Soft tissue sarcomas with complex genomic profiles.

Soft tissue sarcomas (STS) with complex genomic profiles (50% of all STS) are predominantly composed of spindle cell/pleomorphic sarcomas, including leiomyosarcoma, myxofibrosarcoma, pleomorphic liposarcoma, pleomorphic rhabdomyosarcoma, malignant peripheral nerve sheath tumor, angiosarcoma, extraskeletal osteosarcoma, and spindle cell/pleomorphic unclassified sarcoma (previously called spindle cell/pleomorphic malignant fibrous histiocytoma). These neoplasms show, characteristically, gains and losses of numerous chromosomes or chromosome regions, as well as amplifications. Many of them share recurrent aberrations (e.g., gain of 5p13-p15) that seem to play a significant role in tumor progression and/or metastatic dissemination. In this paper, we review the cytogenetic, molecular genetic, and clinicopathologic characteristics of the most common STS displaying complex genomic profiles. Features of diagnostic or prognostic relevance will be discussed when needed.

Ki-67 and CD100 immunohistochemical expression is associated with local recurrence and poor prognosis in soft tissue sarcomas, respectively

Soft tissue sarcomas (STSs) are a heterogeneous group of mesenchymal tumors of >50 subtypes. However, STSs represent <1% of types of cancer. Despite this low frequency, the disease is aggressive and treatment, when possible, is based on traditional chemotherapies. A number of cases of resistance to adjuvant therapies have been reported. Metastases are commonly identified in STS patients during diagnosis and the development of effective clinical parameters is crucial for correct management of the disease. The use of biological markers in cancer is a useful tool to determine patient prognosis. Ki--67 is a protein marker for proliferation of somatic cells and is widely used in prognostic studies of various types of tumor, including STSs. Cluster of differentiation 100 (CD100) is a member of the semaphorin family. The family was initially described as axon guidance molecules important for angiogenesis, organogenesis, apoptosis and neoplasia. CD100 was previously utilized as a prognostic factor in tumors and also in STSs. In the present study, protein expression of Ki--67 and CD100 was analyzed by immunohistochemistry in samples of STS patients of the Barretos Cancer Hospital (Barretos, Brazil) to establish prognostic criteria of the disease. Results demonstrate a correlation between CD100 expression and poor prognosis, consistent with a previous study. Moreover, the expression of Ki-67 was identified to correlate with presence of local or locoregional recurrence. To the best of our knowledge, no large casuistic study has revealed this correlation between Ki--67 and local recurrence in STSs. The use of Ki--67 and CD100 as markers in clinical pathological analysis may be suitable as a prognostic criterion in disease progression.

Characterization of monocarboxylate transporters (MCTs) expression in soft tissue sarcomas: distinct prognostic impact of MCT1 sub-cellular localization

Background: Soft tissue sarcomas (STSs) are a group of neoplasms, which, despite current therapeutic advances, still confer a poor outcome to half of the patients. As other solid tumors, STSs exhibit high glucose consumption rates, associated with worse prognosis and therapeutic response. As highly glycolytic tumors, we hypothesized that sarcomas should present an increased expression of lactate transporters (MCTs).Methods: Immunohistochemical expression of MCT1, MCT2, MCT4 and CD147 was assessed in a series of 86 STSs and the expression profiles were associated with patients' clinical-pathological parameters.Results: MCT1, MCT4 and CD147 were mainly observed in the plasma membrane of cancer cells (around 60% for MCTs and 40% for CD147), while MCT2 was conspicuously found in the cytoplasm (94.2%). Importantly, we observed MCT1 nuclear expression (32.6%). MCT1 and MCT4, alone or co-expressed with CD147 in the plasma membrane, were associated with poor prognostic variables including high tumor grade, disease progression and shorter overall survival. Conversely, we found MCT1 nuclear expression to be associated with low grade tumors and longer overall survival.Conclusions: The present work represents the first report of MCTs characterization in STSs. We showed the original finding of MCT1 expression in the nucleus. Importantly, opposite biological roles should be behind the dual sub-cellular localization of MCT1, as plasma membrane expression of MCT1 is associated with worse patients' prognosis, while nuclear expression is associated with better prognosis.

Inibizione selettiva del gene MYCN mediante PNA (acidi peptido nucleici) anti-gene nel rabdomiosarcoma umano

MYCN oncogene amplification/expression is a feature of many childhood tumors, and some adult tumors, and it is associated with poor prognosis. While MYC expression is ubiquitary, MYCN has a restricted expression after birth and it is an ideal target for an effective therapy. PNAs belong to the latest class of nucleic acid-based therapeutics, and they can bind chromosomal DNA and block gene transcription (anti-gene activity). We have developed an anti-gene PNA that targets specifically the MYCN gene to block its transcription. We report for the first time MYCN targeted inhibition in Rhabdomyosarcoma (RMS) by the anti-MYCN-PNA in RMS cell lines (four ARMS and four ERMS) and in a xenograft RMS mouse model. Rhabdomyosarcoma is the most common pediatric soft-tissue sarcoma, comprising two main subgroups [Alveolar (ARMS) and Embryonal (ERMS)]. ARMS is associated with a poorer prognosis. MYCN amplification is a feature of both the ERMS and ARMS, but the MYCN amplification and expression levels shows a significant correlation and are greater in ARMS, in which they are associated with adverse outcome. We found that MYCN mRNA and protein levels were higher in the four ARMS (RH30, RH4, RH28 and RMZ-RC2) than in the four ERMS (RH36, SMS-CTR, CCA and RD) cell lines. The potent inhibition of MYCN transcription was highly specific, it did not affect the MYC expression, it was followed by cell-growth inhibition in the RMS cell lines which correlated with the MYCN expression rate, and it led to complete cell-growth inhibition in ARMS cells. We used a mutated- PNA as control. MYCN silencing induced apoptosis. Global gene expression analysis (Affymetrix microarrays) in ARMS cells treated with the anti-MYCN-PNA revealed genes specifically induced or repressed, with both genes previously described as targets of N-myc or Myc, and new genes undescribed as targets of N-myc or Myc (mainly involved in cell cycle, apoptosis, cell motility, metastasis, angiogenesis and muscle development). The changes in the expression of the most relevant genes were confirmed by Real-Time PCR and western blot, and their expression after the MYCN silencing was evaluated in the other RMS cell lines. The in vivo study, using an ARMS xenograft murine model evaluated by micro-PET, showed a complete elimination of the metabolic tumor signal in most of the cases (70%) after anti-MYCN-PNA treatment (without toxicity), whereas treatment with the mutated-PNA had no effect. Our results strongly support the development of MYCN anti-gene therapy for the treatment of RMS, particularly for poor prognosis ARMS, and of other MYCN-expressing tumors.

PET-CT-guided interventions in the management of FDG-positive lesions in patients suffering from solid malignancies: initial experiences

Positron emission tomography-computed tomography (PET-CT) has gained widespread acceptance as a staging investigation in the diagnostic workup of malignant tumours and may be used to visualize metabolic changes before the evolution of morphological changes. To make histology of PET findings without distinctive structural changes available for treatment decisions, we developed a protocol for multimodal image-guided interventions using an integrated PET-CT machine. We report our first experience in 12 patients admitted for staging and restaging of breast cancer, non-small cell lung cancer, cervical cancer, soft tissue sarcoma, and osteosarcoma. Patients were repositioned according to the findings in PET-CT and intervention was planned based on a subsequent single-bed PET-CT acquisition of the region concerned. The needle was introduced under CT guidance in a step-by-step technique and correct needle position in the centre of the FDG avid lesion was assured by repetition of a single-bed PET-CT acquisition before sampling. The metabolically active part of lesions was accurately targeted in all patients and representative samples were obtained in 92%. No major adverse effects occurred. We conclude that PET-CT guidance for interventions is feasible and may be promising to optimize the diagnostic yield of CT-guided interventions and to make metabolically active lesions without morphological correlate accessible to percutaneous interventions.

Soft tissue sarcomas in dogs: A study assessing surgical margin, tumour grade and clinical outcome

The purpose of this prospective clinical study was to quantify the surgical margin necessary to maximise local disease control for canine soft tissue sarcoma of various grades. This was achieved via gross and histopathologic studies. Fourteen dogs underwent surgical treatment for 15 localised, measurable, subcutaneous sarcomas. Surgery and histopathologic evaluation were performed to standardised protocols. Regular examinations for local recurrence and distant metastases were performed for at least 12 months postoperatively. One hundred percent local disease control was achieved with deep margins >10mm and 93% one year disease-free survival with wide margins (i.e. >10mm laterally and one fascial plane or >10mm in depth). There was one case of recurrence. Fascial planes appear to act as biological barriers to local tumour invasion but this protective effect may be overcome with high-grade lesions.

Optimal designs for evaluating a series of treatments

Several articles in this journal have studied optimal designs for testing a series of treatments to identify promising ones for further study. These designs formulate testing as an ongoing process until a promising treatment is identified. This formulation is considered to be more realistic but substantially increases the computational complexity. In this article, we show that these new designs, which control the error rates for a series of treatments, can be reformulated as conventional designs that control the error rates for each individual treatment. This reformulation leads to a more meaningful interpretation of the error rates and hence easier specification of the error rates in practice. The reformulation also allows us to use conventional designs from published tables or standard computer programs to design trials for a series of treatments. We illustrate these using a study in soft tissue sarcoma.

The Role Of Fumarase (FH) In Tumorigenesis

In this study, a predisposing gene for a recently characterized cancer syndrome, hereditary leiomyomatosis and renal cell cancer (HLRCC), was identified and the role of the gene was investigated in other familial cancers and in nonsyndromic tumorigenesis. HLRCC is a dominantly inherited disorder predisposing predominantly to uterine and skin leiomyomas, and also to renal cell cancer and uterine leiomyosarcoma. The disease gene was recently localized in Finnish families to 1q42-q43 by a genome-wide linkage search. Independently in the UK, a clinically similar condition, multiple cutaneous and uterine leiomyomata (MCUL), was linked to the same chromosomal region, strongly suggesting that HLRCC and MCUL are actually a single syndrome. Linkage results were confirmed by detecting loss of heterozygosity (LOH) at the disease locus in most of the patients' tumors, suggesting that this predisposing gene acts as a tumor suppressor. Through detailed investigation by genotyping of microsatellite markers and haplotype construction in Finnish and UK HLRCC/MCUL families we were able to narrow the disease locus down to 1.6 Mb. Extensive mutation screening of known and predicted transcripts in the target region resulted in identification of the HLRCC predisposing gene, fumarase (fumarate hydratase, FH). FH is a key enzyme in energy metabolism, catalyzing fumarate to malate in the tricarboxylic acid cycle (TCAC) in mitochondria. Germline alterations in FH segregating with the disease were detected in 25 of 42 HLRCC/MCUL families including whole-gene deletions, truncating small deletions/insertions and nonsense mutations, as well as substitutions or deletions of highly conserved amino acids. Biallelic inactivation was detected in almost all studied tumors of HLRCC patients. Furthermore, FH enzyme activity was reduced in the patients' normal tissues and was completely or virtually absent from tumors. Based on these findings, we extensively demonstrated that mutations in FH underlie the HLRCC/MCUL syndrome. In our studies of other familial cancers, evidence for involvement of FH defects was not found in familial prostate and breast cancers. To investigate the role of FH in sporadic tumorigenesis, we analyzed 652 lesions, including a series of 353 nonsyndromic counterparts of tumor types associated with HLRCC. Mutations in nonsyndromic tumors were rare and appeared to be limited to tumor types observed in the hereditary form of the disease. Biallelic inactivation of FH was detected in a uterine leiomyosarcoma, a cutaneous leiomyoma, a soft-tissue sarcoma, and in two uterine leiomyomas. In the uterine leiomyosarcoma and the cutaneous lesion FH mutations originated from the germline whereas the soft-tissue sarcoma harbored purely somatic changes. In uterine leiomyomas somatic mutations were detected in the two out of five tumors with LOH at the FH locus. Our findings demonstrate that FH inactivation is also involved in nonhereditary tumor development, and further support the hypothesis that FH acts as a tumor suppressor. The role of FH in predisposition to malignancies, renal cell carcinoma and leiomyosarcoma is important in the diagnosis and prevention of cancer among HLRCC patients. This study is of general clinical interest, because prior to our findings, little was known about the molecular genetics of uterine leiomyomas, the most common tumors of women.

Ancylidae (Mollusca, Heterobranchia, Pulmonata, Basommatophora) do Estado do Rio de Janeiro, Brasil: morfologia, sistemática e distribuição geográfica

Os moluscos pateliformes de água doce da região Neotropical são comumente atribuídos a família Ancylidae sensu latum, abrangendo sete gêneros com pelo menos 13 espécies válidas e sete com identificação duvidosa. Os ancilídeos possuem pequenas dimensões, alcançam no máximo 15 mm de comprimento. Sua concha é frágil, composta por duas regiões, a protoconcha e a teleoconcha, as quais apresentam caracteres relevantes para a sistemática. Na parte mole as impressões musculares, a pigmentação do manto, o sistema reprodutor e a rádula são importantes para o estudo da família. Apesar de existirem vários registros de ocorrência para ancílideos no Estado do Rio de Janeiro (ERJ), existem poucos dados morfológicos. O principal objetivo deste estudo foi fornecer e ampliar as informações sobre a morfologia e distribuição geográfica das espécies de Ancylidae encontradas no ERJ. Os materiais utilizados foram procedentes de coletas próprias, material depositado em Coleções Científicas e dados de revisão bibliográfica. O estudo da morfologia comparada das conchas foi realizado com o auxílio de imagens de microscópio óptico e de varredura. Para a comparação das partes moles, os espécimes foram corados e dissecados sob lupa. Através da conquiliometria analisamos as diferenças inter e intrapopulacionais. Com este trabalho, a riqueza conhecida para Ancylidae no ERJ, aumentou de cinco para sete espécies: Burnupia sp., Ferrissia sp., Gundlachia radiata (Guilding, 1828),G. ticaga (Marcus & Marcus, 1962), Gundlachia sp., Hebetancylus moricandi (d`Orbigny, 1837) e Uncancylus concentricus (d`Orbigny, 1837). Gundlachia radiata e U. concentricus constituem novos registros para o ERJ, e G. radiata para a Região Sudeste. As três espécies com o maior número de registros de ocorrência no ERJ foram: G. ticaga (66%), Ferrissia sp. (37%) e Gundlachia sp. (18%). A ampla distribuição de G. ticaga pode ser devido à capacidade de suportar ambientes impactados. Em relação à morfologia, Burnupia sp. difere de Burnupia ingae Lanzer, 1991, única espécie deste gênero descrita para o Brasil, por apresentar diferenças na microescultura da concha e também na forma das impressões musculares. Ferrissia sp. difere de F. gentilis Lanzer, 1991, devido a diferenças na microescultura apical e número de cúspides no dente central da rádula. Gundlachia sp. é diferente de G. ticaga e G. radiata, por apresentar a abertura da concha mais arredondada, ápice mais recurvado, ultrapassando a borda da concha, pontuações irregulares em toda a protoconcha e forma dos músculos adutores anterior direito e posterior mais elíptica. A morfologia interna também mostra diferenças entre Gundlachia sp. e G. ticaga, como o apêndice terminal do útero e o número de folículos do ovoteste. Através das análises conquiliométricas, constatamos para os gêneros Burnupia, Ferrrissia e Gundlachia, que os índices morfométricos se mostraram melhores que as medidas lineares para a discriminação das espécies, provavelmente porque esses índices diminuem o efeito da amplitude de tamanho das conchas, que são fortemente influenciadas pelas variações ecofenotípicas. Contudo, os caracteres diagnósticos das conchas e das partes moles (impressões musculares) são indispensáveis para a identificação dos gêneros e espécies de Ancylidae. Palavras-chave: Mollusca. Moluscos de água doce. Morfologia. Distribuição geográfica. Estado do Rio de Janeiro.uscos.

Estudo temporal de longo prazo da comunidade de moluscos límnicos em um riacho da Vila do Abraão, Ilha Grande, Angra dos Reis, RJ, Brasil

Estudos de longo prazo são essenciais para avaliar efeitos que em pouco tempo não seriam possíveis observá-los. Moluscos límnicos são parte importante dos ecossistemas aquáticos, além se serem vetores de parasitos de importância médica. A comunidade de moluscos do riacho da Vila do Abraão já havia sido estudada, havendo registro de sete espécies, incluindo a exótica Melanoides tuberculata. O objetivo geral do trabalho foi acompanhar a dinâmica das populações de moluscos límnicos, com ênfase em M. tuberculata durante sete anos. Avaliamos as variações na comunidade de moluscos límnicos em um riacho na Vila do Abraão Ilha Grande, Angra dos Reis, RJ, Brasil. Foram realizadas 42 coletas bimestrais no período de julho/2006 a novembro/2013. O trecho estudado foi dividido em oito pontos de coleta, com três coletores em cada, realizando buscas de 15 minutos, totalizando 45 minutos por ponto. Foram mensurados os fatores abióticos: luminosidade, temperatura do ar e da água, umidade, pH, condutividade, profundidade e oxigênio dissolvido. Para o acompanhamento da comunidade, foi calculada a abundância, abundância relativa, constância, índices de diversidade Simpson e Shannon, assim como equitabilidade e uniformidade, para cada espécie em cada expedição de coleta. Para investigar a associação dos moluscos com a helmintofauna, realizou-se o teste de exposição a luz. Para biomassa, de M. tuberculata foi calculado o peso seco da parte mole, e os valores de abundância foram convertidos para densidade. No total do estudo, foram coletados 90.718 espécimes: oito gastrópodes (M. tuberculata, Heleobia australis, Potamolithus sp., Physa acuta, Biomphalaria tenagophila, Gundlachia ticaga, Ferrissia fragilis e Omalonyx matheroni) e um bivalve (Pisidium punctiferum). As duas espécies mais abundantes foram: M. tuberculata, com 80% e P. acuta, com 8% do total dos indivíduos. Melanoides tuberculata foi a espécie mais constante do estudo, já que após Fevereiro/2009 foi encontrada em todos os pontos de coleta, exceto nas últimas expedições. Os índices de diversidade apresentaram valores diferentes entre Simpson e Shannon, mas com variações semelhantes. A equitabilidade e a uniformidade foram muito baixas, indicando uma dominância de M. tuberculata. Apenas M. tuberculata se apresentou parasitado por Centrocestus formosanus, com a maior prevalência em Abril/2013, quando mais de 50% da população estava parasitada. A biomassa foi calculada em 8155 g durante o estudo, e a produção secundária foi estimada em 423 g m-2year-1 no último ano estudado, sofrendo diminuição de acordo com a queda populacional de M. tuberculata que por sua vez, foi possivelmente influenciada pelo parasitismo. Podemos concluir que a riqueza da comunidade aumentou de sete para nove espécies, sendo seis exóticas e três nativas. A comunidade de moluscos da Vila do Abraão encontra-se dominada por M. tuberculata, este sobrepujando as demais, em abundância e biomassa. Tendo em vista o perigo das introduções, reforçamos a importância dos estudos de longo prazo para o acompanhamento de comunidades, sendo importantes para subsidiar estratégias de conservação principalmente em unidades de conservação.

Identification of quantitative trait loci for growth-related traits in the Pacific abalone Haliotis discus hannai Ino

The locations and effects of quantitative trait loci (QTL) were estimated for nine characters for growth-related traits in the Pacific abalone (Haliotis discus hannai Ino) using a randomly amplified polymorphic DNA (RAPD), amplification fragment length polymorphism (AFLP) and SSR genetic linkage map. Twenty-eight putatively significant QTLs (LOD > 2.4) were detected for nine traits (shell length, shell width, total weight, shell weight, weight of soft part, muscle weight, gonad and digestive gland weight, mantle weight and gill weight). The percentage of phenotypic variation explained by a single QTL ranged from 8.0% to 35.9%. The significant correlations (P < 0.001) were found among all the growth-related traits, and Pearson's correlation coefficients were more than 0.81. For the female map, the QTL for growth were concentrated on groups 1 and 4 linkage maps. On the male map, the QTL that influenced growth-related traits gathered on the groups 1 and 9 linkage maps. Genetic linkage map construction and QTL analysis for growth-related traits are the basis for the marker-assisted selection and will eventually improve production and quality of the Pacific abalone.

Angiosarcoma, porphyria cutanea tarda, and probable chloracne in a worker exposed to waste oil contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin

A worker developed angiosarcoma, porphyria cutanea tarda, and skin lesions characteristic of mild chloracne. About 10 years earlier he had been employed at a truck terminal in Saint Louis, Missouri, at a time when it was sprayed with waste oil contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The occurrence of these three rare conditions in a single exposed worker supports the aetiological relation between environmental exposure to TCDD and the subsequent development of soft tissue sarcoma and porphyria cutanea tarda.

Micro-Anatomical Quantitative Imaging Towards Enabling Automated Diagnosis of Thick Tissues at the Point of Care

Histopathology is the clinical standard for tissue diagnosis. However, histopathology has several limitations including that it requires tissue processing, which can take 30 minutes or more, and requires a highly trained pathologist to diagnose the tissue. Additionally, the diagnosis is qualitative, and the lack of quantitation leads to possible observer-specific diagnosis. Taken together, it is difficult to diagnose tissue at the point of care using histopathology.

Several clinical situations could benefit from more rapid and automated histological processing, which could reduce the time and the number of steps required between obtaining a fresh tissue specimen and rendering a diagnosis. For example, there is need for rapid detection of residual cancer on the surface of tumor resection specimens during excisional surgeries, which is known as intraoperative tumor margin assessment. Additionally, rapid assessment of biopsy specimens at the point-of-care could enable clinicians to confirm that a suspicious lesion is successfully sampled, thus preventing an unnecessary repeat biopsy procedure. Rapid and low cost histological processing could also be potentially useful in settings lacking the human resources and equipment necessary to perform standard histologic assessment. Lastly, automated interpretation of tissue samples could potentially reduce inter-observer error, particularly in the diagnosis of borderline lesions.

To address these needs, high quality microscopic images of the tissue must be obtained in rapid timeframes, in order for a pathologic assessment to be useful for guiding the intervention. Optical microscopy is a powerful technique to obtain high-resolution images of tissue morphology in real-time at the point of care, without the need for tissue processing. In particular, a number of groups have combined fluorescence microscopy with vital fluorescent stains to visualize micro-anatomical features of thick (i.e. unsectioned or unprocessed) tissue. However, robust methods for segmentation and quantitative analysis of heterogeneous images are essential to enable automated diagnosis. Thus, the goal of this work was to obtain high resolution imaging of tissue morphology through employing fluorescence microscopy and vital fluorescent stains and to develop a quantitative strategy to segment and quantify tissue features in heterogeneous images, such as nuclei and the surrounding stroma, which will enable automated diagnosis of thick tissues.

To achieve these goals, three specific aims were proposed. The first aim was to develop an image processing method that can differentiate nuclei from background tissue heterogeneity and enable automated diagnosis of thick tissue at the point of care. A computational technique called sparse component analysis (SCA) was adapted to isolate features of interest, such as nuclei, from the background. SCA has been used previously in the image processing community for image compression, enhancement, and restoration, but has never been applied to separate distinct tissue types in a heterogeneous image. In combination with a high resolution fluorescence microendoscope (HRME) and a contrast agent acriflavine, the utility of this technique was demonstrated through imaging preclinical sarcoma tumor margins. Acriflavine localizes to the nuclei of cells where it reversibly associates with RNA and DNA. Additionally, acriflavine shows some affinity for collagen and muscle. SCA was adapted to isolate acriflavine positive features or APFs (which correspond to RNA and DNA) from background tissue heterogeneity. The circle transform (CT) was applied to the SCA output to quantify the size and density of overlapping APFs. The sensitivity of the SCA+CT approach to variations in APF size, density and background heterogeneity was demonstrated through simulations. Specifically, SCA+CT achieved the lowest errors for higher contrast ratios and larger APF sizes. When applied to tissue images of excised sarcoma margins, SCA+CT correctly isolated APFs and showed consistently increased density in tumor and tumor + muscle images compared to images containing muscle. Next, variables were quantified from images of resected primary sarcomas and used to optimize a multivariate model. The sensitivity and specificity for differentiating positive from negative ex vivo resected tumor margins was 82% and 75%. The utility of this approach was further tested by imaging the in vivo tumor cavities from 34 mice after resection of a sarcoma with local recurrence as a bench mark. When applied prospectively to images from the tumor cavity, the sensitivity and specificity for differentiating local recurrence was 78% and 82%. The results indicate that SCA+CT can accurately delineate APFs in heterogeneous tissue, which is essential to enable automated and rapid surveillance of tissue pathology.

Two primary challenges were identified in the work in aim 1. First, while SCA can be used to isolate features, such as APFs, from heterogeneous images, its performance is limited by the contrast between APFs and the background. Second, while it is feasible to create mosaics by scanning a sarcoma tumor bed in a mouse, which is on the order of 3-7 mm in any one dimension, it is not feasible to evaluate an entire human surgical margin. Thus, improvements to the microscopic imaging system were made to (1) improve image contrast through rejecting out-of-focus background fluorescence and to (2) increase the field of view (FOV) while maintaining the sub-cellular resolution needed for delineation of nuclei. To address these challenges, a technique called structured illumination microscopy (SIM) was employed in which the entire FOV is illuminated with a defined spatial pattern rather than scanning a focal spot, such as in confocal microscopy.

Thus, the second aim was to improve image contrast and increase the FOV through employing wide-field, non-contact structured illumination microscopy and optimize the segmentation algorithm for new imaging modality. Both image contrast and FOV were increased through the development of a wide-field fluorescence SIM system. Clear improvement in image contrast was seen in structured illumination images compared to uniform illumination images. Additionally, the FOV is over 13X larger than the fluorescence microendoscope used in aim 1. Initial segmentation results of SIM images revealed that SCA is unable to segment large numbers of APFs in the tumor images. Because the FOV of the SIM system is over 13X larger than the FOV of the fluorescence microendoscope, dense collections of APFs commonly seen in tumor images could no longer be sparsely represented, and the fundamental sparsity assumption associated with SCA was no longer met. Thus, an algorithm called maximally stable extremal regions (MSER) was investigated as an alternative approach for APF segmentation in SIM images. MSER was able to accurately segment large numbers of APFs in SIM images of tumor tissue. In addition to optimizing MSER for SIM image segmentation, an optimal frequency of the illumination pattern used in SIM was carefully selected because the image signal to noise ratio (SNR) is dependent on the grid frequency. A grid frequency of 31.7 mm-1 led to the highest SNR and lowest percent error associated with MSER segmentation.

Once MSER was optimized for SIM image segmentation and the optimal grid frequency was selected, a quantitative model was developed to diagnose mouse sarcoma tumor margins that were imaged ex vivo with SIM. Tumor margins were stained with acridine orange (AO) in aim 2 because AO was found to stain the sarcoma tissue more brightly than acriflavine. Both acriflavine and AO are intravital dyes, which have been shown to stain nuclei, skeletal muscle, and collagenous stroma. A tissue-type classification model was developed to differentiate localized regions (75x75 µm) of tumor from skeletal muscle and adipose tissue based on the MSER segmentation output. Specifically, a logistic regression model was used to classify each localized region. The logistic regression model yielded an output in terms of probability (0-100%) that tumor was located within each 75x75 µm region. The model performance was tested using a receiver operator characteristic (ROC) curve analysis that revealed 77% sensitivity and 81% specificity. For margin classification, the whole margin image was divided into localized regions and this tissue-type classification model was applied. In a subset of 6 margins (3 negative, 3 positive), it was shown that with a tumor probability threshold of 50%, 8% of all regions from negative margins exceeded this threshold, while over 17% of all regions exceeded the threshold in the positive margins. Thus, 8% of regions in negative margins were considered false positives. These false positive regions are likely due to the high density of APFs present in normal tissues, which clearly demonstrates a challenge in implementing this automatic algorithm based on AO staining alone.

Thus, the third aim was to improve the specificity of the diagnostic model through leveraging other sources of contrast. Modifications were made to the SIM system to enable fluorescence imaging at a variety of wavelengths. Specifically, the SIM system was modified to enabling imaging of red fluorescent protein (RFP) expressing sarcomas, which were used to delineate the location of tumor cells within each image. Initial analysis of AO stained panels confirmed that there was room for improvement in tumor detection, particularly in regards to false positive regions that were negative for RFP. One approach for improving the specificity of the diagnostic model was to investigate using a fluorophore that was more specific to staining tumor. Specifically, tetracycline was selected because it appeared to specifically stain freshly excised tumor tissue in a matter of minutes, and was non-toxic and stable in solution. Results indicated that tetracycline staining has promise for increasing the specificity of tumor detection in SIM images of a preclinical sarcoma model and further investigation is warranted.

In conclusion, this work presents the development of a combination of tools that is capable of automated segmentation and quantification of micro-anatomical images of thick tissue. When compared to the fluorescence microendoscope, wide-field multispectral fluorescence SIM imaging provided improved image contrast, a larger FOV with comparable resolution, and the ability to image a variety of fluorophores. MSER was an appropriate and rapid approach to segment dense collections of APFs from wide-field SIM images. Variables that reflect the morphology of the tissue, such as the density, size, and shape of nuclei and nucleoli, can be used to automatically diagnose SIM images. The clinical utility of SIM imaging and MSER segmentation to detect microscopic residual disease has been demonstrated by imaging excised preclinical sarcoma margins. Ultimately, this work demonstrates that fluorescence imaging of tissue micro-anatomy combined with a specialized algorithm for delineation and quantification of features is a means for rapid, non-destructive and automated detection of microscopic disease, which could improve cancer management in a variety of clinical scenarios.

Prevalence of the germline TP53 p.R337H mutation in families with multiple cases of cancer

Germline mutations in TP53 gene are associated with Li-Fraumeni syndrome (LFS) and its variants Li-Fraumeni-like (LFL). They predispose carriers to a wide variety of early onset tumors. In Brazil, there is a high frequency of a germline mutation in this gene (NC_000017.9: c.1010G>A; p.R337H) in Southern and Southeastern regions, due to a founder effect. It is estimated to be present in 0,3% ofthe local population, but only few families have been detected. Due to this significant divergence, the purpose of this study was to verify the effectiveness of wider criteria for detection of these individuals. Herein, clinical criteria were established, DNA samples were collected, analyzed by Restriction Fragment Length Polymorphism (RFLP) and sequenced. Thus, assessing the prevalence of this mutation in families with multiple cases of cancer. Based on our proposed criteria, one out of 31 patients (3,22%) was found to carry p.R337H mutation. The patient developed ductal invasive breast cancer at age 47, invasive adenocarcinoma of the lung at age 48 and soft-tissue sarcoma at age 49. In addition, an extensive cancer family history was referred, atypical for LFS, including a case of Ewing’s sarcoma. These outcomes indicate that the proposed criteria may detect probable carriers who did not fit previous LFS criteria. Nevertheless, additional studies, which might include a larger number of families and more stringent parameters, will be useful to improve screening sensibility