsies and measurements. The primary end point was inhibition of cell proliferation measured by Ki67; the secondary end points included clinical response, adverse events and changes in FOXO3a, FOXM1, p-AKT and HER-3. RESULTS: Overall, there was no significant reduction in Ki67 with treatment (assessment carried out in 28 of 31 subjects enrolled). However, four patients (14%) showed a reduction in Ki67 ≥50%. Four of 25 patients (16%) had a partial response to treatment judged by sequential ultrasound measurements. Response, in terms of either Ki67 or ultrasound, did not relate to changes in any biomarker assessed at baseline, including the estrogen receptor (ER) and epidermal growth factor receptor (EGFR). However, all four clinical responders were HER-3 positive, as were three of four Ki67 responders. CONCLUSIONS: Overall, a pre-surgical course of lapatinib monotherapy had little effect on this group of patients; however, in subsets of patients, especially those with HER-3-positive tumors, we observed either reduction in proliferation (Ki67) or tumor size; EGFR/ER status had no impact.

Pharmacogenetic profiling of CD133 is associated with response rate (RR) and progression-free survival (PFS) in patients with metastatic colorectal cancer (mCRC), treated with bevacizumab-based chemotherapy.

Recent studies suggest CD133, a surface protein widely used for isolation of colon cancer stem cells, to be associated with tumor angiogenesis and recurrence. We hypothesized that gene expression levels and germline variations in CD133 will predict clinical outcome in patients with metastatic colorectal cancer (mCRC), treated in first-line setting with 5-fluorouracil, oxaliplatin and bevacizumab (BV), and we investigated whether there is a correlation with gene expression levels of CD133, vascular endothelial growth factor (VEGF) and its receptors. We evaluated intra-tumoral gene expression levels by quantitative real-time (RT) PCR from 54 patients and three germline variants of the CD133 gene by PCR-restriction-fragment length polymorphism from 91 patients with genomic DNA. High gene expression levels of CD133 (>7.76) conferred a significantly greater tumor response (RR=86%) than patients with low expression levels (7.76, RR=38%, adjusted P=0.003), independent of VEGF or its receptor gene expression levels. Gene expression levels of CD133 were significantly associated with VEGF and its receptors messenger RNA levels (VEGFR-1 (P<0.01), -2 and -3, P<0.05). Combined analyses of two polymorphisms showed a significant association with progression-free survival (PFS) (18.5 months vs 9.8 months, P=0.004) in a multivariate analysis as an independent prognostic factor for PFS (adjusted P=0.002). These results suggest that CD133 is a predictive marker for standard first-line BV-based treatment in mCRC.

Management of complex colonic polyps in the English Bowel Cancer Screening Programme.

BACKGROUND: Large sessile or flat colonic polyps, defined as polyps at least 20 mm in size, are difficult to treat endoscopically and may harbour malignancy. The aim of this study was to describe their current management to provide insight into optimal management. METHODS: This retrospective observational study identified patients with large sessile or flat polyps detected in the English Bowel Cancer Screening Programme between 2006 and 2009. Initial therapeutic modality (surgical or endoscopic), subsequent management and outcomes were recorded. The main outcome measures analysed were: presence of malignancy, need for surgical treatment, complications, and residual or recurrent polyp at 12 months. RESULTS: In total, 557 large sessile or flat polyps with benign appearance or initial histology were identified in 557 patients. Some 436 (78.3 per cent) were initially managed endoscopically and 121 (21.7 per cent) were managed surgically from the outset. Seventy of those initially treated endoscopically subsequently required surgery owing to the presence of malignancy (19) or not being suitable for further endoscopic management (51). Residual or recurrent polyp was present at 12 months in 26 (6.0 per cent) of 436 patients managed endoscopically. There was wide variation between centres in the use of surgery as a primary therapy, ranging from 7 to 36 per cent. Endoscopic complications included bleeding in 13 patients (3.0 per cent) and perforation in two (0.5 per cent). CONCLUSION: Management of large sessile or flat colonic polyps is safe and effective in the English Bowel Cancer Screening Programme. Wide variation in the use of surgery suggests a need for standardized management algorithms. Presented to a meeting of the British Society of Gastroenterology, Birmingham, U.K., March 2011.

Circulating sphingosine-1-phosphate and erythrocyte sphingosine kinase-1 activity as novel biomarkers for early prostate cancer detection.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSION: 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.

The relationship between vitamin D and chemotherapy-induced toxicity - a pilot study.

BACKGROUND: 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. METHODS: From a cohort of over 11 000 individuals, we included those who had vitamin D levels (serum 1,25(OH)(2)D3) 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. RESULTS: 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). CONCLUSION: This pilot study suggests routine vitamin D measurement during treatment does not appear to be necessary in the management of chemotherapy-induced toxicity.

The presence of disseminated tumour cells in the bone marrow is inversely related to circulating free DNA in plasma in breast cancer dormancy.

BACKGROUND: 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. METHODS: 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). RESULTS: 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). CONCLUSION: 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.

The collagen prolyl hydroxylases are novel transcriptionally silenced genes in lymphoma.

BACKGROUND: 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 ß subunit encoded by P4HB. METHODS: 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. RESULTS: 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 Burkitt's lymphoma. CONCLUSIONS: Methylation of C-P3H and C-P4H is common in B lymphomas and may have utility in differentiating disease subtypes.