Prediction of Benefit From Adjuvant Pertuzumab by 80-Gene Signature in the APHINITY (BIG 4-11) Trial.

PURPOSE: At the primary analysis, the APHINITY trial reported a statistically significant but modest benefit of adding pertuzumab to standard adjuvant chemotherapy plus trastuzumab in patients with histologically confirmed human epidermal growth factor receptor 2 (HER2)-positive early-stage breast cancer. This study evaluated whether the 80-gene molecular subtyping signature (80-GS) could identify patients within the APHINITY population who derive the most benefit from dual anti-HER2 therapy. METHODS: In a nested case-control study design of 1,023 patients (matched event to control ratio of 3:1), the 80-GS classified breast tumors into functional luminal type, HER2 type, or basal type. Additionally, 80-GS distinguished tumor subtypes that exhibited a single-dominant functional pathway versus tumors with multiple activated pathways. The primary end point was invasive disease-free survival (IDFS). Hazard ratios (HRs) were evaluated by Cox regression. After excluding patients without appropriate consent and those with missing data, 964 patients were included. RESULTS: The 80-GS classified 50% (n = 479) of tumors as luminal type, 28% (n = 275) as HER2 type, and 22% (n = 209) as basal type. Most luminal-type tumors (86%) displayed a single-activated pathway, whereas 49% of HER2-type and 42% of basal-type tumors were dual activated. There was no significant difference in IDFS among different conventional 80-GS subtypes (single- and dual-activated subtypes combined). However, basal single-subtype tumors were significantly more likely to have an IDFS event (hazard ratio, 1.69 [95% CI, 1.12 to 2.54]) compared with other subtypes. HER2 single-subtype tumors displayed a trend toward greater beneficial effect on the addition of pertuzumab (hazard ratio, 0.56 [95% CI, 0.27 to 1.16]) compared with all other subtypes. CONCLUSION: The 80-GS identified subgroups of histologically confirmed HER2-positive tumors with distinct biological characteristics. Basal single-subtype tumors exhibit an inferior prognosis compared with other subgroups and may be candidates for additional therapeutic strategies. Preliminary results suggest patients with HER2-positive, genomically HER2 single-subtype tumors may particularly benefit from added pertuzumab, which warrants further investigation.

Investigating the concordance in molecular subtypes of primary colorectal tumors and their matched synchronous liver metastasis.

To date, no systematic analyses are available assessing concordance of molecular classifications between primary tumors (PT) and matched liver metastases (LM) of metastatic colorectal cancer (mCRC). We investigated concordance between PT and LM for four clinically relevant CRC gene signatures. Twenty-seven fresh and 55 formalin-fixed paraffin-embedded pairs of PT and synchronous LM of untreated mCRC patients were retrospectively collected and classified according to the MSI-like, BRAF-like, TGFB activated-like and the Consensus Molecular Subtypes (CMS) classification. We investigated classification concordance between PT and LM and association of TGFBa-like and CMS classification with overall survival. Fifty-one successfully profiled matched pairs were used for analyses. PT and matched LM were highly concordant in terms of BRAF-like and MSI-like signatures, (90.2% and 98% concordance, respectively). In contrast, 40% to 70% of PT that were classified as mesenchymal-like, based on the CMS and the TGFBa-like signature, respectively, lost this phenotype in their matched LM (60.8% and 76.5% concordance, respectively). This molecular switch was independent of the microenvironment composition. In addition, the significant change in subtypes was observed also by using methods developed to detect cancer cell-intrinsic subtypes. More importantly, the molecular switch did not influence the survival. PT classified as mesenchymal had worse survival as compared to nonmesenchymal PT (CMS4 vs CMS2, hazard ratio [HR] = 5.2, 95% CI = 1.5-18.5, P = .0048; TGFBa-like vs TGFBi-like, HR = 2.5, 95% CI = 1.1-5.6, P = .028). The same was not true for LM. Our study highlights that the origin of the tissue may have major consequences for precision medicine in mCRC.

Performance Characteristics of the BluePrint® Breast Cancer Diagnostic Test.

The analytical performance of a multi-gene diagnostic signature depends on many parameters, including precision, repeatability, reproducibility and intra-tumor heterogeneity. Here we study the analytical performance of the BluePrint 80-gene breast cancer molecular subtyping test through determination of these performance characteristics. BluePrint measures the expression of 80 genes that assess functional pathways which determine the intrinsic breast cancer molecular subtypes (i.e. Luminal-type, HER2-type, Basal-type). Knowing a tumor's dominant functional pathway can help allocate effective treatment to appropriate patients. Here we show that BluePrint is a highly precise and highly reproducible test with correlations above 98% based on the generated index and subtype concordance above 99%. Therefore, BluePrint can be used as a robust and reliable tool to identify breast cancer molecular subtypes.

MammaPrint and BluePrint Molecular Diagnostics Using Targeted RNA Next-Generation Sequencing Technology.

Next-generation DNA sequencing is rapidly becoming an indispensable tool for genome-directed cancer diagnostics, but next-generation RNA sequencing (RNA-seq) is currently not standardly used in clinical diagnostics for expression assessment. However, multigene RNA diagnostic assays are used increasingly in the routine diagnosis of early-stage breast cancer. Two of the most widely used tests are currently available only as a central laboratory service, which limits their clinical use. We evaluated the use of RNA-seq as a decentralized method to perform such tests. The MammaPrint and BluePrint RNA-seq tests were found to be equivalent to the clinically validated microarray tests. The RNA-seq tests were highly reproducible when performed in different locations and were stable over time. The MammaPrint RNA-seq test was clinically validated. Our data demonstrate that RNA-seq can be used as a decentralized platform, yielding results substantially equivalent to results derived from the predicate diagnostic device.

Smartphone Application Monitoring of Acceleration Forces During Pneumatic Tube System Transport of Emergency Department Patient Samples.

BACKGROUND: The use of pneumatic tube system (PTS) transport has gained considerable popularity in modern hospitals but is also associated with sample hemolysis. The potential contribution of PTS-associated acceleration forces to high hemolysis rates observed in the emergency department (ED) has not been investigated before and can be easily examined nowadays using smartphone applications. The first aim of our study was to investigate whether our PTS induces hemolysis of patient samples obtained from our ED. We also explored a potential correlation between hemolysis index (HI) on the one hand and acceleration forces during PTS transport or other potential causes of hemolysis related to patient characteristics on the other for two different blood sampling techniques. METHODS: Blood samples from 100 ED patients were collected in one Sarstedt S-Monovette® serum tube (PTStransported to laboratory) and two BD Vacutainer® serum tubes (one PTS-transported and one hand-carried). For all serum samples HI was measured. A smartphone was sent along with the samples in order to register accelerations during transport. Patient's erythrocyte sedimentation rate (ESR), mean corpuscular volume (MCV), hematocrit, total cholesterol, low density lipoprotein (LDL), and high-density lipoprotein (HDL) concentration were determined as well. RESULTS: Hemolysis rate was only 1 - 4% and 5% for PTS and hand-carried transport, respectively. Calculated acceleration vector sums for PTS transport from the ED to laboratory reached up to 131.49 m/second2 (13.40 g). No correlation could be demonstrated between HI on the one hand and acceleration forces acting on the samples during PTS transport or ESR, MCV, hematocrit, and HDL concentration on the other. However, an inverse correlation was noted between HI and cholesterol (total and LDL) concentration in serum tubes transported via PTS, though not in those carried by hand. CONCLUSIONS: We demonstrated that our PTS does not induce or contribute to hemolysis of ED patient samples, even at high acceleration vector sums up to 13 g. Technological advancements such as the development of smartphone applications offer the ability to regularly monitor acceleration forces during PTS transport of patient samples. Low total cholesterol and LDL concentrations may affect the erythrocyte membrane fluidity, making erythrocytes more prone to hemolysis.

A breast cancer gene signature for indolent disease.

PURPOSE: Early-stage hormone-receptor positive breast cancer is treated with endocrine therapy and the recommended duration of these treatments has increased over time. While endocrine therapy is considered less of a burden to patients compared to chemotherapy, long-term adherence may be low due to potential adverse side effects as well as compliance fatigue. It is of high clinical utility to identify subgroups of breast cancer patients who may have excellent long-term survival without or with limited duration of endocrine therapy to aid in personalizing endocrine treatment. METHODS: We describe a new ultralow risk threshold for the 70-gene signature (MammaPrint) that identifies a group of breast cancer patients with excellent 20 year, long-term survival prognosis. Tumors of these patients are referred to as "indolent breast cancer." We used patient series on which we previously established and assessed the 70-gene signature high-low risk threshold. RESULTS: In an independent validation cohort, we show that patients with indolent breast cancer had 100% breast cancer-specific survival at 15 years of follow-up. CONCLUSIONS: Our data indicate that patients with indolent disease may be candidates for limited treatment with adjuvant endocrine therapy based on their very low risk of distant recurrences or death of breast cancer.

Prognostic Value of MammaPrint® in Invasive Lobular Breast Cancer.

BACKGROUND: MammaPrint® is a microarray-based gene expression test cleared by the US Food and Drug Administration to assess recurrence risk in early-stage breast cancer, aimed to guide physicians in making neoadjuvant and adjuvant treatment decisions. The increase in the incidence of invasive lobular carcinomas (ILCs) over the past decades and the modest representation of ILC in the MammaPrint development data set calls for a stratified survival analysis dedicated to this specific subgroup. STUDY AIM: The current study aimed to validate the prognostic value of the MammaPrint test for breast cancer patients with early-stage ILCs. MATERIALS AND METHODS: Univariate and multivariate survival associations for overall survival (OS), distant metastasis-free interval (DMFI), and distant metastasis-free survival (DMFS) were studied in a study population of 217 early-stage ILC breast cancer patients from five different clinical studies. RESULTS AND DISCUSSION: A significant association between MammaPrint High Risk and poor clinical outcome was shown for OS, DMFI, and DMFS. A subanalysis was performed on the lymph node-negative study population. In the lymph node-negative study population, we report an up to 11 times higher change in the diagnosis of an event in the MammaPrint High Risk group. For DMFI, the reported hazard ratio is 11.1 (95% confidence interval = 2.3-53.0). CONCLUSION: Study results validate MammaPrint as an independent factor for breast cancer patients with early-stage invasive lobular breast cancer. Hazard ratios up to 11 in multivariate analyses emphasize the independent value of MammaPrint, specifically in lymph node-negative ILC breast cancers.

Equivalence of MammaPrint array types in clinical trials and diagnostics.

MammaPrint is an FDA-cleared microarray-based test that uses expression levels of the 70 MammaPrint genes to assess distant recurrence risk in early-stage breast cancer. The prospective RASTER study proved that MammaPrint Low Risk patients can safely forgo chemotherapy, which is further subject of the prospective randomized MINDACT trial. While MammaPrint diagnostic results are obtained from mini-arrays, clinical trials may be performed on whole-genome arrays. Here we demonstrate the equivalence and reproducibility of the MammaPrint test. MammaPrint indices were collected for breast cancer samples: (i) on both customized certified array types (n = 1,897 sample pairs), (ii) with matched fresh and FFPE tissues (n = 552 sample pairs), iii) for control samples replicated over a period of 10 years (n = 11,333), and iv) repeated measurements (n = 280). The array type indicated a near perfect Pearson correlation of 0.99 (95 % CI: 0.989-0.991). Paired fresh and FFPE samples showed an excellent Pearson correlation of 0.93 (95 % CI 0.92-0.94), in spite of the variability introduced by intratumoral tissue heterogeneity. Control samples showed high consistency over 10 year's time (overall reproducibility of 97.4 %). Precision and repeatability are overall 98.2 and 98.3 %, respectively. Results confirm that the combination of the near perfect correlation between array types, excellent equivalence between tissue types, and a very high stability, precision, and repeatability demonstrate that results from clinical trials (such as MINDACT and I-SPY 2) are equivalent to current MammaPrint FFPE and fresh diagnostics, and can be used interchangeably.

Schoolchildren as BLS instructors for relatives and friends: Impact on attitude towards bystander CPR.

INTRODUCTION: We investigated the impact of Cardio-Pulmonary Resuscitation (CPR) instruction by children on the attitude of people to perform bystander CPR. METHODS: In 2012, children from primary and secondary school (age span 11­13 years) received a free individual CPR training package containing an inexpensive manikin and a training video. After a CPR training session by their class teacher, they were invited to teach their relatives and friends. After the training, the trainees of the children were invited to participate in a web survey, containing a test and questions about prior CPR training and about their attitude towards bystander CPR (BCPR) before and after the training. We measured the impact on the attitude to perform BCPR and the theoretical knowledge transfer by the children. RESULTS: A total of 4012 training packages were distributed to 72 schools of which 55 class teachers subscribed their students (n = 822) for the training programme for relatives and friends. After a validation procedure, 874 trainees of 290 children were included in the study. In comparison to trainees of secondary schoolchildren, trainees of primary schoolchildren scored better for the test as well as for a positive change of attitude towards future BCPR (P < 0.001). For every child-instructor 1.7 people changed their attitude towards BCPR positively. CONCLUSIONS: Instructing schoolchildren to teach their relatives and friends in Basic Life Support (BLS) led to a more positive attitude towards BCPR. The results were more positive with trainees from primary schoolchildren than with trainees from secondary schoolchildren.

Gene expression signature to improve prognosis prediction of stage II and III colorectal cancer.

PURPOSE: This study aims to develop a robust gene expression classifier that can predict disease relapse in patients with early-stage colorectal cancer (CRC). PATIENTS AND METHODS: Fresh frozen tumor tissue from 188 patients with stage I to IV CRC undergoing surgery was analyzed using Agilent 44K oligonucleotide arrays. Median follow-up time was 65.1 months, and the majority of patients (83.6%) did not receive adjuvant chemotherapy. A nearest mean classifier was developed using a cross-validation procedure to score all genes for their association with 5-year distant metastasis-free survival. RESULTS: An optimal set of 18 genes was identified and used to construct a prognostic classifier (ColoPrint). The signature was validated on an independent set of 206 samples from patients with stage I, II, and III CRC. The signature classified 60% of patients as low risk and 40% as high risk. Five-year relapse-free survival rates were 87.6% (95% CI, 81.5% to 93.7%) and 67.2% (95% CI, 55.4% to 79.0%) for low- and high-risk patients, respectively, with a hazard ratio (HR) of 2.5 (95% CI, 1.33 to 4.73; P = .005). In multivariate analysis, the signature remained one of the most significant prognostic factors, with an HR of 2.69 (95% CI, 1.41 to 5.14; P = .003). In patients with stage II CRC, the signature had an HR of 3.34 (P = .017) and was superior to American Society of Clinical Oncology criteria in assessing the risk of cancer recurrence without prescreening for microsatellite instability (MSI). CONCLUSION: ColoPrint significantly improves the prognostic accuracy of pathologic factors and MSI in patients with stage II and III CRC and facilitates the identification of patients with stage II disease who may be safely managed without chemotherapy.