Implementation Of Molecular Tests Research Articles

Supporting Clinical Decisions with Rapid Molecular Diagnostic Pneumonia Panel in Pediatric Intensive Care Unit: Single Center Experience in Turkiye.

Lower respiratory tract infections are the leading cause of morbidity and mortality in children worldwide. It is crucial to promptly conduct diagnostic investigations in order to determine the microbiological cause of pneumonia, since this is necessary to ensure the appropriate delivery of antibiotic therapy to each individual patient. We evaluated the results of a rapid molecular diagnostic pneumonia panel in children with LRTI in a pediatric intensive care unit (PICU). Rapid molecular diagnostic pneumonia panel (BioFire®, FilmArray Pneumonia Panel plus; FA-PP) findings (71 results from 46 children) in a tertiary care PICU between 2019 and 2023 were retrospectively reviewed. At least one bacterial pathogen was detected in 57 cases. A total of 77% of children had underlying conditions. A total of 70.4% of children needed invasive mechanical ventilation and 54.4% had ventilator-associated pneumonia. Pseudomonas aeruginosa (50.8%), Acinetobacter calcoaceticus baumannii complex (42%), and Klebsiella pneumoniae (38.6%) were the most common pathogens detected with the FA-PP. Of the 33 cases diagnosed with VAP, more than one pathogen was identified in 65.9% of cases, with the most commonly identified bacteria being K. pneumoniae (43.1%), P. aeruginosa (38.6%), and Acinetobacter calcoaceticus baumannii complex (31.8%). According to the FA-PP results, the same antibiotic therapy was continued in 39.4% of cases, escalated in 54.5%, and de-escalated in 6.1%. The utilization of the FA-PP has some beneficial effects, including more prompt delivery of findings compared to conventional approaches. Additionally, this approach enables the identification of resistance profiles in children diagnosed with pneumonia in the PICU. Consequently, these test results facilitate the organization of antibiotic treatment strategies, including escalation and de-escalation approaches. The detection of resistance patterns was exclusively determined via the implementation of molecular testing, prompting a reevaluation of the isolation technique in accordance with the obtained data.

Prospective Validation of ThyroSPEC Molecular Testing of Indeterminate Thyroid Nodule Cytology Following Diagnostic Pathway Optimization.

Background: Molecular testing for cytologically indeterminate thyroid nodules (ITNs) is often reported with incomplete data on clinical assessment and ultrasound malignancy risk (USMR) stratification. This study aimed to clinically validate the diagnostic accuracy of a novel molecular test, assess the incremental preoperative malignancy risk of other clinical factors, and measure the impacts of introducing molecular testing at the population level. Methods: Comprehensive clinical data were collected prospectively for the first 615 consecutive patients with ITNs in a centralized health care system following implementation of a reflexive molecular test. Clinical data include patient history, method of nodule discovery, clinical assessment, USMR, cytology, molecular testing, and surgery or follow-up along with surgeon notes on surgical decision-making. Accuracy of molecular testing and the impact of the introduction of molecular testing were calculated. A multivariable regression model was developed to identify which clinical factors have the most diagnostic significance for ITNs. Results: A locally developed, low-cost molecular test achieved a negative predictive value (NPV) of 76-91% [confidence interval, CI 66-95%] and a positive predictive value (PPV) of 46-65% [CI 37-75%] in ITNs using only residual material from standard liquid cytology fine-needle aspiration (FNA). Sensitivity was highest (80%; [CI 63-92%]) in the American Thyroid Association (ATA) intermediate-suspicion ultrasound category, and lowest (46%; [CI 19-75%]) in the ATA high-suspicion ultrasound category. Following implementation of molecular testing, diagnostic yield increased by 14% (p = 0.2442) and repeat FNAs decreased by 24% (p = 0.05). Mutation was the primary reason for surgery in 76% of resected, mutation-positive patients. High-risk mutations were associated with a 58% (p = 0.0001) shorter wait for surgery. Twenty-six percent of patients with a negative molecular test result underwent surgery. Multivariable regression highlighted molecular testing and USMR as significantly associated with malignancy. Conclusions: Molecular testing improves preoperative risk stratification but requires further stratification for intermediate-risk mutations. Incorporation of clinical factors (especially USMR) with molecular testing may increase the sensitivity for detection of malignancy. Introduction of molecular testing offers some clinical benefits even in a low resection rate setting, and directly influences surgical decision-making. This study illustrates the importance of the local diagnostic pathway in ensuring appropriate integrated use of molecular testing for best outcomes.

Molecular testing of endometrial carcinoma in real-world clinical practice.

Molecular classification has brought significant changes in the management of endometrial cancer (EC). In this article, we aim to analyze our first experience with an implementation of molecular testing into daily clinical practice. In all newly diagnosed EC, the status of mismatch repair (MMR) and p53 proteins has been evaluated immunohistochemically as a part of the routine histopathological examination since May 2021. In tumors that do not meet clinical criteria for a low risk and those with MMR deficiency or p53 mutation, the molecular genetic testing of the POLE gene is performed as well. Recommendations for adjuvant treatment or follow-up are subsequently made based on the risk of recurrence. Genetic counselling is proposed to all patients with MMR-deficient tumors or family history of cancer. A total of 85 patients with newly diagnosed EC between May 2021 and May 2022 were enrolled in the analysis. The median age was 66 years. The results of molecular testing were as follows: 22 (26%) MMR-deficient, 8 (9%) p53-mutated and none POLE-ultramutated of those 40 tumors with performed POLE sequencing. A total of 46 (51%) patient had a low risk, 2 (2%) intermediate, 14 (16%) high-intermediate and 20 (24%) patients had a high risk of recurrence. Advanced or metastatic diseases were diagnosed in 6 (7%) patients. The median time between surgery and multidisciplinary tumor board decision was 21 days (8-36). A total of 76 (90%) patients underwent the whole treatment plan according to the recurrence risk. At the time of analysis, the results of genetic testing were available in 18 patients and revealed 4 (22%) carriers of a pathogenic variant in any of the genes associated with Lynch syndrome. Molecular testing combining immunohistochemical analyses of MMR and p53 proteins in all newly diagnosed EC patients with sequencing analysis of POLE in those with non-low-risk disease is feasible and does not prolong the time needed for treatment decision.

The Use of Xpert MTB/RIF Ultra Testing for Early Diagnosis of Tuberculosis: A Retrospective Study from a Single-Center Database.

Tuberculosis (TB) is a multisystemic contagious disease produced by Mycobacterium tuberculosis complex bacteria (MTBC), with a prevalence of 65:100,000 inhabitants in Romania (six times higher than the European average). The diagnosis usually relies on the detection of MTBC in culture. Although this is a sensitive method of detection and remains the "gold standard", the results are obtained after several weeks. Nucleic acid amplification tests (NAATs), being a quick and sensitive method, represent progress in the diagnosis of TB. The aim of this study is to assess the assumption that NAAT using Xpert MTB/RIF is an efficient method of TB diagnosis and has the capacity to reduce false-positive results. Pathological samples from 862 patients with TB suspicion were tested using microscopic examination, molecular testing and bacterial culture. The results show that the Xpert MTB/RIF Ultra test has a sensitivity of 95% and a specificity of 96.4% compared with 54.8% sensitivity and 99.5% specificity for Ziehl-Neelsen stain microscopy, and an average of 30 days gained in the diagnosis of TB compared with bacterial culture. The implementation of molecular testing in TB laboratories leads to an important increase in early diagnostics of the disease and the prompter isolation and treatment of infected patients.

Evolution of pancreatic ductal adenocarcinoma diagnosis during the last decade.

670 Background: Pancreatic Ductal Adenocarcinoma (PDAC) has historically been an important diagnostic and therapeutic challenge. The multidisciplinary approach and new diagnostic techniques’ implementation have modified this process. Methods: We conducted a retrospective analysis based on clinical data of patients with PDAC between the years 2010 to 2021, analyzing the diagnosis and initial treatment evolution. Results: 673 patients between 2010-2021 with a suspected diagnosis of pancreatic adenocarcinoma were reviewed. Most of them were metastatic (n=362; 53.8%), followed by locally advanced unresectable (n=166; 24.7%) and resectable or borderline resectable (n=145; 21.5%). Regarding the pathological diagnosis, it was not possible in 62 patients (9.2%), varying over time from 21.2% in 2010-2012 to 1% in 2019-2021 (p<0,0001). Moreover, the number of biopsies has decreased with a mean number of biopsies to obtain a pathological diagnosis of 1.55 (2010-2012) vs 1.31 (2019-2021). During this last period, most of the diagnoses were made by cytological analysis (61.4%; n=121). Specifically in the 2019-2021 patients subgroup, we found that 18 NGS (9,1%) were performed in this period (solid tumor), with 4 patients having actionable mutations (22.2%; 3 KRAS G12C). Germline (g) mutational panels were carried out in 89 patients, finding only 9 positive cases (10.1%), being 3 of them gBRCA1/2 mutated (3,4%). In our study, a decrease in palliative management was evidenced over time. In 2010-2012, 28,8% of patients received exclusively palliative care against 9,6% in 2019-21 (p< 0.0001). An increase in PDAC diagnosis was observed since 2010, 44 patients/year in 2010-12 vs. 66 patients/year in 2019-21 (including COVID-19 pandemic period). All previous results are summarized. Conclusions: The diagnosis of PDAC has changed throughout the last decade, increasing the percentage of patients with a pathological diagnosis without increasing the number of invasive procedures. The number of patients suitable for anti-cancer therapy has also increased among time. In our cohort, the implementation of molecular testing would change the therapeutic approach in more than 20% of patients.[Table: see text]

The 10-year journey of non–small cell lung cancer: A real-world experience.

9133 Background: Over the past 10 years, the treatment of non-small cell lung cancer (NSCLC) has been continuously revolutionized. This study evaluated how this revolution improved outcomes in the real-world. Methods: We collected clinical, pathological, and molecular data of patient with NSCLC using an in house real-time updated cohort (ROOT-HEALTH). The primary objective was the 3-year survival (OS3) rate by clinical stage, histology, and driver mutations including EGFR, ALK, ROS1, RET, MET exon 14 skipping, BRAF V600E, KRAS G12C, and NTRK between two time periods (Period I: 2010-2015 vs. Period II: 2016-2021). Results: We included data on 22,060 patients who received any anti-cancer treatment between January 2010 and December 2020. Adenocarcinoma (AD) was the predominant histology (70.2% in Period I vs. 74.2% in Period II) (Standardized mean difference [SMD] = 0.09, P < 0.001). In Period I, 36.8%, 10.4%, 15.9%, and 34.6% patients were in stage I, II, III, and IV respectively. In Period II, 44.8%, 9.6%, 15.1%, 29.9% were in each stage, respectively (SMD = 0.15, P < 0.001). Patients during Period II were more likely to perform molecular tests for EGFR, ALK, and/or others than those during Period I in both of AD (79.1% in Period I vs. 95.1% in Period II) and non-AD (53.5% in Period I and 85.0% in Period II). In patients with AD, 49.4% in Period I and 56.4% in Period II had any druggable mutations. Among non-AD patients, 4.9% and 6.2% of patients had any druggable mutations in Period I and II, respectively. In patients with AD, in Period I, the OS3 rates were 92.9 %, 72.6%, 56.6%, and 29.0% for each stage (I, II, III, and IV), respectively. In Period II, the OS3 rates of AD were 96.4 %, 86.8%, 70.2%, and 52.1% for each stage, respectively (P < 0.001). In stage IV AD with EGFR mutation, the OS3 rates were 42.5% and 65.4% in Period I and II, respectively (P < 0.001). In stage IV AD with ALK rearrangement, the OS3 rates were 53.2% and 73.6% in Period I and II, respectively (P < 0.001). In patients with non-AD, the OS3 rates were 71.6%, 60.2%, 38.8%, and 10.2% for each stage in Period I. In Period II, the OS3 rates of non-AD were 83.1%, 73.7%, 58.2%, and 29.6% for each stage (P < 0.001). In stage IV non-AD with any druggable mutations, the OS3 rates were 27.1% and 46.0% in Period I and II, respectively (P = 0.05). Conclusions: Our study is meaningful as a large-scale study that reflects changes of survival outcome in a timely manner.Through our real-world experience of over 10 years, the proportions of early stage and were found to be increased. The implementation of molecular testing has also been expanded. Of note, the survival outcomes were remarkably improved across all stages, especially in patients with stage III-IV. Further improvement of outcomes through effect of emerging driver mutations in the next five years is expected.

Unlocking SARS-CoV-2 detection in low- and middle-income countries

SummaryLow- and middle-income countries (LMICs) are significantly affected by SARS-CoV-2, partially due to their limited capacity for local production and implementation of molecular testing. Here, we provide detailed methods and validation of a molecular toolkit that can be readily produced and deployed using laboratory equipment available in LMICs. Our results show that lab-scale production of enzymes and nucleic acids can supply over 50,000 tests per production batch. The optimized one-step RT-PCR coupled to CRISPR-Cas12a-mediated detection showed a limit of detection of 102 ge/μL in a turnaround time of 2 h. The clinical validation indicated an overall sensitivity of 80%–88%, while for middle and high viral load samples (Cq ≤ 31) the sensitivity was 92%–100%. The specificity was 96%–100% regardless of viral load. Furthermore, we show that the toolkit can be used with the mobile laboratory Bento Lab, potentially enabling LMICs to implement detection services in unattended remote regions.

EPEN-45. NORMALIZING AND FACILITATING GENOMIC TESTING AT DIAGNOSIS IN PEDIATRIC EPENDYMOMA

The current consensus is that diagnosis and treatment of ependymoma should be based upon clinical and molecular classification. As we move into this paradigm, it is important all ependymoma cases undergo tumor collection, preservation, and molecular profiling at diagnosis. Our group of 6 sites gathered data on a cohort of 72 ependymoma cases. Sites were asked to report known molecular findings; 60/68 eligible cases (88%) did not include genetic findings. The low number of cases with molecular findings was surprising and since cases were diagnosed from as early as 2004, we asked collaborators to share their current practice in profiling (e.g., how frequently; in what setting were ependymomas sent for testing) to try and better understand current practice at sites. Since the publication of ependymoma molecular data, sites with a neuro-oncology program report sending almost all newly diagnosed ependymomas for molecular testing, whereas current practices at sites without dedicated neuro-oncology were less consistent. Profiling in the setting of relapse was more frequently reported at all centers. The implementation of molecular testing at diagnosis may need support at sites without dedicated neuro-oncology. Lead investigators for upcoming ependymoma clinical trials will need to think carefully about the logistics of profiling at centers where this is not standard practice at diagnosis.

The Role of Molecular Testing in Identifying Carbapenemase Producing Organisms: Implications for Infection Control and Prevention Strategies

<h3>Background</h3> As multi-drug resistance proliferates, healthcare institutions must implement antimicrobial stewardship processes to quickly identify and disarm the microscopic enemy. Infection control (IC) surveillance should consider incorporating molecular testing to rapidly identify resistance threats and implement appropriate infection control measures. <h3>Methods</h3> In 2014, a large metropolitan teaching hospital implemented gram-negative blood culture nucleic acid testing (BC-GN). A retrospective review by a multidisciplinary team consisting of an IC practitioner and an infectious disease (ID) pharmacist assessed timeliness of organism identification, the prevalence of Carbapenemase-producing (CP) markers and collaboration opportunities for improvement of antimicrobial stewardship efforts. <h3>Results</h3> BC-GN testing has increased the speed of identifying epidemiologically significant organisms by 81% from the time of laboratory receipt. Of identified Carbapenem-resistant isolates from blood cultures, 41% also expressed CP mechanisms. Additionally, 50% of patients identified with CP organisms had a previously confirmed Carbapenem-resistant isolate from non-blood specimens. In collaboration with ID pharmacist, specific types of CP mechanisms were found to be associated with specific antibiotic resistance patterns. <h3>Conclusions</h3> Implementation of molecular testing provides quick identification of isolates and epidemiologically significant genetic markers. Understanding the CP prevalence assists ID professionals with the information necessary to treat patients appropriately while enhancing antimicrobial stewardship efforts. Trending antibiotic resistance patterns in BC-GN specimens with confirmed CP mechanisms can aid in developing a proxy marker for non-blood specimens where molecular testing is not yet available.

MON-525 ATA and ACR TIRADS Classification Systems as Additional Predictors of Malignancy in Afirma GSC Suspicious Thyroid Nodules

Objective: Evaluation and management of thyroid nodules with cytologically indeterminate results remain challenging in clinical practice. Despite the implementation of molecular testing in an attempt to avoid surgical intervention, diagnostic thyroidectomy still occurs due to the relatively low positive predictive value of these molecular testing. We conducted a study to analyze whether combining US characteristics and results of molecular testing would better elucidate predicting true positive results. Methods: We retrospectively reviewed thyroid ultrasound images of 172 nodules in 162 patients (mean age, 55 years +/- 14) with indeterminate cytology results (Bethesda III and IV) that underwent Afirma Gene Sequencing Classifier (GSC) testing at a single academic medical center between 2017–2019. All nodules were classified according to 2015 American Thyroid Association (ATA) and 2017 American College of Radiology Thyroid Imaging Reporting and Data System (ACR-TIRADS). Results: A total of 172 with subsequent Afirma GSC molecular testing were included in the study. There were 127 nodules with Bethesda III (AUS/FLUS) (73.8%), and 45 nodules with Bethesda IV (SFN/HCN) (26.2%) results. The mean nodule volume was 5.4 +/- 10 cm3. Afirma GSC identified 129 nodules (75%) as benign and 43 nodules (25%) as suspicious. Per ATA classification, 10.4% (18) of nodules were classified as very low risk, 40.7% (70) as low risk, 36.5% (62) as intermediate and 12.8% (22) as high risk for malignancy. There was a significant association between ATA classification and Afirma benign nodules (P=0.002). All nodules were also classified per TIRADS system with the following distribution: 5 (1.16%) TIRADS 1, 7 (4%) TIRADS 2, 54 (31%) TIRADS 3, 90 (52%) TIRADS 4, and 16 (9.3%) TIRADS 5. We did not observe the similar association between TIRADS system and benign nodules as we did with ATA classification (P=0.4).35 patients (79.5%) with Afirma suspicious results underwent surgery, of which 18 (51.4%) surgical pathology were malignant. 8 patients with Afirma suspicious results decided to proceed with ultrasound surveillance. The malignancy rates of nodules with low, intermediate and high suspicion for malignancy classified by the ATA guidelines were 44% (9 of 18), 33% (56 of 18) and 22% (4 of 18). The malignancy rates of TIRADS category 3, 4 and 5 nodules were 44% (8 of 18 nodules), 44% (8 of 18 nodules) and 11% (2 of 18 nodules). Subset analysis of surgical pathology benign and cancerous nodules did not show significant association between ATA (P=0.5) or TIRADS (P=0.4) classification systems. Conclusion: Our study showed that Afirma benign nodules were associated with a lower risk of malignancy per ATA classification but not with TIRADS system. We did not find a significant association between pathology proven cancer cases and high-risk ATA or TIRADS ultrasound classification systems.

Impact of the introduction of a nucleic acid amplification test for Clostridium difficile diagnosis on stool rejection policies

BackgroundThe change from non-molecular to nucleic acid amplification tests (NAATs) is known to increase the detection of Clostridium difficile infection (CDI); however, the impact on stool rejection policies in clinical laboratories is unclear. The current guidelines have reinforced the importance of respecting strict conditions for performing tests on stool samples for CDI diagnosis. The purpose of this study was to estimate whether the implementation of molecular tests has resulted in changes in stool rejection policies between clinical laboratories that introduced NAATs and those that did not.ResultsA survey was conducted to evaluate the change in the number of stool samples rejected and the rejection criteria among 12 hospital laboratories in southwestern France before and after the switch from non-molecular tests to NAATs using retrospective data from June 1 till September 30, 2013 and the same period 2014. Four laboratories introduced NAATs as a second or third step in the process. A total of 1378 and 1297 stools samples were collected in 2013 and 2014, respectively. The mean number of rejected stool samples significantly increased (p < 0.001, Chi square test), with a total of 99 (7.1%) and 147 (11.3%) specimens rejected in 2013 and 2014, respectively. Notably, these laboratories had more stringent criteria and were no longer testing the stool samples of patients with CDI-positive results within 7 days. In contrast, there was a significant decrease in the rate of rejected stool samples (p < 0.001, Chi square test) in the five laboratories that did not adopt NAATs and a less stringent stool rejection policy.ConclusionNucleic acid amplification test implementation improved compliance with recommended stool rejection policies. Laboratories should follow the recommended laboratory algorithm for the CDI diagnosis combined with the correct stool rejection policy.

Sensibilidad y Especificidad de la Citología Cervicouterina y la prueba PCR-hrHPV con el diagnóstico Histopatológico, en el Hospital “Solon Espinosa Ayala”, Solca-Quito.

Introducción: La implementación de las pruebas moleculares para la detección de la infección por hrHPV ha generado cambios en las directrices del tamizaje en la detección oportuna del carcinoma cervicouterino. El objetivo del estudio es presentar la sensibilidad y especificidad de los estudios citológicos y las pruebas moleculares con los estudios histológicos. Métodos: Se realizó un estudio transversal, retrospectivo en el hospital de Solca-Quito de enero a diciembre 2014. Se recolectaron los casos con los diagnósticos citológicos cervicouterinos, los resultados de la prueba de PCR tiempo real de hrHPV (Hibribio®) y los diagnósticos histopatológicos en las pacientes a las que se realizó biopsia. El análisis realizado fue “de prueba diagnóstica” para medir la sensibilidad y especificidad de las pruebas. Resultados: 730 estudios moleculares de hrHPV conjuntamente con estudios citológicos fueron realizados. Los casos positivos para hrHPV fueron 301/730 casos (41.2 %). La mayoría de casos hrHPV positivos corresponde a los genotipos 16/18 (59.5 %) y se encuentra en los rangos de edad entre 30 y 49 años (58.8 %). En 168 casos se realizó además estudio histopatológico, en los que se determinó la sensibilidad (S) de la citología Vs Histología la cual fue de 76 %, la especificidad (E) fue de 48 %, con un valor predictivo positivo (VPP) de 90 %. La S de HrHPV vs Histología fue de 74%, E 39 %, VPP 89 %; la S de Citología + HrHVP vs Histología fue de 91 %, E 40 %, VPP 90 %. Conclusión: La mayor sensibilidad para el diagnóstico de cáncer cervicouterino la realización de la Citología y la presencia de HrHVP. La mayor especificidad se consiguió con el estudio de Citología.

Implementation of molecular testing for EGFR mutations and ALK rearrangement in lung adenocarcinoma in Brazil.

e18002 Background: Lung cancer is the leading cause of cancer death worldwide. In Brazil, cancer is the second most common cause of death and there were an estimated 27,330 new cases of lung cancer in 2014. Targeted therapies have changed disease prognosis and current clinical guidelines advocate for EGFR/ALK molecular testing for all patients with advanced-stage lung adenocarcinoma. Access to this testing is often limited in the developing world. In the case of Brazil, there is limited data regarding the frequency of testing and the changes in patterns of testing overtime. Methods: De-identified data was obtained from a commercial database that surveys approximately 2,000 cancer physicians in Brazil regarding practice patterns. On average 300 physicians responded to the survey, which is conducted every 6 months, and provided information on about 18,000 patients. Subsequently, we identified patients with lung cancer and calculated the frequency of testing for EGFR mutations and ALK rearrangement from 2011 to 2016. Results: 11,684 patients with lung adenocarcinoma were analyzed from 2011 to 2016. The frequency of testing for EGFR mutations increased significantly: 12.8% (287/2,228) in 2011, 34.4% (738/2,142) in 2012, 39.2% in 2013 (822/2,092), 43.9% in 2014 (866/1,972), 53.3% (1165/2,184) in 2015, and 58% (626/1,066) for the first semester of 2016. Testing for ALK rearrangement also increased noticeably over the same period: 0% in 2011 (0/2,228) and 2012 (0/2,142), 0.9% (19/2,092) in 2013, 2.99% (59/1,972) in 2014, 5.5% (121/2,184) in 2015, and 4.8% (52/1,066) for the first semester of 2016. Conclusions: To our knowledge this is the largest data analysis regarding changing practice patterns of molecular testing for lung adenocarcinoma over time in Brazil and Latin America. The frequency of testing for EGFR mutations and ALK rearrangement has increased over the last 5 years but is still below the current guidelines recommending that all patients with advanced disease be tested. Further understanding of the barriers to testing, will hopefully lead to national strategies for universal implementation of molecular testing.

Europe Does It Better: Molecular Testing across a National Health Care System—The French Example

Drug approvals for molecularly stratified tumor subgroups make molecular testing mandatory and require that molecular diagnostics be performed nationwide. To this end, the French National Cancer Institute (INCa) and the French Ministry of Health have set up a national network of 28 regional molecular genetics centers. Selective molecular tests are performed in these facilities. They are free of charge for all patients in their region, irrespective of the type of establishment in which they are receiving treatment. A specific program has also been implemented to anticipate the launch of new targeted therapies and to accelerate the time-to-access to new drugs and experimental therapies. The initiative has been operational for 5 years and has been successful in meeting its initial aims of uniform nationwide test provision and fast implementation of molecular tests for new tumor biomarkers.

Metastatic esophagogastric adenocarcinoma: trends in first-line treatment and predictive factors for the implementation of HER2 testing in clinical practice during the first year after trastuzumab market approval

Little is known about the use of first-line chemotherapy in clinical practice in patients with advanced esophagogastric adenocarcinoma, and no data have been published regarding potential obstacles for the implementation of molecular testing for targeted agents in this patient group. Here, we sought to evaluate factors influencing treatment decisions with special focus on the implementation of HER2 testing during the first year after trastuzumab market approval in Germany. A total of 754 patients undergoing treatment decisions for palliative first-line therapy in 2010 were documented using Therapiemonitor(®). Drug use and intensity of first-line treatment were analyzed. Data on HER2 testing and test algorithm are described, and variables influencing HER2 testing were selected using bivariate analysis. Significant factors were included in a multivariate logistic regression analysis. Compared with previous years, treatment intensity has further increased. The use of chemotherapy triplets rose from 10.1 % in 2006 to 60.3 % in 2010. In 2010, 49.1 % of patients were tested for HER2 and in 52.2 % of these patients the currently proposed test algorithm was used. Using multivariate logistic regression analysis age ≥67 years and "initiating institution: practice" were found to negatively impact the likelihood of HER2 testing, while treatment goal "prevention of progression", multiple metastases and a Karnofsky status >80 % showed positive correlation with HER2 testing. The tendency to use more intensive first-line chemotherapy regimens in patients with advanced esophagogastric adenocarcinoma continued in 2010. Only a minority of patients had an access to the appropriate molecular diagnostics and therefore to treatment with trastuzumab. The access was limited due to the preselection following individual, clinical and institutional factors.

Imported submicroscopic malaria in Madrid

BackgroundSubmicroscopic malaria (SMM) can be defined as low-density infections of Plasmodium that are unlikely to be detected by conventional microscopy. Such submicroscopic infections only occasionally cause acute disease, but they are capable of infecting mosquitoes and contributing to transmission. This entity is frequent in endemic countries; however, little is known about imported SMM.The goals of this study were two-fold: a) to know the frequency of imported SMM, and b) to describe epidemiological, laboratorial and clinical features of imported SMM.MethodsA retrospective study based on review of medical records was performed. The study population consisted of patients older than 15 years attended at the Tropical Medicine Unit of Hospital Carlos III, between January 1, 2002 and December 31, 2007. Routinely detection techniques for Plasmodium included Field staining and microscopic examination through thick and thin blood smear. A semi-nested multiplex malaria PCR was used to diagnose or to confirm cases with low parasitaemia.ResultsSMM was diagnosed in 104 cases, representing 35.5% of all malaria cases. Mean age (IC95%) was 40.38 years (37.41-43.34), and sex distribution was similar. Most cases were in immigrants, but some cases were found in travellers. Equatorial Guinea was the main country where infection was acquired (81.7%). Symptoms were present only in 28.8% of all SMM cases, mainly asthenia (73.3% of symptomatic patients), fever (60%) and arthromialgias (53.3%). The associated laboratory abnormalities were anaemia (27.9%), leukopaenia (15.4%) and thrombopaenia (15.4%). Co-morbidity was described in 75 cases (72.1%).ConclusionsResults from this study suggest that imported SMM should be considered in some patients attended at Tropical Medicine Units. Although it is usually asymptomatic, it may be responsible of fever, or laboratory abnormalities in patients coming from endemic areas. The possibility of transmission in SMM has been previously described in endemic zones, and presence of vector in Europe has also been reported. Implementation of molecular tests in all asymptomatic individuals coming from endemic area is not economically feasible. So re-emergence of malaria (Plasmodium vivax) in Europe may be speculated.

General morphological and biological features of neoplasms: integration of molecular findings

This review highlights the importance of morphology-molecular correlations for a proper implementation of new markers. It covers both general aspects of tumorigenesis (which are normally omitted in papers analysing molecular pathways) and the general mechanisms for the acquired capabilities of neoplasms. The mechanisms are also supported by appropriate diagrams for each acquired capability that include overlooked features such as mobilization of cellular resources and changes in chromatin, transcription and epigenetics; fully accepted oncogenes and tumour suppressor genes are highlighted, while the pathways are also presented as activating or inactivating with appropriate colour coding. Finally, the concepts and mechanisms presented enable us to understand the basic requirements for the appropriate implementation of molecular tests in clinical practice. In summary, the basic findings are presented to serve as a bridge to clinical applications. The current definition of neoplasm is descriptive and difficult to apply routinely. Biologically, neoplasms develop through acquisition of capabilities that involve tumour cell aspects and modified microenvironment interactions, resulting in unrestricted growth due to a stepwise accumulation of cooperative genetic alterations that affect key molecular pathways. The correlation of these molecular aspects with morphological changes is essential for better understanding of essential concepts as early neoplasms/precancerous lesions, progression/dedifferentiation, and intratumour heterogeneity. The acquired capabilities include self-maintained replication (cell cycle dysregulation), extended cell survival (cell cycle arrest, apoptosis dysregulation, and replicative lifespan), genetic instability (chromosomal and microsatellite), changes of chromatin, transcription and epigenetics, mobilization of cellular resources, and modified microenvironment interactions (tumour cells, stromal cells, extracellular, endothelium). The acquired capabilities defining neoplasms are the hallmarks of cancer, but they also comprise useful tools to improve diagnosis and prognosis, as well as potential therapeutic targets. The application of these concepts in oncological pathology leads to consideration of the molecular test requirements (Molecular Test Score System) for reliable implementation; these requirements should cover biological effects, molecular pathway, biological validation, and technical validation. Sensible application of molecular markers in tumour pathology always needs solid morphological support.

Molecular markers of gliomas: a clinical approach

Over the last decade, the knowledge on the molecular genetic background of gliomas has dramatically increased. This information provides the basis for the molecular target therapies and molecular tests serve to complement the subjective nature of histopathologic criteria and add useful data regarding response to treatments and prognosis. In particular, the use of loss of heterozygosity (LOH) and methylation specific polymerase chain reaction (PCR) (MSP) based testing of gliomas is already in place and used clinically in several centers. This paper provides a brief overview of these molecular genetic aberrations and discusses the clinical utility, as well as the advantages and disadvantages of such approach.Newly developed molecular techniques, such as LOH testing, fluorescence in situ hybridization (FISH), DNA sequencing and MSP, are currently being employed in assessment of gliomas in some laboratories. However, the clinical use of some markers and the context in which the information obtained should be used are still not entirely understood. Therefore, this paper will focus on validation and implementation of molecular testing in gliomas, with emphasis on LOH on chromosomes 1p, 19q, 17p and 10q and O6-methylguanine-DNA methyltransferase (MGMT) methylation status.

Detection of HBV DNA by nested-PCR in a HBsAg and anti-HBc negative blood bank donor

Background The criteria and protocol adopted in serological screening of blood bank donors have significantly reduced the possibility of HBV transmission. However, it is possible that, in a very recent phase of HBV infection, HBsAg seronegative donors be able to transmit the virus. This study reports a case of a donor with this serological profile who was involved in the viral transmission to a seronegative receptor. Case report A blood donor had her sample tested for HBsAg and anti-HBc, which resulted negative. At the second donation the sample demonstrated to be seropositive for anti-HBc, anti-HBs and seronegative for HBsAg. The first stored sample was tested for the presence of HBV DNA. Two fragments could be identified in the genomic region corresponding to HBV core and precore. Only one individual was involved in the transfusion of hemo-derivatives originating from the processing of this bag, and was seropositive for HBsAg, HBeAg and anti-HBc markers and seronegative for the anti-HBe and anti-HBs markers. Conclusion This case illustrates the possibility of the occurrence of HBV transmission from blood bank donors seronegative for HBsAg and anti-HBc. This fact could be associated with the possibility of the donor to be in the pre-seroconversion phase of a recent infection, when the levels of HBsAg present in the circulation are below the limits of detection. The implementation of molecular tests or higher sensitivity HBsAg assays could further reduce the risk of HBV transmission via blood transfusion.