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PURPOSE: Tumor surgery is aimed at complete resection of the lesion while ensuring a sufficient tumor-specific safety distance. Nevertheless, in many cases the most peripheral part - the invasion front - remains in situ. Tumor cells at the tumor margin have been reported to lose their epithelial properties and acquire features of mesenchymal cells. The process of epithelial-to-mesenchymal transition (EMT) is believed to be of prime importance for tissue and vessel invasion. Furthermore, the detection of tumor-infiltrating lymphocytes in the microenvironment of breast cancer might serve as a reliable prognostic marker. METHODS: We investigated tissue microarrays of 352 breast cancer patients with regard to the presence and distribution of the EMT factor Snail, and the presence of FoxP3, CD3 and CD8 in the immune microenvironment. RESULTS: The expression of the transcription factor Snail is strongly associated with longer disease-free and overall survival. The presence of CD3, CD8 or FoxP3 is associated with a better outcome, although statistically significant results were noted only for FoxP3. The prognostic significance of FoxP3 and Snail were also proven in multivariate analysis. CONCLUSIONS: Based on previous studies concerning the intratumoral heterogeneity of EMT, our results suggest that Snail and FoxP3 are possible prognostic markers for breast cancer. The diverse presence of lymphocytes in the tumor microenvironment (CD3 and CD8) was confirmed. Although the importance of these markers is known, their specific role in tumor invasion and metastasis as well as their hierarchical organization in these tumors remain unclear.
Assuntos
Neoplasias da Mama/diagnóstico , Transição Epitelial-Mesenquimal , Linfócitos do Interstício Tumoral/citologia , Fatores de Transcrição da Família Snail/metabolismo , Microambiente Tumoral , Biomarcadores Tumorais , Neoplasias da Mama/patologia , Complexo CD3/genética , Complexo CD3/metabolismo , Antígenos CD8/genética , Antígenos CD8/metabolismo , Linhagem Celular Tumoral , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Marcadores Genéticos , Humanos , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Modelos de Riscos Proporcionais , Fatores de Transcrição da Família Snail/genética , Fatores Socioeconômicos , Análise Serial de TecidosRESUMO
Tumor-infiltrating lymphocytes (TILs) in breast cancer are a key representative of the tumor immune microenvironment and have been shown to provide prognostic and predictive biomarkers. The extent of lymphocytic infiltration in tumor tissues can be assessed by evaluating hematoxylin and eosin (H&E)-stained tumor sections. We investigated tissue microarrays of 31 invasive breast cancer patients, looking at quantity and topological distribution of CD3+, CD8+, CD20+, Ki67+, FoxP3+ TILs and CD3+/FoxP3+, CD8+/FoxP3+ cell ratios. We separately evaluated TILs at the invasive edge and at the center of the tumor, to find any clinical implications of tumor heterogeneity. No statistically significant difference was found in quantity and distribution of both TIL subsets and TIL ratios, by comparing patients who suffered from a local or distant recurrence of the tumor (relapse group: 13 patients) with patients not showing cancer relapse (non-relapse group: 18 patients). In the whole sample, we observed three main statistically significant positive correlations: (1) between CD3+ and CD8+ T-cells; (2) between FoxP3+ and Ki67+ lymphocyte infiltration; (3) between CD3+/FoxP3+ cell ratio (C3FR) and CD8+/FoxP3+ cell ratio (C8FR). Tumor heterogeneity and stronger positive TIL associations were found in the non-relapse group, where both CD3-CD8 and FoxP3-Ki67 inter-correlations were found to be significant at the center of the tumor, while the correlation between C3FR and C8FR was significant at the invasive edge. No correlations between TIL subsets were detected in the relapse group. Our findings suggest the existence of stronger inter-subtype lymphocytic networks in invasive breast cancer not showing recurrence. Further evaluations of clinical and topological correlations between and within TIL subsets are needed, in addition to the assessment of TIL quantification and distribution, in order to follow up on whether morphological evaluation of TILs might reveal the underlying lymphocytic functional connectivity and help relapse prediction.
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Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Comunicação Celular/imunologia , Linfócitos do Interstício Tumoral/imunologia , Microambiente Tumoral/imunologia , Biomarcadores , Neoplasias da Mama/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Imunofenotipagem , Contagem de Linfócitos , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Invasividade Neoplásica , Recidiva Local de Neoplasia , Prognóstico , Estudos Retrospectivos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
BACKGROUND: Breast cancer screening programs have been established worldwide and early detection of breast cancer has increased steadily. The most common way to confirm dignity of non-palpable and sonographically-occult suspicious findings on mammography is the stereotactically-guided vacuum-assisted breast biopsy PURPOSE: To compare two stereotactically guided vacuum-assisted breast biopsy systems measuring time effectiveness and quality of harvested material in clinical practice. MATERIAL AND METHODS: One hundred and forty-six patients presenting with suspicious microcalcifications on mammography were included in the study. Biopsies were carried out with either the Mammotome(®) system (11-gauge and 8-gauge) or the ATEC(®) system (12-gauge and 9-gauge). Lesions with a diameter <15 mm on mammography were biopsied with 11-gauge or 12-gauge devices whereas lesions >15 mm were targeted with 8-gauge and 9-gauge. Mammotome(®) system 8-gauge device was used in 34 patients, the 11-gauge system in 37 patients. The ATEC(®) system 9-gauge system was used in 37 patients and 12-gauge in 38 patients. Time was taken, focusing on preparing the system, time of collecting the samples, preparing the samples, and cleaning the site. During the biopsies 24 samples were taken. The histologic quality of the tissue samples was judged by a pathologist in a blinded fashion according to a specimen grading classification concerning tissue fragmentation, artefacts, and the adequacy of the tissue for diagnosis. RESULTS: The median overall time for the Mammotome(®) system was 879 s (11-gauge) and 934 s (8-gauge) and for the ATEC(®) system 671 s (12-gauge) and 673 s (9-gauge). The ATEC(®) system displays a significantly shorter overall time for small and large biopsy devices (U-test, P < 0.001). Concerning the mean time difference of the overall time comparing small and large systems the ATEC(®) system was 267.6 s faster using the small and 244.8 s faster using the large system. Comparing the histologic quality of tissue samples the Mammotome(®) system shows significantly higher values for the large and the small system (Chi-square test, P < 0.001). CONCLUSION: Both biopsy systems meet all requirements for daily practice and confirm the diagnosis of suspicious microcalcifications. The ATEC(®) system was observed to be faster but this difference of about 250 s might not be relevant in daily practice. The Mammotome(®) system provides a better histologic quality of tissue samples.
Assuntos
Biópsia/instrumentação , Mama/patologia , Técnicas Estereotáxicas/instrumentação , Biópsia/métodos , Calcinose/patologia , Feminino , Humanos , Pessoa de Meia-Idade , VácuoRESUMO
BACKGROUND: Hamartomas of the breast are rare benign tumors. Pre- and also postoperative differentiation from other benign or even malignant tumors is challenging. CASE PRESENTATION: A 36-year-old female presented with a giant tumor of the left breast. The patient had suffered from an early breast cancer of the contralateral right breast the year before, which was treated with breast-conserving therapy, radiation, and endocrine therapy ever since. The hamartoma was classified as BI-RADS 2 in mammography and BI-RADS 4 in ultrasound. On clinical examination, a tumor of nearly 15 cm in size led to an abstruse deformity of the breast and the nipple-areola complex. We found an indolent, grand bulging tumor with an elastic texture directly beneath the skin. A biopsy that had been performed before was compatible with the suspected hamartoma. Because of the remaining diagnostic uncertainties after contralateral breast cancer and the progressive malformation of the left breast, a tumor extirpation utilizing a reduction mammaplasty was performed without complications. Subsequent genetic analyses excluded a loss of PTEN in this patient. CONCLUSION: We presented the rare case of a 36-year-old woman with a history of breast cancer and a 700-g breast hamartoma. The preoperative and even the postoperative specification of a hamartoma remains challenging, and associations with genetic alterations should be considered.
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Human chromosome 8q24.21 has been implicated as a susceptibility region for colorectal cancer (CRC) as a result of genome-wide association and candidate gene studies. To assess the impact of molecular variants at 8q24.21 upon the CRC risk of German individuals and to refine the disease-associated region, a total of 2,713 patients with operated CRC (median age at diagnosis: 63 years) were compared with 2,718 sex-matched control individuals (median age at inclusion: 65 years). Information on microsatellite instability in tumors was available for 901 patients. Association analysis of SNPs rs10505477 and rs6983267 yielded allelic p-values of 1.42 x 10(-7) and 2.57 x 10(-7), respectively. For both polymorphisms, the odds ratio was estimated to be 1.50 (95% CI: 1.29-1.75) under a recessive disease model. The strongest candidate interval, outside of which significance dropped by more than 4 orders of magnitude, was delineated by SNPs rs10505477 and rs7014346 and comprised 17 kb. In a subgroup analysis, the disease association was found to be more pronounced in MSI-stable tumors (odds ratio: 1.71). Our study confirms the role of genetic variation at 8q24.21 as a risk factor for CRC and localizes the corresponding susceptibility gene to a 17 kb candidate region.
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Cromossomos Humanos Par 8 , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/genética , Predisposição Genética para Doença , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Neoplasias Colorretais/diagnóstico , Feminino , Alemanha , Humanos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
In HNSCC, protein- and mRNA-expression of the antileukoproteinase SLPI are significantly inverse correlated with HPV-infection suggesting that elevated expression of SLPI protects against HPV-infections. Moreover, SLPI-expression is up-regulated in HNSCC-patients reporting a smoking habit. Here, we investigate the described correlation in other HPV-driven cancers, namely vulvar squamous cell carcinoma (VSCC). FFPE samples of 99 VSCC were analyzed by PCR for HPV-DNA-expression and by RT-qPCR for SLPI-mRNA-expression. Of 99 VSCC 10 (10.1%) are HPV-positive; 9 were HPV16; 1 HPV18; all were E6/E7 mRNA-positive. 33 of the 99 patients (33.3%) reported a smoking habit; 7 (21.1%) of these were HPV-positive. Of 66 (66.7%) non-smokers 3 (4.5%) were HPV-positive. SLPI-expression was 4.0-fold lower in HPV-positive than HPV-negative patients. Smoking resulted in 2.3-fold higher SLPI expression. The data presented here indicate that SLPI plays a pivotal role in HPV-infection not only in HNSCC but also in VSCC and possibly also in other HPV-driven cancers. This however, needs to be analyzed in future studies. Furthermore these data lead to the hypothesis that the smoking induced SLPI-increase is systemic rather than local, as assumed based on the HNSCC data.
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The Ashcroft scale for the evaluation of bleomycin-induced lung fibrosis is the analysis of stained histological samples by visual assessment. Based on the knowledge that this procedure is not standardized in animals and results are highly variable, we hypothesized that modification of this method may improve quantification of lung fibrosis in small animals. To prove our hypothesis, we evaluated pulmonary fibrosis in Lewis rats induced by a single intratracheal injection of 0.3 mg/kg body weight bleomycin (n = 13) compared with the same amount of saline in a control group (n = 4). We modified the Ashcroft scale by precisely defining the assignment of grades from 0 to 8 for the increasing extent of fibrosis in lung histological samples. Thirty-two observers were randomly assigned to evaluate 108 photographs of slides using either the Ashcroft scale or the modified scale. Consistent with our hypothesis, there was a significant reduction in the variability of standard deviations with the modified scale compared with the Ashcroft scale (mean of variability 0.25 versus 0.62, P < 0.0001). Applying the kappa index, the Ashcroft scale showed only a fair to moderate agreement (0.23-0.59) between the observers and a low intra-observer agreement (0.51-0.74) in contrast to the modified scale, which demonstrated a moderate to good agreement between the observers (0.65-0.93, P < 0.0001) and a high intra-observer agreement (0.87-0.91, P < 0.05). To test the modified scale in vivo, we compared both scales with the results of computed tomography (CT) of the lungs obtained from the same mice. In agreement, the modified scale demonstrated a better correlation to CT scans (R = 0.58) compared with the Ashcroft scale (R = 0.33). In summary, quantification of lung fibrosis in histological lung sections using the modified scale is reliable and reproducible.
Assuntos
Fibrose Pulmonar/diagnóstico , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/toxicidade , Bleomicina/administração & dosagem , Bleomicina/toxicidade , Exposição por Inalação , Variações Dependentes do Observador , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/diagnóstico por imagem , Fibrose Pulmonar/patologia , Radiografia , Ratos , Ratos Endogâmicos Lew , Reprodutibilidade dos TestesRESUMO
BACKGROUND: While standard RNA expression tests stratify patients into risk groups, RNA-Seq can guide personalized drug selection based on expressed mutations, fusion genes, and differential expression (DE) between tumor and normal tissue. However, patient-matched normal tissue may be unavailable. Additionally, biological variability in normal tissue and technological biases may confound results. Therefore, we present normal expression reference data for two sequencing methods that are suitable for breast biopsies. RESULTS: We identified breast cancer related and drug related genes that are expressed uniformly across our normal samples. Large subsets of these genes are identical for formalin fixed paraffin embedded samples and fresh frozen samples. Adipocyte signatures were detected in frozen compared to formalin samples, prepared by surgeons and pathologists, respectively. Gene expression confounded by adipocytes was identified using fat tissue samples. Finally, immune repertoire statistics were obtained for healthy breast, tumor and fat tissues. CONCLUSIONS: Our reference data can be used with patient tumor samples that are asservated and sequenced with a matching aforementioned method. Coefficients of variation are given for normal gene expression. Thus, potential drug selection can be based on confidently overexpressed genes and immune repertoire statistics. MATERIALS AND METHODS: Normal expression from formalin and frozen healthy breast tissue samples using Roche Kapa RiboErase (total RNA) (19 formalin, 9 frozen) and Illumina TruSeq RNA Access (targeted RNA-Seq, aka TruSeq RNA Exome) (11 formalin, 1 frozen), and fat tissue (6 frozen Access). Tumor DE using 10 formalin total RNA tumor samples and 1 frozen targeted RNA tumor sample.
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Microsatellite instable gastric cancer (MSI-GC) is a specific molecular subtype of GC. We studied the phenotypes, genotypes, and clinicopathologic characteristics of MSI-GC in a white GC cohort and compared our findings with an extended literature review. The study cohort consisted of 482 patients. Specimens were available from 452 cases and were used for immunostaining (MLH1, PMS2, MSH2, MSH6) and molecular biological analyses (BAT-25, BAT-26, NR-21, NR-24, NR-27; Epstein-Barr virus in situ hybridization). Thirty-four (7.5%) GCs were MSI. Loss of MLH1 and/or PMS2 was found in 30 (88%) MSI-GC, 3 (9%) showed loss of MSH2 and/or MSH6. One (3%) MSI-GC was identified only by molecular biological testing. A single case was heterogeneous and contained microsatellite-stable and instable tumor areas. Twenty-one (62%) MSI-GCs showed unusual histologic features. MSI-GC was not found in diffuse-type or Epstein-Barr virus-positive GC. MSI-GC was significantly more prevalent in elderly patients, distal stomach, and was associated with a significantly lower number of lymph node metastases and a significantly better overall and tumor-specific survival. MSI-GC constitutes a small but relevant subgroup of GC with distinct clinicopathologic characteristics. Our literature review illustrates the shortcomings of missing standardized testing algorithms with prevalences of MSI-GC ranging from 0% to 44.5%. Future studies should test the hypothesis that patients with MSI-GCs may not need adjuvant/perioperative chemotherapy. However, this will require a standardized, quality-controlled diagnostic algorithm of MSI for GC.
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Instabilidade de Microssatélites , Neoplasias Gástricas/patologia , Idoso , Feminino , Humanos , Imuno-Histoquímica , Masculino , Neoplasias Gástricas/genéticaRESUMO
BACKGROUND: Inadvertently retained sponges and instruments still constitute a major but preventable complication in surgery. Given the high geographic mobility of patients, the fluctuation of physician-patient contact, and communication problems due to language barriers, the conscientious use of structured safety protocols in clinical routine is an essential aspect of quality in health care. CASE PRESENTATION: We report the case of a 24-year-old refugee from Syria who presented at our gynecological outpatient department with a tumor in the lower abdomen, suspected to be a lump in the ovary or the uterus. Language barriers hindered exact recording of the patient's medical history. We knew she had undergone three Caesarean sections several years ago. The diagnostic laparoscopy unexpectedly revealed a tumor suspected to be a retained surgical sponge. The lesion was removed completely and the patient discharged from the clinic five days later. CONCLUSION: In ambiguous cases, the diagnostic and therapeutic potential of minimally invasive surgery ensures safe and effective treatment of the patient, a short hospital stay, and low rates of complications. Especially in cases of language and/or cultural barriers, structured safety protocols should be a part of clinical routine in order to prevent unnecessary complications.
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Targeting the PD-1/PD-L1 immune checkpoint signaling is a novel promising treatment strategy in several tumor entities, and it is suggested that PD-L1/PD-1 expression is predictive for a PD-1/PD-L1 checkpoint inhibitor treatment response. We investigated the expression of PD-L1 and PD-1 by immunohistochemistry in a large and well characterized gastric cancer (GC) cohort of Caucasian patients, consisting of 465 GC samples and 15 corresponding liver metastases. Staining results were correlated with clinico-pathological characteristics and survival. PD-L1 expression was found in tumor cells of 140 GCs (30.1%) and 9 liver metastases (60%) respectively in immune cells of 411 GCs (88.4%) and 11 liver metastases (73.3%). PD-1 was expressed in tumor infiltrating lymphocytes in 250 GCs (53.8%) and in 11 liver metastases (73.3%). PD-L1 expression was significantly more prevalent in men, GCs of the proximal stomach, unclassified, papillary, Her2/neu-positive, Epstein-Barr-virus-positive, microsatellite instable, and PIK3CA-mutated GCs. A high PD-L1/PD-1 expression was associated with a significantly better patient outcome, and PD-L1 turned out to be an independent survival prognosticator. The correlation of PD-L1/PD-1 expression with distinct clinico-pathological patient characteristics may serve as a surrogate marker of PD-L1-positive GCs and may direct the use of immune checkpoint treatment strategies.
Assuntos
Antígeno B7-H1/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Hepáticas/secundário , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Gástricas/patologia , Idoso , Estudos de Coortes , Feminino , Seguimentos , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Masculino , Prognóstico , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/metabolismo , Taxa de Sobrevida , População BrancaRESUMO
The application of Trastuzumab on gastric cancer patients is based on Her2/neu immunostaining. The testing method relies on visual estimation of both membranous staining intensity, and positive tumor ratio with respect to a 10% cutoff. We evaluated the effect of inter- and intraobserver variations of both factors on therapeutic decision, especially if the positive tumor ratio hovers around the 10% cutoff. Ten pathologists scored 12 Her2/neu immunohistologically stained whole sections of gastric cancer. Applying the common rules for Her2/neu testing for gastric cancer, they separately noted the strongest identifiable staining intensity and the corresponding positive tumor ratio. Scoring was done repeatedly using the microscope, plain virtual microscopy, and virtual microscopy with a manual outline drawing function. Agreements on the strongest identified staining intensities were moderate. Overall concordance correlation coefficients of positive tumor ratios ranged from 0.55 to 0.81. Reproducibility was not improved by virtual microscopy. Pathologists have a good ability to estimate ratios of clearly demarcated areas, but gradients in staining intensities hinder reproducible visual demarcation of positive tumor areas. When hovering around the 10% positive tumor ratio cutoff there is a risk of misinterpretation of the staining results. This could lead to a denial of Trastuzumab therapy. Assessment of Her2/neu expression should be carried out by experienced pathologists because they can more reproducibly rate membranous staining intensities. The low reproducibility of positive tumor ratio is inherent in the testing method and cannot be improved by virtual microscopy. Therefore, we propose to reconsider the 10% cut-off limit.
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Biomarcadores Tumorais/análise , Receptor ErbB-2/análise , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Reações Falso-Positivas , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Microscopia/instrumentação , Reprodutibilidade dos Testes , Neoplasias Gástricas/diagnóstico , TrastuzumabRESUMO
Sebaceous gland neoplasias are the cutaneous manifestation of the Muir-Torre syndrome, which is known to be a phenotypical variant of hereditary nonpolyposis colorectal cancer. Both hereditary nonpolyposis colorectal cancer and Muir-Torre syndrome are caused by inherited DNA mismatch repair defects. As a prominent molecular genetic feature, all tumors associated with a DNA mismatch repair defect exhibit high microsatellite instability. So far, the frequency of DNA mismatch repair defects in patients selected solely on the basis of a sebaceous gland tumor has never been determined. In order to estimate this frequency, we assessed microsatellite instability with up to 10 microsatellite markers in a newly collected unselected series of 25 sebaceous gland neoplasias (six sebaceous adenomas, 16 sebaceous epitheliomas, three sebaceous carcinomas) in comparison to 32 sebaceous gland hyperplasias from unrelated patients. As many as 15 of the 25 sebaceous gland neoplasias (60%), but only one of the 32 sebaceous gland hyperplasias (3%), exhibited high microsatellite instability. Thus, in our study, the majority of patients with a sebaceous gland neoplasia in contrast to patients with a sebaceous gland hyperplasia are highly suspicious for an inherited DNA mismatch repair defect. On the basis of the subsequently collected tumor histories, nine of the 15 patients with a high microsatellite unstable sebaceous gland neoplasia were identified to have Muir-Torre syndrome. In none of these cases, however, were the clinical Amsterdam criteria for diagnosing hereditary nonpolyposis colorectal cancer fulfilled. In the sebaceous tumors of the remaining six patients, high microsatellite instability was an incidental finding. In two of these six patients, single relatives were known to be affected with internal cancer; however, their family histories were not suggestive of Muir-Torre syndrome or hereditary nonpolyposis colorectal cancer. In comparison with microsatellite instability screening studies in a variety of other randomly selected tumors, our study identifies sebaceous gland neoplasias as tumors with the highest frequency of high microsatellite instability reported so far, whereas sebaceous gland hyperplasia rarely exhibits high microsatellite instability. Therefore, screening for microsatellite instability in sebaceous gland neoplasias will be of great value in the detection of an inherited DNA mismatch repair defect, which predisposes to various types of internal cancers.
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Fibroblastos/metabolismo , Hiperplasia/patologia , Repetições de Microssatélites , Neoplasias das Glândulas Sebáceas/genética , Neoplasias das Glândulas Sebáceas/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Pareamento Incorreto de Bases , Biomarcadores Tumorais , Neoplasias Colorretais/metabolismo , Reparo do DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Hiperplasia/metabolismo , Masculino , Pessoa de Meia-Idade , Fenótipo , Fatores de RiscoRESUMO
Hereditary non-polyposis colorectal cancer (HNPCC) accounts for approximately 2 to 4% of the total colorectal cancer burden. For economic reasons a diagnostic "stepladder" is recommended. After evaluation of the family history, diagnostic microsatellite instability (MSI) analysis has found its place as a valuable screening tool for HNPCC. Immunohistochemical analysis can help to pinpoint the affected gene. The detection of a mutation in one of the responsible mismatch repair gene confirmed the diagnosis HNPCC. Here we demonstrate our experience of some important pitfalls that will be discussed in this study. In MSI testing, one potential source for false-negative results is intralesional heterogeneity. We demonstrate examples of a flat adenoma and a carcinoma, which required laser microdissection to correctly determine the microsatellite status. In these lesions manual microdissection, the most frequently applied method, was not sufficient. However, the number of cells obtained by using laser microdisssection can fall below a necessary minimum, which can also cause false-negative results of MSI analysis, as shown here in a mucinous carcinoma. In addition, evaluation of immunohistochemically stained tissue slides requires experience to avoid false-positive or false-negative interpretation. A case with two synchronous colorectal cancers revealed loss of MSH2 expression in one carcinoma, whereas the second carcinoma stained positively leading to a false-negative interpretation. In some cases, false-positive results can be obtained, if a perinuclear-staining pattern is interpreted as positive. In summary, there are several potential pitfalls in the molecular screening for HNPCC. Therefore the importance of correct interpretation of clinical data, immunohistochemistry, and microsatellite analysis in combination, performed by a pathologist with experience in molecular genetics is essential. In addition, laser microdissection of tumor areas that have been chosen by a pathologist is highly recommended in cases that cannot be resolved with manual microdissection.
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Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Repetições de Microssatélites , Adenoma/diagnóstico , Adenoma/genética , Biomarcadores Tumorais/biossíntese , Carcinoma/diagnóstico , Carcinoma/genética , Diferenciação Celular , Núcleo Celular/metabolismo , DNA de Neoplasias/análise , Proteínas de Ligação a DNA/metabolismo , Reações Falso-Negativas , Feminino , Humanos , Imuno-Histoquímica , Lasers , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Proteína 2 Homóloga a MutS , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Muir-Torre syndrome (MTS) is a rare autosomal-dominant disorder characterized by the predisposition to both sebaceous skin tumors (or multiple keratoacanthomas) and internal malignancies. A subtype of MTS is allelic to hereditary nonpolyposis colorectal cancer and is caused by germline mutations in the DNA mismatch repair genes MSH2 or MLH1. In these cases both internal and skin tumors show characteristic microsatellite instability (MSI). The aim of the present study was to determine whether immunohistochemical examination of MSH2 or MLH1 protein expression in MTS-associated skin tumors can be used as a diagnostic screening tool to identify patients with germline mutations in MSH2 or MLH1. In the present study 28 skin lesions from 17 patients (20 sebaceous gland tumors, 4 sebaceous hyperplasias, 3 keratoacanthomas, and 1 squamous cell carcinoma) were tested immunohistochemically with antibodies against MSH2 and MLH1. Eighteen of these tumors were from eight patients with known MSH2 germline mutations, two tumors were from a patient with a germline mutation in MLH1, and eight microsatellite stable sporadic skin tumors served as controls. One sample had to be excluded because of a lack of immunoreactivity. All eight microsatellite stable tumors expressed both DNA repair proteins. In 15 of the tumors from MSH2 germline mutation carriers, loss of MSH2 expression was observed, one tumor showed reduced MSH2 expression, and one tumor displayed positive immunoreactivity to MSH2. Both tumors of the MLH1 germline mutation carrier showed loss of the MLH1 protein. In conclusion, our findings demonstrate that immunohistochemical testing of MTS-related skin tumors is a reliable screening method with high predictive value for the diagnosis of the DNA mismatch repair-deficient MTS.
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Pareamento Incorreto de Bases , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Reparo do DNA , DNA de Neoplasias/genética , Proteínas de Ligação a DNA , Mutação em Linhagem Germinativa , Proteínas de Neoplasias/análise , Proteínas Proto-Oncogênicas/análise , Neoplasias das Glândulas Sebáceas/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Humanos , Imuno-Histoquímica , Repetições de Microssatélites , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Proteínas Nucleares , Proteínas Proto-Oncogênicas/genética , Neoplasias das Glândulas Sebáceas/patologiaRESUMO
Microsatellite instability (MSI) is a characteristic feature of up to 15% of colorectal cancers (CRC) and is associated with better response to adjuvant chemotherapy with 5-fluorouracil (5-FU). In this study we have investigated the association between the MSI status and the mRNA expression as well as the polymorphisms of the cellular target of 5-FU therapy, thymidylate synthase. Polymorphisms in the 3'- and the 5'-UTR of the TS gene were determined by a PCR assay in 53 colorectal cancer tissues. TS mRNA was quantified by real-time RT-PCR. Data were correlated with the MSI phenotype. There was neither a significant correlation between the polymorphisms in the TS gene and the MSI phenotype nor between the mRNA expression and MSI status. CRC with a 3R/3R or 2R/3R genotype showed a significantly higher TS mRNA expression than those with 2R/2R genotype (p=0.001 and p=0.026, respectively). No association was seen between the polymorphism of the 3'-UTR and mRNA expression. From our results, we conclude that there is no association between MSI status and TS expression. Samples containing the 3R/3R or 2R/3R genotype of TS seem to have higher mRNA levels perhaps due to a higher mRNA stability. Polymorphic variants of the 3'-UTR do not influence the TS mRNA level. Genotyping of the 5'-UTR and quantitation of TS mRNA levels might serve as predictors for the response to 5-FU based chemotherapy.
Assuntos
Neoplasias Colorretais/genética , Polimorfismo Genético , Timidilato Sintase/genética , Regiões 5' não Traduzidas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Quimioterapia Adjuvante , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/metabolismo , Feminino , Fluoruracila/uso terapêutico , Expressão Gênica , Humanos , Masculino , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Timidilato Sintase/metabolismoRESUMO
BACKGROUND: The purpose of this single-center study was to determine the practicability of the intra-operative use of one-step nucleic acid amplification (OSNA) as the only method for detection of SLN. The OSNA system has been well described and is supposed to be as accurate as conventional histology. METHODS: Three hundred and thirty SLNs from 143 breast cancer patients were analyzed in an intra-operative setting. The CK19-copy number was determined by OSNA and divided into 3 results ("-" no metastasis; "+" micrometastasis; "++" marcometastasis). If OSNA gave a positive result, an axillary lymph node dissection was carried out during the same session. The central 1-mm slice of each node was obtained for permanent histology. Additionally, the results were correlated to clinicopathological factors, and the time for the intra-operative use was evaluated. RESULTS: Thirty-nine of the 143 patients were OSNA positive, 22 with macrometastatic and 17 with micrometastatic spread. The mean time for the OSNA run with one SLN was 34.4 min. We could show a correlation between the tumor size and OSNA positivity as well as between the numbers of OSNA positive SLNs with the tumor load of associated non-SLNs. Furthermore, we found that a cutoff CK19 copy number of 7,900/µL indicates a positive non-SLN result with the highest sensitivity (91 %) and specificity (61 %). CONCLUSION: We found OSNA to be very helpful for the intra-operative determination of the tumor load of a SLN as a basis for decision-making concerning further surgical axillary intervention. OSNA allows precise differentiation of micro- from macrometastasis, and the CK19 copy number predicts the probability of tumor load in other axillary lymph nodes and might help to find adequate adjuvant treatment options. This objective method is well suitable for everyday use and may reduce the pathologic workload and the risk of secondary operative interventions with all associated costs and stress for the patients.
Assuntos
Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/secundário , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Adulto , Idoso , Idoso de 80 Anos ou mais , Axila , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/cirurgia , Carcinoma Ductal de Mama/diagnóstico , Carcinoma Ductal de Mama/cirurgia , Feminino , Humanos , Período Intraoperatório , Queratina-19/genética , Linfonodos/patologia , Metástase Linfática , Pessoa de Meia-Idade , Micrometástase de Neoplasia , Kit de Reagentes para Diagnóstico , Carga TumoralRESUMO
Microsatellite analysis (MSA) in tumour tissue is useful for pre-selection of hereditary non-polyposis colorectal cancer (HNPCC) patients for mutation screening, but is time-consuming and cost-intensive. Immunohistochemistry (IHC) for expression of MLH1 and MSH2 proteins is simple, fast, and indicates the affected gene. IHC has therefore been proposed as an alternative pre-screening method. However, some authors report a lower sensitivity of IHC compared with MSA. The present study reports IHC results for MSH2 and MLH1 performed in 82 tumours with high microsatellite instability (MSI-H) from 81 carriers of pathogenic mutations in MSH2 or MLH1. One hundred per cent (38/38) of the tumours from MSH2 mutation carriers showed loss of MSH2 staining; in all cases, the affected MSH2 gene was predicted correctly by IHC. Complete loss of MLH1 expression was observed in 66% (29/44) of MLH1 mutation carriers. Weak positive MLH1 staining was observed in 14 (32%) cases and, in one case, normal MLH1 staining was seen. The pathologist was aware of the weak staining pattern as an indicator of an MLH1 mutation; 98% of the MLH1 mutations were predicted correctly. To evaluate whether weak positive MLH1 staining is observed more often with in-frame or missense mutations, IHC data from 23 MSI-H tumours from carriers of unspecified variants were added and mutations were grouped into truncating mutations, large non-truncating deletions, and small non-truncating mutations. Weak MLH1 staining was observed in all three categories and it is postulated that other factors, such as mutation of the second allele, also influence protein expression. In conclusion, IHC can be regarded as a very useful method for selecting HNPCC patients for mutation analysis, as long as it is interpreted by an experienced pathologist. The high specificity of IHC in terms of indicating the affected gene is useful for evaluating unspecified variants. However, the staining pattern does not predict whether the underlying germ-line mutation is truncating or not.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Mutação em Linhagem Germinativa , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/genética , Proteínas Adaptadoras de Transdução de Sinal , Biomarcadores Tumorais/genética , Proteínas de Transporte/metabolismo , Neoplasias Colorretais Hereditárias sem Polipose/metabolismo , Análise Mutacional de DNA , Heterozigoto , Humanos , Repetições de Microssatélites , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismoRESUMO
Mutations in DNA MMR genes, mainly MSH2 and MLH1, account for the majority of HNPCC, an autosomal dominant predisposition to colorectal cancer and other malignancies. The evaluation of many questions regarding HNPCC requires clinically and genetically well-characterized HNPCC patient cohorts of reasonable size. One main focus of this multicenter study is the evaluation of the mutation spectrum and mutation frequencies in a large HNPCC cohort in Germany; 1,721 unrelated patients, mainly of German descent, who met the Bethesda criteria were included in the study. In tumor samples of 1,377 patients, microsatellite analysis was successfully performed and the results were applied to select patients eligible for mutation analysis. In the patients meeting the strict Amsterdam criteria (AC) for HNPCC, 72% of the tumors exhibited high microsatellite instability (MSI-H) while only 37% of the tumors from patients fulfilling the less stringent criteria showed MSI-H; 454 index patients (406 MSI-H and 48 meeting the AC of whom no tumor samples were available) were screened for small mutations. In 134 index patients, a pathogenic MSH2 mutation, and in 118 patients, a pathogenic MLH1 mutation was identified (overall detection rate for pathogenic mutations 56%). One hundred sixty distinct mutations were detected, of which 86 are novel mutations. Noteworthy is that 2 mutations were over-represented in our patient series: MSH2,c.942+3A>T and MLH1,c.1489_1490insC, which account for 11% and 18% of the MSH2 and MLH1 mutations, respectively. A subset of 238 patients was screened for large genomic deletions. In 24 (10%) patients, a deletion was found. In 72 patients, only unspecified variants were found. Our findings demonstrate that preselection by microsatellite analysis substantially raises mutation detection rates in patients not meeting the AC. As a mutation detection strategy for German HNPCC patients, we recommend to start with screening for large genomic deletions and to continue by screening for common mutations in exon 5 of MSH2 and exon 13 of MLH1 before searching for small mutations in the remaining exons.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Proteínas de Ligação a DNA/genética , Mutação em Linhagem Germinativa , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Bases , Proteínas de Transporte , Humanos , Repetições de Microssatélites , Dados de Sequência Molecular , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutSRESUMO
Hereditary nonpolyposis colorectal cancer (HNPCC) is often caused by a deficiency in DNA mismatch repair. By using conventional methods of mutation analysis, point mutations in the DNA mismatch repair genes MSH2 and MLH1 have been detected in up to 64% of patients suspected of HNPCC. However, large genomic deletions cannot be detected by these methods. In our study, we applied a semiquantitative multiplex PCR to detect the proportion of large deletions in patients meeting the Bethesda criteria whose tumours exhibited microsatellite instability (MSI). Of 368 unrelated patients, 180 exhibited MSI. In these patients, 68 disease-causing point mutations (38%) had previously been detected in the MSH2 and MLH1 genes by SSCP, heteroduplex analysis or DHPLC followed by direct sequencing. The remaining 112 patients (including 24 patients with rare missense or other unclarified variants) were examined for large deletions. We identified deletions in 19 patients (10.6%); 11/19 (58%) deletions were located in MSH2 and 8/19 (42%) in MLH1, respectively. The size of deletions ranged from 1 exon to a deletion of a whole gene. Five breakpoints of deletions were sequenced; Alu-repetitive elements were involved in all of them. In patients meeting the Amsterdam criteria the proportion of large deletions was 12.6%. A similar proportion of deletions was found in the group of patients with a positive family history for colorectal cancer and MSI tumours, not meeting the Amsterdam criteria. The results of our study suggest that large genomic deletions in both MSH2 and MLH1 genes play a considerable role in the pathogenesis of HNPCC and should be part of the routine HNPCC mutation detection protocols.