Correction to: PiggyBac mutagenesis and exome sequencing identify genetic driver landscapes and potential therapeutic targets of EGFR-mutant gliomas.

An amendment to this paper has been published and can be accessed via the original article.

CRISPR and transposon in vivo screens for cancer drivers and therapeutic targets.

Human cancers harbor substantial genetic, epigenetic, and transcriptional changes, only some of which drive oncogenesis at certain times during cancer evolution. Identifying the cancer-driver alterations amongst the vast swathes of "passenger" changes still remains a major challenge. Transposon and CRISPR screens in vivo provide complementary methods for achieving this, and each platform has its own advantages. Here, we review recent major technological breakthroughs made with these two approaches and highlight future directions. We discuss how each genetic screening platform can provide unique insight into cancer evolution, including intra-tumoral heterogeneity, metastasis, and immune evasion, presenting transformative opportunities for targeted therapeutic intervention.

PiggyBac mutagenesis and exome sequencing identify genetic driver landscapes and potential therapeutic targets of EGFR-mutant gliomas.

BACKGROUND: Glioma is the most common intrinsic brain tumor and also occurs in the spinal cord. Activating EGFR mutations are common in IDH1 wild-type gliomas. However, the cooperative partners of EGFR driving gliomagenesis remain poorly understood. RESULTS: We explore EGFR-mutant glioma evolution in conditional mutant mice by whole-exome sequencing, transposon mutagenesis forward genetic screening, and transcriptomics. We show mutant EGFR is sufficient to initiate gliomagenesis in vivo, both in the brain and spinal cord. We identify significantly recurrent somatic alterations in these gliomas including mutant EGFR amplifications and Sub1, Trp53, and Tead2 loss-of-function mutations. Comprehensive functional characterization of 96 gliomas by genome-wide piggyBac insertional mutagenesis in vivo identifies 281 known and novel EGFR-cooperating driver genes, including Cdkn2a, Nf1, Spred1, and Nav3. Transcriptomics confirms transposon-mediated effects on expression of these genes. We validate the clinical relevance of new putative tumor suppressors by showing these are frequently altered in patients' gliomas, with prognostic implications. We discover shared and distinct driver mutations in brain and spinal gliomas and confirm in vivo differential tumor suppressive effects of Pten between these tumors. Functional validation with CRISPR-Cas9-induced mutations in novel genes Tead2, Spred1, and Nav3 demonstrates heightened EGFRvIII-glioma cell proliferation. Chemogenomic analysis of mutated glioma genes reveals potential drug targets, with several investigational drugs showing efficacy in vitro. CONCLUSION: Our work elucidates functional driver landscapes of EGFR-mutant gliomas, uncovering potential therapeutic strategies, and provides new tools for functional interrogation of gliomagenesis.

PiggyBac transposon tools for recessive screening identify B-cell lymphoma drivers in mice.

B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.

Evolutionary routes and KRAS dosage define pancreatic cancer phenotypes.

The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest KrasMUT levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous KrasMUT in driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgfß-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer.

Disentangling PTEN-cooperating tumor suppressor gene networks in cancer.

We have recently performed a whole-body, genome-wide screen in mice using a single-copy inactivating transposon for the identification of Pten (phosphatase and tensin homolog)-cooperating tumor suppressor genes (TSGs). We identified known and putative TSGs in multiple cancer types and validated the functional and clinical relevance of several promising candidates for human prostate cancer.

A single-copy Sleeping Beauty transposon mutagenesis screen identifies new PTEN-cooperating tumor suppressor genes.

The overwhelming number of genetic alterations identified through cancer genome sequencing requires complementary approaches to interpret their significance and interactions. Here we developed a novel whole-body insertional mutagenesis screen in mice, which was designed for the discovery of Pten-cooperating tumor suppressors. Toward this aim, we coupled mobilization of a single-copy inactivating Sleeping Beauty transposon to Pten disruption within the same genome. The analysis of 278 transposition-induced prostate, breast and skin tumors detected tissue-specific and shared data sets of known and candidate genes involved in cancer. We validated ZBTB20, CELF2, PARD3, AKAP13 and WAC, which were identified by our screens in multiple cancer types, as new tumor suppressor genes in prostate cancer. We demonstrated their synergy with PTEN in preventing invasion in vitro and confirmed their clinical relevance. Further characterization of Wac in vivo showed obligate haploinsufficiency for this gene (which encodes an autophagy-regulating factor) in a Pten-deficient context. Our study identified complex PTEN-cooperating tumor suppressor networks in different cancer types, with potential clinical implications.

CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice.

Here, we show CRISPR/Cas9-based targeted somatic multiplex-mutagenesis and its application for high-throughput analysis of gene function in mice. Using hepatic single guide RNA (sgRNA) delivery, we targeted large gene sets to induce hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). We observed Darwinian selection of target genes, which suppress tumorigenesis in the respective cellular/tissue context, such as Pten or Cdkn2a, and conversely found low frequency of Brca1/2 alterations, explaining mutational spectra in human ICC/HCC. Our studies show that multiplexed CRISPR/Cas9 can be used for recessive genetic screening or high-throughput cancer gene validation in mice. The analysis of CRISPR/Cas9-induced tumors provided support for a major role of chromatin modifiers in hepatobiliary tumorigenesis, including that of ARID family proteins, which have recently been reported to be mutated in ICC/HCC. We have also comprehensively characterized the frequency and size of chromosomal alterations induced by combinatorial sgRNA delivery and describe related limitations of CRISPR/Cas9 multiplexing, as well as opportunities for chromosome engineering in the context of hepatobiliary tumorigenesis. Our study describes novel approaches to model and study cancer in a high-throughput multiplexed format that will facilitate the functional annotation of cancer genomes.

Histerectomía abdominal en un servicio de cirugía general / Abdominal hysterectomy in a general surgery service

Introducción: la histerectomía abdominal es un proceder quirúrgico de práctica frecuente cuya principal indicación es el fibroma uterino. Consiste en una operación potencialmente complicaciones, en especial las sépticas. Objetivo: describir los resultados obtenidos con la práctica de la histerectomía abdominal en el Servicio de Cirugía General del Hospital Universitario "Amalia Simoni" de Camagüey. Métodos: se realizó estudio descriptivo, longitudinal y retrospectivo de 98 pacientes expuestas a histerectomía abdominal en el mencionado centro hospitalario, desde enero de 2008 hasta diciembre de 2010, de las cuales se escogió una muestra de 91 que cumplían los criterios de inclusión y de exclusión establecidos. Las historias clínicas fueron la fuente de obtención de los datos primarios. Resultados: se obtuvo que el fibroma uterino resultó ser la causa más frecuente para realizar el proceder quirúrgico, no hubo complicaciones peroperatorias, reintervenciones ni mortalidad y la estadía posoperatoria promedio fue corta. Conclusiones: el método clínico resultó de gran valor para diagnosticar el fibroma uterino; asimismo, el hematoma de la herida quirúrgica fue la complicación posoperatoria más común y predominó el diagnóstico histopatológico de fibroleiomioma uterino en correspondencia con el diagnóstico preoperatorio.

Introduction: abdominal hysterectomy is a surgical procedure of frequent practice, which main indication is the uterine fibroma. It consists potentially in a polluted operation, where technical principles should be fulfilled to avoid complications, mainly septic ones. Objective: to describe the results obtained with the practice of the abdominal hysterectomy in the General Surgery Service of "Amalia Simoni" University Hospital in Camagüey. Methods: a descriptive, longitudinal and retrospective study was carried out in 91 female patients who had abdominal hysterectomy in the mentioned hospital, from January, 2008 to December, 2010, who fulfilled the established inclusion and exclusion criteria. The medical records were the source for the primary data. Results: it was obtained that the uterine fibroma turned out to be the most frequent cause to carry out the surgical procedure, there were neither peroperative complications, reinterventions nor mortality and the average posoperative stay was short. Conclusions: the clinical method was of great value to diagnose the uterine fibroma; likewise, the hematoma of the surgical wound was the most common postoperative complication and the histopathological diagnosis of uterine fibroleiomioma prevailed in correspondence to the preoperative diagnosis.

Prelamin A causes progeria through cell-extrinsic mechanisms and prevents cancer invasion.

Defining the relationship between ageing and cancer is a crucial but challenging task. Mice deficient in Zmpste24, a metalloproteinase mutated in human progeria and involved in nuclear prelamin A maturation, recapitulate multiple features of ageing. However, their short lifespan and serious cell-intrinsic and cell-extrinsic alterations restrict the application and interpretation of carcinogenesis protocols. Here we present Zmpste24 mosaic mice that lack these limitations. Zmpste24 mosaic mice develop normally and keep similar proportions of Zmpste24-deficient (prelamin A-accumulating) and Zmpste24-proficient (mature lamin A-containing) cells throughout life, revealing that cell-extrinsic mechanisms are preeminent for progeria development. Moreover, prelamin A accumulation does not impair tumour initiation and growth, but it decreases the incidence of infiltrating oral carcinomas. Accordingly, silencing of ZMPSTE24 reduces human cancer cell invasiveness. Our results support the potential of cell-based and systemic therapies for progeria and highlight ZMPSTE24 as a new anticancer target.

Novel germline CDKN2A mutation associated with head and neck squamous cell carcinomas and melanomas.

BACKGROUND: The ability to identify individuals at increased risk of cancer is of immediate clinical relevance. Germline mutations in the CDKN2A locus, encoding the key tumor suppressor proteins p16/INK4A and p14/ARF, are frequently present in kindreds with hereditary cutaneous melanoma but have seldom been reported in families with genetic susceptibility to head and neck squamous cell carcinomas (HNSCC). METHODS: We report the pedigree of a patient with an unusually high incidence of HNSCC and melanomas. CDKN2A mutation analysis was performed with standard capillary sequencing and multiplex ligation-dependent probe amplification. RESULTS: A previously unreported germline CDKN2A mutation affecting only the p16/INK4A open reading frame, c.106delG (p.Ala36ArgfsX17), was detected in the proband. This mutation causes a premature termination codon. CONCLUSIONS: Our report emphasizes the need to consider germinal CDKN2A mutations in the differential diagnosis of familial HNSCC and the importance of awareness of these tumors in carriers of CDKN2A mutations.

Aging and chronic DNA damage response activate a regulatory pathway involving miR-29 and p53.

Aging is a multifactorial process that affects most of the biological functions of the organism and increases susceptibility to disease and death. Recent studies with animal models of accelerated aging have unveiled some mechanisms that also operate in physiological aging. However, little is known about the role of microRNAs (miRNAs) in this process. To address this question, we have analysed miRNA levels in Zmpste24-deficient mice, a model of Hutchinson-Gilford progeria syndrome. We have found that expression of the miR-29 family of miRNAs is markedly upregulated in Zmpste24(-/-) progeroid mice as well as during normal aging in mouse. Functional analysis revealed that this transcriptional activation of miR-29 is triggered in response to DNA damage and occurs in a p53-dependent manner since p53(-/-) murine fibroblasts do not increase miR-29 expression upon doxorubicin treatment. We have also found that miR-29 represses Ppm1d phosphatase, which in turn enhances p53 activity. Based on these results, we propose the existence of a novel regulatory circuitry involving miR-29, Ppm1d and p53, which is activated in aging and in response to DNA damage.

Novel germline SDHD deletion associated with an unusual sympathetic head and neck paraganglioma.

BACKGROUND: Paragangliomas (PGLs) are rare tumors arising either from sympathetic or parasympathetic-associated chromaffin tissue. PGLs can occur either sporadically or as part of a hereditary syndrome. Sympathetic head and neck PGLs are extremely rare tumors and only a few cases have been reported to date. METHODS: We report the pedigree of a patient with a head and neck PGL arising from the right sympathetic trunk. SDHD mutation analysis was performed using standard sequencing, multiplex ligation-dependent probe amplification, chromosome 11-specific comparative genome hybridization, and long-range/short-range polymerase chain reaction (PCR) approaches. RESULTS: A previously unreported chromosome 11q deletion encompassing 5 annotated genes (SDHD, DLAT, PIH1D2, C11Orf57, and TIMM8B) was detected in the proband. CONCLUSION: PGL families considered "mutation-negative" may be attributable to large gene deletions not detectable by standard sequencing methods. Therefore, deletion analysis should be offered to families or individuals at risk for hereditary PGLs.

PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice.

Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here, we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon-transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed that PiggyBac is capable of genome-wide mutagenesis. The PiggyBac screen uncovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 21 mouse lines to be compatible with both transposon systems in constitutive, tissue- or temporal-specific mutagenesis. Mice with different transposon types, copy numbers, and chromosomal locations support wide applicability.

Metastasectomía pulmonar: experiencia de once años / Lung metastasectomy: experience of eleven years

INTRODUCCIÓN. Las metástasis pulmonares son frecuentes tanto en tumores primarios epiteliales como en los mesenquimatosos, y se explican por el hecho de que todo el gasto cardíaco transita por la circulación menor. La indicación quirúrgica es un problema de relación costo-beneficio y está condicionada entre otros aspectos por el intervalo libre de enfermedad, el estado general del paciente, el número de metástasis y el tipo de resección. El objetivo del presente estudio fue presentar nuestra experiencia y resultados con dicho tratamiento. MÉTODOS. Se realizó un estudio descriptivo transversal retrospectivo con 6 pacientes con metástasis en pulmón, tratados en el Hospital Amalia Simoni, de Camagüey, entre 1997 y 2007. Las variables analizadas fueron sexo, edad, histopatología y localización del tumor primario, operación inicial, tiempo transcurrido entre la operación inicial y la metastasectomía, número de metástasis pre y transoperatorias, vía de abordaje, localización de los nódulos, tipo de resección quirúrgica y supervivencia. RESULTADOS. La media de edad fue de 56 años y no hubo diferencias en cuanto al sexo. En tres pacientes las tumoraciones primarias dependían del sistema digestivo, la mayoría fueron nódulos únicos y la lobectomía pulmonar fue el procedimiento más utilizado. La supervivencia promedio fue de 13 meses. No hubo morbilidad importante, ni fallecidos en la serie(AU)

INTRODUCTION. Lung metastases are frequent both in primary epithelial tumors and mesenchymatous tumors due to the fact that the whole cardiac output passes through the minor circulation. The surgical indication is a problem of cost-benefit relationship, and it is conditioned among other aspects by the disease free interval, the general state of the patient, the number of metastases and the type of resection. The objective of this study was to present our experience and results with this treatment. METHODS. A descriptive cross-sectional retrospective study was conducted in 6 patients with lung metastasis treated at Amalia Simoni Hospital in Camaguey between 1997 and 2007. The variables analyzed were sex, age, histopathology and localization of the primary tumor, initial operation, time elapsed between the initial operation and metastasectomy, number of pre- and transoperative metastases, approach route, localization of the nodules, type of surgical resection and survival. RESULTS. Average age was 56 years old and there was no difference as regards sex. The primary tumors depended on the digestive system in 3 patients. Most of them were unique nodules and pulmonary lobectomy was the most used procedure. Average survival was 13 months. No important morbidity was reported and there were no deaths in the series(AU)

Schwannoma benigno de mediastino: a propósito de un caso / Mediastinal bening schwannoma: a propos of a case

Se presenta un caso interesante de una tumoración de mediastino posterior, localizada hacia el hemitórax izquierdo, en una mujer de 50 años de edad que solo refería disnea en los grandes esfuerzos y, con el tiempo, en los medianos. Resultó ser un schwannoma benigno voluminoso, de 15 cm, encapsulado, con áreas quísticas y hemorragia interior, y zonas de tejido blando, de color blanquecino amarillento, con colapso del pulmón. Esta tumoración se asoció a una bulla subpleural del lóbulo inferior del pulmón izquierdo, de 8 cm. El tumor y la bulla fueron estudiados, tratados quirúrgicamente y resecados, a través de una toracotomía vertical axilar izquierda. En la actualidad la paciente se encuentra asintomática.

An interesting case of posterior mediastinal tumor toward the left hemithorax in a 50-year-old female that only referred dyspnea at big efforts and, with the time, at moderate efforts, was presented. It proved to be a voluminous benign encapsulated schwannoma of 15 cm with cystic areas and internal hemorrhage, and zones of whitish yellow soft tissue with lung collapse. This tumor was associated with a subpleural bulla of 8 cm of the lower lobule of the left lung. The tumor and the bulla were studied, surgically treated and resected by left axillary vertical thoracotomy. The patient is asymptomatic at present.

Liposarcoma gigante de mediastino / Giant mediastinal liposarcoma

El liposarcoma es, entre los sarcomas, el tumor maligno de los tejidos blandos más frecuente en el adulto. Se presenta un caso de liposarcoma situado en el mediastino, localización infrecuente, y que resultó ser un liposarcoma bien diferenciado. El paciente fue un hombre de 48 años de edad que ingresa en la Sala de Neumotisiología con disnea y una masa mediastínica situada hacia el hemitórax izquierdo. Se estudió con radiografía de tórax anteroposterior y lateral y, además, con tomografía axial computadorizada. Fue necesaria una toracotomía con urgencia relativa por la agudización del cuadro clínico mediastínico compresivo. La evolución fue buena durante la intervención quirúrgica y después de ella y actualmente ha concluido su tratamiento adyuvante (radioterapia y quimioterapia) y se siente bien(AU)

Liposarcoma is the most frequent malignant soft tissue tumor. This article presented a case of well-differentiated liposarcoma located in the mediastinum, which is a rare location. The patient was a 48 years-old man that was admitted to the pneumothysiology service because he was short of breath and had a mediastinal mass located near left hemithorax. He was studied using anteroposterior and lateral thoracic radiography in addition to computerized tomography. It was necessary to urgently perform thorachotomy due to his acute clinical picture with mediastinal compression. The patient evolved positively during surgery and afterwards; at present, he has finished his adjuvant treatment based on radiotherapy and chemotherapy and he feels good(AU)

Infectious diseases in Mexico. A survey from 1995-2000.

Data obtained at a central laboratory for emerging, re-emerging, and other infectious diseases in Mexico from 1995-2000 are presented. An outstanding increase of DEN-3 circulation was identified. Aedes aegypti, the dengue vector, is widely distributed. Leptospirosis has become the most important differential diagnosis for dengue. Identification of rabies virus variants allowed cataloging of new transmitters of rabies. Rotavirus showed a clear seasonal distribution, while different proportions of pathogenic classes of Escherichia coli under endemic and outbreak conditions were seen. Serotypes of several bacteria are reported as well as the sources of isolation and frequency of Shigella, Salmonella, and Vibrio cholerae. Rise and disappearance of cholera could be followed along the past decade. Influenza strains were identified, as were several pathogens causing sexually transmitted infections. Laboratory support was important for surveillance after Hurricane Mitch. Multidrug-resistant strains of Mycobacterium tuberculosis are emerging and primary resistance is very high. It is now mandatory to search for antibodies to Trypanosoma cruzi in blood banks. Triatoma barberi, a peridomestic bug, is the main vector of Chagas disease. Localized cutaneous leishmaniosis increased in regions having a guerrilla element in Chiapas. Modern immunodiagnostic techniques are used for control studies of cysticercosis and similar techniques were recently standardized for Trichinella spiralis detection. Low iodine values in children's urine were found in several Mexican states; therefore, use of iodized salt should be encouraged.

Búsqueda de anticuerpos contra trichinella spiralis en roedores de libre desplazamiento capturados en un zoológico de la Ciudad de México / Search for antibodies against trichinella spiralis in free roaming rodents caught in a zoological park from Mexico City

A serological survey to search for antibodies against T. spiralis was performed in free roaming rats (n=64) and mice (n=35) caught in zoological park from Mexico City. Serum samples were analyzed by ELISA and immunoelectrotransfer blot assay (EIBT). None serum show positive absorbance values in ELISA nor recognized T. spiralis specific antigenic fractions in EIBT. However, two rat samples recognized three antigens of 31,37 y 55 kDa, while one of them reacted with two additional antigens of 64 and 67 kDa. As it is known that the antigen epitope profiles varied among trichinella species, it could be possible that in rats, there is 3 percent of antibody prevalence agains trichinella sp.; however, due that other organisms could induce the production of cross-reacting antibodies, such conclusion can not be supported at all. These results suggest that T. spiralis was not part of helminthological fauna in these rodents