Ultra-micro samples can be used for mRNA quantification of lung cancer biomarkers

Background Isolating sufficient material for molecular testing remains challenging in non-small cell lung cancer (NSCLC). The use of new ultra-microsamples (uMS) is proven sufficient for DNA and mRNA detection, but whether uMS are useful for quantifying mRNA expression is unknown. We investigated if uMS from lung cancer patients can be used to generate quantitative data on mRNA expression. Methods uMS were collected from primary tumors and lymph nodes from patients suspected of having lung cancer. mRNA was isolated, reverse-transcribed into cDNA and quantified with quantitative PCR assays for hepatocyte growth factor receptor (MET), hepatocyte growth factor (HGF), epidermal growth factor receptor (EGFR) and amphiregulin (AREG) mRNA. The fraction of tumor cells to normal cells was estimated in each sample. Results MET, HGF, EGFR, and AREG expression were evaluated in 90 samples (30 containing cancer cells and 60 without cancer cells). MET and EGFR expression were negligible in samples without cancer cells. In samples containing cancer cells, MET and EGFR could be quantified in 13 samples each. Adjustment for tumor-cell fraction made it possible to obtain a quantitative result for the tumor-cell mRNA expression of MET and EGFR. In contrast, AREG and HGF were expressed in samples without tumor cells. These samples were used to establish the AREG and HGF mRNA expression in normal cells. Seven out of 14?AR-positive and two out of eight HGF-positive samples with tumor cells were above a cut-off of the mean?+?2SD established in samples without tumor cells. Conclusion We demonstrate that uMS contain high-quality mRNA, and quantitative studies can be performed when the tumor-cell fraction is considered.     

Temporal trends in hip fracture incidence,mortality, and morbidity in Denmark from 1999 to 2012

Background and purpose — While development in hip fracture incidence and mortality is well examined, none has yet looked at the temporal trends regarding prevalence of co-morbidities. Therefore we investigated changes in incidence of first hip fracture, co-morbidity prevalence, 30?day- and 1-year mortality in hip fracture patients in the Danish population during the period 1999 to 2012.

Patients and methods — Patients >18 years admitted with a fractured hip in Denmark between 1996 and 2012 were identified with data for the period 1999–2012 being analyzed regarding prevalence of co-morbidities, incidence, and mortality.

Results — 122,923 patients were identified. Incidence in the whole population declined but sex-specific analysis showed no changes for men. For the whole study population, 30-day and 1-year mortality remained unchanged. Age at time of first hip fracture also remained unchanged. Of the included co-morbidities a decrease in prevalence of malignancy and dementia in women was found while there was an increase in the prevalence of all remaining co-morbidities, except hemi- or paraplegia for both sexes, rheumatic diseases for women, and for men diabetes with complications, myocardial infarction, AIDS/HIV, and malignancy.

Interpretation — While hip fracture incidence declined for women it was unchanged for men; likewise, 30-day and 1-year mortality rates together with age at first fracture remained unchanged. When these results are compared with the relatively large increase in the prevalence of co-morbidities, it does not seem likely that the increased disease burden is affecting either the incidence or the mortality.     

Rapid Selection of Plasmodium falciparum Chloroquine Resistance Transporter Gene and Multidrug Resistance Gene-1 Haplotypes Associated with Past Chloroquine and Present Artemether-Lumefantrine Use in Inhambane District,Southern Mozambique

Chloroquine (CQ) use in Mozambique was stopped in 2002 and artemether-lumefantrine (AL) was implemented in 2008. In light of no use of CQ and extensive use of AL, we determined the frequency of molecular markers of Plasmodium falciparum drug resistance/tolerance to CQ and AL in persons living in Linga-Linga, an isolated peninsula and in Furvela village, which is located 8 km inland. The P. falciparum chloroquine resistance transporter gene CVMNK wild type increased in frequency from 43.9% in 2009 to 66.4% in 2010 (P ≤ 0.001), and combined P. falciparum multidrug resistance gene 1 N86-184F-D1246 haplotype increased significantly between years (P = 0.039). The combination of P. falciparum chloroquine resistance transporter gene CVMNK and P. falciparum multidrug resistance gene NFD increased from 24.3% (2009) to 45.3% in (2010, P = 0.017). The rapid changes observed may largely be caused by decreased use of CQ and large-scale use of AL. In the absence of a clear AL-resistance marker and the (almost) continent-wide use of AL in sub-Saharan Africa, and when considering CQ reintroduction, continued monitoring of these markers is needed.     

Re-interpretation of PAM50 gene expression as quantitative tumor dimensions shows utility for clinical trials: application to prognosis and response to paclitaxel in breast cancer

Background

We recently showed PAM50 gene expression data can be represented by five quantitative, orthogonal, multi-gene breast tumor traits. These novel tumor ‘dimensions’ were superior to categorical intrinsic subtypes for clustering in high-risk breast cancer pedigrees, indicating potential to represent underlying genetic susceptibilities and biological pathways. Here we explore the prognostic and predictive utility of these dimensions in a sub-study of GEICAM/9906, a Phase III randomized prospective clinical trial of paclitaxel in breast cancer.

Methods

Tumor dimensions, PC1–PC5, were calculated using pre-defined coefficients. Univariable and multivariable Cox proportional hazards (PH) models for disease-free survival (DFS) were used to identify associations between quantitative dimensions and prognosis or response to the addition of paclitaxel. Results were illustrated using Kaplan–Meier curves.

Results

Dimensions PC1 and PC5 were associated with DFS (Cox PH p = 6.7 \(\times\) 10⁻⁷ and p = 0.036), remaining significant after correction for standard clinical–pathological prognostic characteristics. Both dimensions were selected in the optimal multivariable model, together with nodal status and tumor size (Cox PH p = 1.4 \(\times\) 10⁻¹²). Interactions with treatment were identified for PC3 and PC4. Response to paclitaxel was restricted to tumors with low PC3 and PC4 (log-rank p = 0.0021). Women with tumors high for PC3 or PC4 showed no survival advantage.

Conclusions

Our proof-of-concept application of quantitative dimensions illustrated novel findings and clinical utility beyond standard clinical–pathological characteristics and categorical intrinsic subtypes for prognosis and predicting chemotherapy response. Consideration of expression data as quantitative tumor dimensions offers new potential to identify clinically important patient subsets in clinical trials and advance precision medicine.

     

Non-response for health-related quality of life outcomes in ICU patients: A systematic review of the reporting in randomised trials

Background

Health-related quality of life (HRQoL) is frequently assessed in randomised clinical trials (RCTs) in the intensive care unit (ICU), but data are limited regarding the proportions of patients without responses or not surviving to HRQoL follow-up and the handling of this. We aimed to describe the extent and pattern of missing HRQoL data in intensive care trials and describe how these data and deaths were handled statistically.

Methods

We conducted a systematic review and meta-analysis following a published protocol. We searched PubMed, EMBASE, CINAHL and Cochrane Library for RCTs involving adult ICU patients reporting HRQoL as an outcome and excluded RCTs unobtainable in full text. We performed risk of bias assessment independently and in duplicate.

Results

We included 196 outcomes from 88 RCTs published in the years 2002–2022; the numbers of patients alive and eligible to respond HRQoL were reported in 76% of trials. At follow-up, median 27% (interquartile range 14%–39%) of patients had died, and median 20% (9%–38%) of survivors did not respond across outcomes. Analyses of 80% of outcomes were restricted to complete cases only. The handling of non-survivors in analyses were reported for 46% of outcomes, with 26% of all outcomes reported as including non-survivors (using the value zero or the worst possible score).

Conclusion

For HRQoL outcomes in ICU trials, we found that mortality at time of follow-up was high and non-response among survivors frequent. The reporting and statistical handling of these issues were insufficient, which may have biased results.