Whole-exome sequencing and clinical interpretation of FFPE tumor samples to guide precision cancer medicine

Translating whole exome sequencing (WES) for prospective clinical use may impact the care of cancer patients; however, multiple innovations are necessary for clinical implementation. These include: (1) rapid and robust WES from formalin-fixed paraffin embedded (FFPE) tumor tissue, (2) analytical output similar to data from frozen samples, and (3) clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival FFPE tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a “long tail” of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15/16 patients. In one patient, previously undetected findings guided clinical trial enrollment leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine

Assessing the clinical utility of cancer genomic and proteomic data across tumor types

Molecular profiling of tumors promises to advance the clinical management of cancer, but the benefits of integrating molecular data with traditional clinical variables have not been systematically studied. Here we retrospectively predict patient survival using diverse molecular data (somatic copy-number alteration, DNA methylation and mRNA, miRNA and protein expression) from 953 samples of four cancer types from The Cancer Genome Atlas project. We found that incorporating molecular data with clinical variables yielded statistically significantly improved predictions (FDR < 0.05) for three cancers but those quantitative gains were limited (2.2–23.9%). Additional analyses revealed little predictive power across tumor types except for one case. In clinically relevant genes, we identified 10,281 somatic alterations across 12 cancer types in 2,928 of 3,277 patients (89.4%), many of which would not be revealed in single-tumor analyses. Our study provides a starting point and resources, including an open-access model evaluation platform, for building reliable prognostic and therapeutic strategies that incorporate molecular data

Seed characteristics and asymbiotic germination of Galeandra batemanii Rolfe and G. greenwoodiana Warford

The genus Galeandra Lindl. is a member of subtribe Cyrtopodiinae (Dressler 1993). It is represented in the Americas, from Florida through Mexico and Central America and as far south as Argentina. In Mexico, suitable habitat of Galeandra batemanii Rolfe and of Galeandra greenwoodiana Warford is sparse, patchy and widely separated with sites 300 km or more apart (Warford 1994). Galeandra batemanii has been report- ed to be occasionally lithophytic (Pollard 1974). The genus Galeandra Lindl. is a member of subtribe Cyrtopodiinae (Dressler 1993). It is represented in the Americas, from Florida through Mexico and Central America and as far south as Argentina. In Mexico, suitable habitat of Galeandra batemanii Rolfe and of Galeandra greenwoodiana Warford is sparse, patchy and widely separated with sites 300 km or more apart (Warford 1994). Galeandra batemanii has been report- ed to be occasionally lithophytic (Pollard 1974).

Potential impact of insect herbivores on orchid conservation

When an orchid is deliberately or inadvertently relocated, it is likely to encounter a range of biological challenges to long term survival including a complex and dynamic insect community which could alter conservation expectations yet there have been few studies of the phytophagous insects associated with wild orchids. We have investigated the assemblage of such insects associated with terrestrial orchids being monitored in our long term studies in Gatineau Park, Québec, Canada. Aphids, leafminers, moths, thrips, weevils, and whiteflies were found to be injurious to orchids although in different combinations and with varying impact according to the orchid host, habitat and year. Loss of seeds and even complete desiccation of plants was observed. Where the leafminer, Parallelomma vittatum Meigen (Diptera: Scathophagidae) infested Cypripedium reginae Walter randomly, this was not the case with Cypripedium parviflorum var. pubescens (Willd.) Knight where specific plants were repeatedly infested. Some infestations such as with the leafminers seemed to be in equilibrium with parasitoids thus minimizing potential impact. A likely climate-related asynchrony of parasitoid and leafminer led to an outbreak in 2009 which heavily impacted the introduced Epipactis helleborine (L.) Crantz while a phenological shift in a thrips primary host, Trillium grandiflorum (Michaux) Salisb. (Melanthiaceae), in 2010, contributed to severe herbivory in habitats where both E. helleborine and trillium occurred. If climate change can lead to changes in insect abundance and impact on orchids, it would be useful to investigate the potential impact of phytophagous insects before assisted migration is considered as a conservation measure

Effects of trampling on a terrestrial orchid environment

Orchids and soil communities that support them may be affected when we monitor or conduct inves- tigative procedures, or through tourism especially during the blooming season, but this has never been investigated or quantified (Light 2004). The immedi- ate and direct effect of human disturbance including crushing of flowering plants and seedlings is obvious: broken stems are unlikely to resume growth and a season’s reproductive effort can be lost. Orchids and soil communities that support them may be affected when we monitor or conduct inves- tigative procedures, or through tourism especially during the blooming season, but this has never been investigated or quantified (Light 2004). The immedi- ate and direct effect of human disturbance including crushing of flowering plants and seedlings is obvious: broken stems are unlikely to resume growth and a season’s reproductive effort can be lost.

The role of common orchids in appreciating the complexity of biodiversity conservation

Para conservar especies se debe comprender su biología, ecología y su relativa vulnerabilidad a cambios. Para conservar la biodiversidad, necesitamos un entendimiento profundo del impacto de los disturbios naturales y antropogénicos, de las interacciones y, del rol que juega el ecosistema en la supervivencia de las especies. Las suposiciones de abundancia y declinación y por consiguiente el estatus de conservación puede ser totalmente erróneo si no se toman en cuenta aspectos críticos de la historia natural o las relaciones con otros organismos. Por más de dos décadas hemos monitoreado las poblaciones de dos orquídeas terrestres que crecen en la cercanía del Parque Gatineau, Québec, Canadá. La abundancia relativa y disponibilidad de poblaciones grandes de Cypripedium parviflorum var. pubescens y de la especie introducida Epipactis helleborine nos dio la oportunidad de realizar experimentos in situ y de desarrollar y evaluar aspectos de conservación los cuales no son ejecutables con orquídeas poco comunes o raras. Hemos aprendido que la distribución, congregación y persistencia de E. helleborine está relacionada con la de diversidad de árboles y también que las semillas de las orquídeas germinan mejor en tierra apisonada que en aquella que no presenta disturbio, sin embargo, la localidad sería una variable preponderante. Apisonamiento experimental ocasionado por el tráfico de transeúntes dentro de las localidades de colonias de orquídeas comunes ha revelado un impacto negativo en las comunidades de nemátodos fungívoros, lo que podría reflejar ligeros cambios en el ensamblaje fúngico del cual los nemátodos se alimentan. Entre tanto, las plantas maduras de C. parviflorum var. pubescens no parecen ser afectadas por los efectos que tienen los transeúntes en el sendero. Sin embargo, la germinación y la supervivencia de las plántulas podrían haber sido alteradas, pero no nos daremos cuenta de estos cambios en algún tiempo. To conserve a species, we must understand its biology, ecology, and relative vulnerability to change. To conserve biodiversity, we need a profound understanding of the relative impact of natural and anthropogenic disturbances and species interactions and of the role of the ecosystem in species survival. Assumptions of the basis for abundance and decline and therefore conservation status might be ill founded if we miss critical aspects of life history or of inter-relationships with other organisms. For more than two decades we have monitored populations of two common terrestrial orchids that grow in close proximity in Gatineau Park, Québec, Canada. The relative abundance and availability of large populations of Cypripedium parviflorum var. pubescens and of the introduced Epipactis helleborine presented us with an opportunity to conduct in situ experimentation, and to develop and evaluate conservation approaches not possible with uncommon or rare orchids. We have learned that the distribution, patchiness, and persistence of E. helleborine is related to the presence and diversity of trees and that seeds of this orchid in trampled soil may germinate better than those in undisturbed soil but that location is a likely overriding variable. Experimental trampling within colonies of common orchids has revealed that the fungivorous nematode community is negatively impacted by foot traffic, which could be reflecting subtle changes in the soil fungal assemblage upon which the nematodes feed. While mature plants of C. parviflorum var. pubescens do not seem to have been affected by nearby foot traffic, seed germination and seedling survival could have been altered, but we may not become aware of such changes for some time

High-throughput mutation profiling of CTCL samples reveals KRAS and NRAS mutations sensitizing tumors toward inhibition of the RAS/RAF/MEK signaling cascade

Cutaneous T-cell lymphomas (CTCLs) are malignancies of skin-homing lymphoid cells, which have so far not been investigated thoroughly for common oncogenic mutations. We screened 90 biopsy specimens from CTCL patients (41 mycosis fungoides, 36 Sézary syndrome, and 13 non-mycosis fungoides/Sézary syndrome CTCL) for somatic mutations using OncoMap technology. We detected oncogenic mutations for the RAS pathway in 4 of 90 samples. One mycosis fungoides and one pleomorphic CTCL harbored a KRAS(G13D) mutation; one Sézary syndrome and one CD30(+) CTCL harbored a NRAS(Q61K) amino acid change. All mutations were found in stage IV patients (4 of 42) who showed significantly decreased overall survival compared with stage IV patients without mutations (P = .04). In addition, we detected a NRAS(Q61K) mutation in the CTCL cell line Hut78. Knockdown of NRAS by siRNA induced apoptosis in mutant Hut78 cells but not in CTCL cell lines lacking RAS mutations. The NRAS(Q61K) mutation sensitized Hut78 cells toward growth inhibition by the MEK inhibitors U0126, AZD6244, and PD0325901. Furthermore, we found that MEK inhibitors exclusively induce apoptosis in Hut78 cells. Taken together, we conclude that RAS mutations are rare events at a late stage of CTCL, and our preclinical results suggest that such late-stage patients profit from MEK inhibitors