Sensitive molecular testing methods can demonstrate NSCLC driver mutations in malignant pleural effusion despite non-malignant cytology.

Malignant pleural effusion (MPE) may be diagnosed by cytologic evaluation of pleural fluid, though false negative results can occur. Pleural effusions may provide a source of tumour material for genotyping in lung cancer patients. Detection of MPE may be improved through use of highly sensitive molecular techniques. We identified five patients with non-small cell lung cancer (NSCLC) with initial pleural fluid samples that were non-malignant on cytology, but were subsequently clinically confirmed to have MPE. Tumour mutation status was confirmed via routine testing of diagnostic clinical specimens. Cytologically negative pleural fluid cell-block specimens were analysed by amplicon-based parallel sequencing (APS) for somatic mutations commonly detected in NSCLC, and selected cases by improved and complete enrichment CO-amplification at lower denaturation temperature PCR (ICECOLD PCR) for known mutations. Mutations were detected in three out of three (sensitivity 100%) cytologically non-malignant pleural fluids from patients with a known mutation: two patients with known Kirsten rat sarcoma (KRAS) mutation demonstrated the same KRAS mutation in their pleural fluids by APS, both at approximately 2% mutant allele frequency. In one patient with a known KRAS mutation, ICECOLD PCR detected the same KRAS variant at 0.7% frequency. No mutations were detected in patients with wild-type findings from reference samples (specificity 100%). Sensitive DNA sequencing methods can detect cancer-driver mutations in cytologically non-malignant pleural fluid specimens from NSCLC patients with MPE. Our findings demonstrate the feasibility of sensitive molecular diagnostic techniques for improvement of diagnostic assessment of pleural effusions in patients with lung cancer.

Clinical Utility of Real-Time Targeted Molecular Profiling in the Clinical Management of Ovarian Cancer: The ALLOCATE Study.

PURPOSE: The ALLOCATE study was designed as a pilot to demonstrate the feasibility and clinical utility of real-time targeted molecular profiling of patients with recurrent or advanced ovarian cancer for identification of potential targeted therapies. PATIENTS AND METHODS: A total of 113 patients with ovarian cancer of varying histologies were recruited from two tertiary hospitals, with 99 patient cases suitable for prospective analysis. Targeted molecular and methylation profiling of fresh biopsy and archived tumor samples were performed by screening for mutations or copy-number variations in 44 genes and for promoter methylation of BRCA1 and RAD51C. RESULTS: Somatic genomic or methylation events were identified in 85% of all patient cases, with potentially actionable events with defined targeted therapies (including four resistance events) detected in 60% of all patient cases. On the basis of these findings, six patients received molecularly guided therapy, three patients had unsuspected germline cancer-associated BRCA1/2 mutations and were referred for genetic counseling, and two intermediate differentiated (grade 2) serous ovarian carcinomas were reclassified as low grade, leading to changes in clinical management. Additionally, secondary reversion mutations in BRCA1/2 were identified in fresh biopsy samples of two patients, consistent with clinical platinum/poly (ADP-ribose) polymerase inhibitor resistance. Timely reporting of results if molecular testing is done at disease recurrence, as well as early referral for patients with platinum-resistant cancers, were identified as factors that could improve the clinical utility of molecular profiling. CONCLUSION: ALLOCATE molecular profiling identified known genomic and methylation alterations of the different ovarian cancer subtypes and was deemed feasible and useful in routine clinical practice. Better patient selection and access to a wider range of targeted therapies or clinical trials will further enhance the clinical utility of molecular profiling.

No increase in protease resistance and a decrease in reverse transcriptase resistance mutations in primary HIV-1 infection: 1992-2001.

Rates of antiretroviral resistance in recently transmitted virus in Sydney, Australia fluctuated over the past decade, influenced by treatment trends. Current rates of drug resistance are not high in historical terms or compared with those reported. Rates of resistance to reverse transcriptase inhibitors peaked in the mid-1990s, fell dramatically with the introduction of combination therapy and appear to have plateaued at 10-15% over the past 3 years. Primary resistance mutations in the protease gene are still rare.

Rates of transmission of antiretroviral drug resistant strains of HIV-1.

BACKGROUND: It is clear that transmission of drug resistant HIV-1 is possible and occurs regularly. However, there is a lack of clarity concerning the true rate of this transmission in a given population, the impact of combination therapies on this rate, and the contribution of transmitted resistant virus to treatment failure either in an individual or on a population basis. OBJECTIVES: To provide a review of our current understanding of rates of transmission of drug resistant HIV-1 in various populations and to report the results of a study conducted to determine this rate in Sydney, Australia in the years 1992-2000. STUDY DESIGN: A review of the literature combined with a prospective study of antiretroviral drug resistance in 130 individuals who were diagnosed with symptomatic primary infection at St. Vincent's Hospital, Sydney, Australia between 1992 and 2000. Sequencing of reverse transcriptase (RT) and protease (PR) was performed by the TruGene HIV-1 genotyping kit (Visible Genetics Inc.). RESULTS: The results found in the Sydney population contrast with much of the literature. The prevalence of mutations that conferred primary resistance to protease inhibitors (PIs) was only 0.8% at position V82I. Secondary mutations/polymorphisms were seen in the PR at position L10I/V, K20R, M36I, L63P, A71T/V, or V77I in 60%. L63P was the most frequently found mutation (46.3%). The incidence of protease-resistant strains of HIV in primary HIV-1 infection did not change after the introduction of PIs in 1996. The distribution of the most common resistance mutations in the RT was as follows; M41L (8.5%) and T215Y (8.5%) and K70R (4.8%). The frequency of mutations associated with NRTI resistance was significantly lower in the post 1995 samples (43.9 vs. 19.1%, P < 0.05). Moreover, both M41L and K70R, but not T215Y, occurred with significantly decreased frequency in the post 1995 samples. CONCLUSIONS: In contrast to other studies we found no increase in the rate of PR resistance and a decrease in the rate of RT resistance in recently transmitted virus over the period 1992-2000. The reasons for the differences between these results and those reported from elsewhere may relate to treatment regimens used in the transmitting population and may have implications for treatment policies in this country.