Diagnostic value of increased sensitivity by massively parallel sequencing

Routine molecular diagnostics modalities are unable to confidently detect low frequency mutations (<5-15%) that may indicate response to targeted therapies. We confirm the presence of a low frequency NRAS mutation in a rectal cancer patient using massively parallel sequencing when previous Sanger sequencing results proved negative and Q-PCR testing inconclusive. There is increasing evidence that these low frequency mutations may confer resistance to anti-EGFR therapy. In view of negative/inconclusive Sanger sequencing and Q-PCR results for NRAS mutations in a KRAS wt rectal case, the diagnostic biopsy and 4 distinct subpopulations of cells in the resection specimen after conventional chemo/radiotherapy were massively parallel sequenced using the Ion Torrent PGM. DNA was derived from FFPE rectal cancer tissue and amplicons produced using the Cancer Hotspot Panel V2 and sequenced using semiconductor technology. NRAS mutations were observed at varying frequencies in the patient biopsy (12.2%) and all four subpopulations of cells in the resection with an average frequency of 7.3% (lowest 2.6%). The results of the NGS also provided the mutational status of 49 other genes that may have prognostic or predictive value, including KRAS and PIK3CA. NGS technology has been postulated in diagnostics because of its capability to generate results in large panels of clinically meaningful genes in a cost-effective manner. This case illustrates another potential advantage of this technology: its use for detecting low frequency mutations that may influence therapeutic decisions in cancer treatment.

Expression of proteins associated with hypoxia and Wnt pathway activation is of prognostic significance in hepatocellular carcinoma

In the development and progression of hepatocellular carcinoma, tumor hypoxia plays an important role, as does activation of the Wnt pathway. The aim of this study was to characterize the expression and interrelationship between hypoxia and Wnt-pathway-associated proteins as prognostic factors for hepatocellular carcinoma. Expression of HIF-1α, CA-IX, E-cadherin, β-catenin, and Ki-67 was assessed by immunohistochemistry in 179 primary hepatocellular carcinoma cases. Univariate and multivariate analyses were performed to assess the relationship between the clinicopathological factors, protein expression, overall survival (OS), and recurrence-free survival (RFS). By univariate analysis, tumor stage, size, satellitosis, and vascular invasion were confirmed as prognostic factors for worse OS and RFS. High expression of HIF-1α, CA-IX, β-catenin, Ki-67, and E-cadherin was observed in 60, 15, 64, 8, and 64 % of tumors, respectively, and this was significantly associated with poor OS. CA-IX, HIF-1α, and E-cadherin were independent predictors of poor prognosis. We stratified 169 patients into four groups according to the expression level of hypoxia and Wnt pathway markers. The group with high expression of both hypoxia and Wnt-pathway-associated proteins showed worst OS. The poor survival of this group was also significant in patients with early stage disease and tumor size of less than 5 cm (p < 0.05). We identified a subgroup of hepatocellular carcinoma patients with high expression of both hypoxia and Wnt pathway proteins and found this predictive of poor survival. The therapeutic options for this group might need to be revisited.

Guidance for laboratories performing molecular pathology for cancer patients

Molecular testing is becoming an important part of the diagnosis of any patient with cancer. The challenge to laboratories is to meet this need, using reliable methods and processes to ensure that patients receive a timely and accurate report on which their treatment will be based. The aim of this paper is to provide minimum requirements for the management of molecular pathology laboratories. This general guidance should be augmented by the specific guidance available for different tumour types and tests. Preanalytical considerations are important, and careful consideration of the way in which specimens are obtained and reach the laboratory is necessary. Sample receipt and handling follow standard operating procedures, but some alterations may be necessary if molecular testing is to be performed, for instance to control tissue fixation. DNA and RNA extraction can be standardised and should be checked for quality and quantity of output on a regular basis. The choice of analytical method(s) depends on clinical requirements, desired turnaround time, and expertise available. Internal quality control, regular internal audit of the whole testing process, laboratory accreditation, and continual participation in external quality assessment schemes are prerequisites for delivery of a reliable service. A molecular pathology report should accurately convey the information the clinician needs to treat the patient with sufficient information to allow for correct interpretation of the result. Molecular pathology is developing rapidly, and further detailed evidence-based recommendations are required for many of the topics covered here.

Endoscopic tri-modal imaging improves detection of gastric intestinal metaplasia among a high-risk patient population in Singapore

BACKGROUND: Detection of pre-neoplastic gastric mucosal changes and early gastric cancer (EGC) by white-light endoscopy (WLE) is often difficult. In this study we investigated whether combined autofluorescence imaging (AFI) and narrow band imaging (NBI) can improve detection of pre-neoplastic lesions and early gastric cancer in high-risk patients.

PATIENTS AND METHODS: Chinese patients who were 50-years-old or above with dyspepsia were examined by both high-resolution WLE and combined AFI followed by NBI (AFI-NBI), consecutively in a prospective randomized cross-over setting, by two experienced endoscopists. The primary outcome was diagnostic ability of the two methods for patients with pre-neoplastic lesions such as intestinal metaplasia (IM) and mucosal atrophy.

RESULTS: Sixty-five patients were recruited. One patient with large advanced gastric cancer was found and excluded from the analysis. Among the remaining 64 patients, 38 (59%) had IM; of these, 26 (68%) were correctly identified by AFI-NBI (sensitivity 68%, specificity 23%) and only 13 (34%) by WLE (sensitivity 34%, specificity 65%). AFI-NBI detected more patients with IM than did WLE (p=0.011). Thirty-one patients (48%) had mucosal atrophy. Ten patients (32%) were identified by AFI-NBI (sensitivity 32%, specificity 79%) and four patients (13%) by WLE (sensitivity 13%, specificity 88%) (p=0.100). No dysplasia or EGC was found.

CONCLUSION: AFI-NBI identified significantly more patients with IM than did WLE. Our result warrants further studies to define the role of combined AFI-NBI endoscopy for detection of precancerous conditions.

POU2F1 activity regulates HOXD10 and HOXD11 promoting a proliferative and invasive phenotype in head and neck cancer

HOX genes are master regulators of organ morphogenesis and cell differentiation during embryonic development, and continue to be expressed throughout post-natal life. To test the hypothesis that HOX genes are dysregulated in head and neck squamous cell carcinoma (HNSCC) we defined their expression profile, and investigated the function, transcriptional regulation and clinical relevance of a subset of highly expressed HOXD genes. Two HOXD genes, D10 and D11, showed strikingly high levels in HNSCC cell lines, patient tumor samples and publicly available datasets. Knockdown of HOXD10 in HNSCC cells caused decreased proliferation and invasion, whereas knockdown of HOXD11 reduced only invasion. POU2F1 consensus sequences were identified in the 5' DNA of HOXD10 and D11. Knockdown of POU2F1 significantly reduced expression of HOXD10 and D11 and inhibited HNSCC proliferation. Luciferase reporter constructs of the HOXD10 and D11 promoters confirmed that POU2F1 consensus binding sites are required for optimal promoter activity. Utilizing patient tumor samples a significant association was found between immunohistochemical staining of HOXD10 and both the overall and the disease-specific survival, adding further support that HOXD10 is dysregulated in head and neck cancer. Additional studies are now warranted to fully evaluate HOXD10 as a prognostic tool in head and neck cancers.

LNK mutations and myeloproliferative disorders

The lymphocyte adaptor protein (LNK) is one of a family of adaptor proteins involved cell signalling and control of B cell populations. It has a critical role in regulation of signalling in hematopoiesis. Lnk negatively regulates cytokine initiated cell signalling and it functions as a negative regulator of the mutant protein in myeloproliferative neoplasms JAK2V617F. A number of mutations in LNK have been described in a variety of myeloproliferative neoplasms some of which have been demonstrated to cause increased cellular proliferation. The majority of mutations occur in exon 2. In a small number of cases idiopathic erythrocytosis with subnormal erythropoietin levels LNK mutations have been found which may account for the clinical phenotype. Thus investigation for LNK mutations should be considered in the investigation of idiopathic erythrocytosis and perhaps other myeloproliferative neoplasms.

Low-grade malignant Triton tumor of the oral cavity:a case report

Malignant Triton tumor (MTT) is a malignant peripheral nerve sheath tumor showing rhabdomyoblastic differentiation. It is considered a high-grade neoplasm with poor outcome. This report describes an MTT appearing in the oral cavity. On histologic examination the encapsulated lesion was composed of interlacing fascicles of spindle cells and scattered, large, strap-like pleomorphic cells with abundant eosinophilic cytoplasm. No cross striations were seen. Examination of levels through the tissue showed a total of only 4 normal mitoses and no necrosis. Immunohistochemistry demonstrated diffuse S100 positivity in the spindle cells. The large pleomorphic cells were weakly positive for alpha-sarcomeric actin and myoglobin, although variably but strongly positive for desmin. Management involved a small en bloc resection of the maxilla. After 33 months there was no sign of recurrence or distant metastasis. It was concluded that low-grade variants of MTT occur that do not have an aggressive clinical course.

DNA-Repair Defects and Olaparib in Metastatic Prostate Cancer

BACKGROUND: Prostate cancer is a heterogeneous disease, but current treatments are not based on molecular stratification. We hypothesized that metastatic, castration-resistant prostate cancers with DNA-repair defects would respond to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibition with olaparib.

METHODS: We conducted a phase 2 trial in which patients with metastatic, castration-resistant prostate cancer were treated with olaparib tablets at a dose of 400 mg twice a day. The primary end point was the response rate, defined either as an objective response according to Response Evaluation Criteria in Solid Tumors, version 1.1, or as a reduction of at least 50% in the prostate-specific antigen level or a confirmed reduction in the circulating tumor-cell count from 5 or more cells per 7.5 ml of blood to less than 5 cells per 7.5 ml. Targeted next-generation sequencing, exome and transcriptome analysis, and digital polymerase-chain-reaction testing were performed on samples from mandated tumor biopsies.

RESULTS: Overall, 50 patients were enrolled; all had received prior treatment with docetaxel, 49 (98%) had received abiraterone or enzalutamide, and 29 (58%) had received cabazitaxel. Sixteen of 49 patients who could be evaluated had a response (33%; 95% confidence interval, 20 to 48), with 12 patients receiving the study treatment for more than 6 months. Next-generation sequencing identified homozygous deletions, deleterious mutations, or both in DNA-repair genes--including BRCA1/2, ATM, Fanconi's anemia genes, and CHEK2--in 16 of 49 patients who could be evaluated (33%). Of these 16 patients, 14 (88%) had a response to olaparib, including all 7 patients with BRCA2 loss (4 with biallelic somatic loss, and 3 with germline mutations) and 4 of 5 with ATM aberrations. The specificity of the biomarker suite was 94%. Anemia (in 10 of the 50 patients [20%]) and fatigue (in 6 [12%]) were the most common grade 3 or 4 adverse events, findings that are consistent with previous studies of olaparib.

CONCLUSIONS: Treatment with the PARP inhibitor olaparib in patients whose prostate cancers were no longer responding to standard treatments and who had defects in DNA-repair genes led to a high response rate. (Funded by Cancer Research UK and others; ClinicalTrials.gov number, NCT01682772; Cancer Research UK number, CRUK/11/029.).

ThermoSpots to detect hypothermia in children with severe acute malnutrition

INTRODUCTION: Hypothermia is a risk factor for increased mortality in children with severe acute malnutrition (SAM). Yet frequent temperature measurement remains unfeasible in under-resourced units in developing countries. ThermoSpot is a continuous temperature monitoring sticker designed originally for neonates. When applied to skin, its liquid crystals are designed to turn black with hypothermia and remain green with normothermia.

AIMS: To (i) estimate the diagnostic accuracy of ThermoSpots for detecting WHO-defined hypothermia (core temperature <35.5°C or peripheral temperature <35.0°C) in children with SAM and (ii) determine their acceptability amongst mothers.

METHODS: Children with SAM in a malnutrition unit in Malawi were enrolled during March-July 2010. The sensitivity and specificity of ThermoSpots were calculated by comparing the device colour against 'gold standard' rectal temperatures taken on admission and follow up peripheral temperatures taken until discharge. Guardians completed a questionnaire to assess acceptability.

RESULTS: Hypothermia was uncommon amongst the 162 children enrolled. ThermoSpot successfully detected the one rectal temperature and two peripheral temperatures recorded that met the WHO definition of hypothermia. Overall, 3/846 (0.35%) temperature measurements were in the WHO-defined hypothermia range. Interpreting the brown transition colour (between black and green) as hypothermia improved sensitivities. For milder hypothermia definitions, sensitivities declined (<35.4°C, 50.0%; <35.9°C, 39.2%). Specificity was consistently above 94%. From questionnaires, 40/43 (93%) mothers reported they were 90-100% happy with the device overall. Free-text answers revealed themes of "Skin Rashes", "User-satisfaction" and "Empowerment".

CONCLUSION: Although hypothermia was uncommon in this study, ThermoSpots successfully detected these episodes in malnourished children and were acceptable to mothers. Research in settings where hypothermia is common is needed to determine performance with certainty. Instructing users to act when the device's transition colour appears could improve accuracy. If reliable, ThermoSpots may offer simple, acceptable and continuous temperature measurement for high-burden areas and reduce the workload of over-stretched staff.

Address inaccuracy in health registration data: determinants and impacts

Access to demographic data that are complete, accurate and up-to-date is fundamental to many aspects of public health, government and academic work and for accurate interpretation of other databases. Health registration data are the prime source of demographic information for health and social care systems; for example, as an indicator of need, as a source of denominators to convert number of events into rates, or in the case of the residential address information as the basis for generating the call-recall invitation letters that are used for most screening programs (e.g. breast, colo-rectal and AAA screening). However, list inflation (ghosts, duplicates or emigrants) and a degree of address inaccuracy are recognised caveats with the health registration data and a recent NILS-related study on breast screening suggests that improved address accuracy might be a fast and efficient means of increasing screening uptake rates in cities and amongst deprived populations. In NI these data are collated by the BSO who uniquely in the UK also have access to data relating to prescribing, dental registrations and use of A&E services. These can be used to supplement the standard demographic and address information by (i) indicating patients who are alive and resident in NI and (ii) providing an independent source of probably improved address information. This study will use the NI Unique Property Reference Number (UPRN), rather than the addresses per se which are difficult to work with, to compare the addresses registered in the BSO with those addresses in the enumerated 2011 census. Assuming that the census is a more accurate source of address information for individuals, a comparison of the health registration addresses with those recorded at the census, the aim of the proposed study will be to (i) characterise the amount and distributions of these differences, (ii) to see what proportion of those who do not attend for screening did not actually receive an invitation letter because the addresses were incorrect, (iii) to determine how much of the social gradient (and urban/rural differences) in screening uptake are due to address inaccuracies, (iv) a comparison of timing of address changes at the BSO will provide information on the delays in updating of addresses.

Clopidogrel use and cancer-specific mortality: a population-based cohort study of colorectal, breast and prostate cancer patients

PURPOSE: Concerns were raised about the safety of antiplatelet thienopyridine derivatives after a randomized control trial reported increased risks of cancer and cancer deaths in prasugrel users. We investigate whether clopidogrel, a widely used thienopyridine derivative, was associated with increased risk of cancer-specific or all-cause mortality in cancer patients.

METHODS: Colorectal, breast and prostate cancer patients, newly diagnosed from 1998 to 2009, were identified from the National Cancer Data Repository. Cohorts were linked to the UK Clinical Practice Research Datalink, providing prescription records, and to the Office of National Statistics mortality data (up to 2012). Unadjusted and adjusted hazard ratios (HRs) for cancer-specific and all-cause mortality in post-diagnostic clopidogrel users were calculated using time-dependent Cox regression models.

RESULTS: The analysis included 10 359 colorectal, 17 889 breast and 13 155 prostate cancer patients. There was no evidence of an increase in cancer-specific mortality in clopidogrel users with colorectal (HR = 0.98 95% confidence interval (CI) 0.77, 1.24) or prostate cancer (HR = 1.03 95%CI 0.82, 1.28). There was limited evidence of an increase in breast cancer patients (HR = 1.22 95%CI 0.90, 1.65); however, this was attenuated when removing prescriptions in the year prior to death.

CONCLUSIONS: This novel study of large population-based cohorts of colorectal, breast and prostate cancer patients found no evidence of an increased risk of cancer-specific mortality among colorectal, breast and prostate cancer patients using clopidogrel.

Statin use after diagnosis of breast cancer and survival: a population-based cohort study

BACKGROUND: Preclinical studies have shown that statins, particularly simvastatin, can prevent growth in breast cancer cell lines and animal models. We investigated whether statins used after breast cancer diagnosis reduced the risk of breast cancer-specific, or all-cause, mortality in a large cohort of breast cancer patients.

METHODS: A cohort of 17,880 breast cancer patients, newly diagnosed between 1998 and 2009, was identified from English cancer registries (from the National Cancer Data Repository). This cohort was linked to the UK Clinical Practice Research Datalink, providing prescription records, and to the Office of National Statistics mortality data (up to 2013), identifying 3694 deaths, including 1469 deaths attributable to breast cancer. Unadjusted and adjusted hazard ratios (HRs) for breast cancer-specific, and all-cause, mortality in statin users after breast cancer diagnosis were calculated using time-dependent Cox regression models. Sensitivity analyses were conducted using multiple imputation methods, propensity score methods and a case-control approach.

RESULTS: There was some evidence that statin use after a diagnosis of breast cancer had reduced mortality due to breast cancer and all causes (fully adjusted HR = 0.84 [95% confidence interval = 0.68-1.04] and 0.84 [0.72-0.97], respectively). These associations were more marked for simvastatin 0.79 (0.63-1.00) and 0.81 (0.70-0.95), respectively.

CONCLUSIONS: In this large population-based breast cancer cohort, there was some evidence of reduced mortality in statin users after breast cancer diagnosis. However, these associations were weak in magnitude and were attenuated in some sensitivity analyses.

Diagnostic value of surgical lung biopsy: comparison with clinical and radiological diagnosis

OBJECTIVE: To determine overall and disease-related accuracy of the clinical/imagiological evaluation for pulmonary infiltrates of unknown aetiology, compared with the pathological result of the surgical lung biopsy (SLB) and to evaluate the need for the latter in this setting. METHODS: We conducted a retrospective review of the experiences of SLB in 366 consecutive patients during the past 5 years. The presumptive diagnosis was based on clinical, imagiological and non-invasive or minimally invasive diagnostic procedures and compared with the gold standard of histological diagnosis by SLB. We considered five major pathological groups: diffuse parenchymal lung disease (DPLD), primitive neoplasms, metastases, infectious disease and other lesions. Patients with previous histological diagnosis were excluded. RESULTS: In 56.0% of patients (n=205) clinical evaluation reached a correct diagnosis, in 42.6% a new diagnosis was established (n=156) by the SLB, which was inconclusive in 1.4% (n=5). The pre-test probability for each disease was 85% for DPLD, 75% for infectious disease, 64% for primitive neoplasms and 60% for metastases. Overall sensitivity, specificity, positive and negative predictive values for the clinical/radiological diagnosis were 70%, 90%, 62% and 92%, respectively. For DPLD: 67%, 90%, 76% and 85%; primitive neoplasms: 47%, 90%, 46% and 90%; metastases: 99%, 79%, 60% and 99%; infectious disease 38%, 98%, 53% and 96%. CONCLUSIONS: Despite a high sensitivity and specificity of the clinical and imagiological diagnosis, the positive predictive value was low, particularly in the malignancy group. SLB should be performed in pulmonary infiltrates of unknown aetiology because the clinical/imagiological assessment missed and/or misdiagnosed an important number of patients.

Ajustamento psicossocial e adesão terapêutica em doentes oncológicos: estudo em doentes com cancro colorectal

O cancro do cólon e reto (CCR) representa uma das neoplasias malignas mais relevantes, não só pela morbilidade mas também pelas altas taxas de mortalidade. Paralelamente, esta neoplasia representa uma elevada sobrecarga psicossocial, para o indivíduo, que se reflete na existência de sintomatologia depressiva e ansiosa e de uma qualidade de vida deficitária e que se estima influenciar o processo de adesão terapêutica. Assim, o presente estudo tem como objetivo estudar a associação das variáveis sóciodemográficas, clínicas e de ajustamento psicossocial, com a adesão terapêutica em doentes com cancro colo-rectal. Trinta e cinco pacientes com cancro colo-rectal, acompanhados na Liga Portuguesa Contra o Cancro, foram selecionados de acordo com o método de amostragem de conveniência. Os instrumentos de avaliação psicossocial utilizados consistiram nos seguintes: questionário de variáveis sociodemográficas e clínicas; na Escala de Ansiedade e Depressão Hospitalar (EADH); no questionário de Qualidade de Vida da Organização Europeia de Investigação e Tratamento de Cancro (QLQC30) e na Escala de Adesão Geral (EAG). Os resultados obtidos revelam a existência de relações significativas entre sintomatologia psicopatológica, qualidade de vida e adesão ao tratamento em pacientes com CCR, mais espeficamente, encontrou-se que: a sintomatologia psicopatológica correlacionou-se negativamente com adesão ao tratamento e com a qualidade de vida, e a adesão ao tratamento associou-se positivamente com a qualidade de vida. Concluimos que o presente estudo contribui com a clarificação do sofrimento psicossocial do paciente com CCR e da interação das variáveis demográficas, clínicas e de ajustamento psicossocial com a adesão terapeutica destes doentes. Implicações clinicas e de investigação são apontadas neste contexto.

Caracterização dos mecanismos de transporte epitelial no intestino da dourada, Sparus auratus

Dissertação de mest., Aquacultura e Pescas, Faculdade de Ciências do Mar e do Ambiente, Univ. do Algarve, 2010