Giant cell tumor of bone. Chromosomal analysis of 48 specimens and review of the literature.

Giant cell tumor of bone (GCT) is a distinct clinical, radiographic, and pathologic benign entity that constitutes 5% of all primary bone tumors. For a 5-year period, 47 benign GCTs and 1 malignant GCT from 34 different patients were cytogenetically characterized. Analysis showed clonal karyotypic abnormalities in 16 specimens. Clonal structural abnormalities detected in more than one patient included translocations involving 11p15, fus(14p;21p), and fus(15p;21p). None of the clonal numerical abnormalities observed occurred in more than one patient. Thirty-seven of the 44 successfully analyzed specimens (84%) demonstrated telomeric fusion, with most frequent involvement of chromosomal telomeres 11p, 13p, 15p, 18p, 19p, and 21p. We also compared the presence or absence of random and/or clonal karyotypic abnormalities with clinical behavior to determine if a relationship existed. Most notably, chromosomal abnormalities were detected in all 13 successfully analyzed recurrent lesions, five of which were clonally aberrant. This study summarizes the cytogenetic findings and their relevance in 48 specimens analyzed at our institution and reviews the findings of the 18 other published cases.     

Plexiform fibrohistiocytic tumor. Report of a case involving preoperative aspiration cytology and immunohistochemical and ultrastructural analysis of surgical specimens.

A typical case of plexiform fibrohistiocytic tumor (Enzinger and Zhang) occurring in the skin and subcutis of the abdominal wall in a 7-year-old girl is reported. Preoperative fine-needle aspiration cytology revealed a benign lesion with fibroblastic-histiocytic features which also contained bi- and multinucleated giant cells. The surgical specimen showed a tumor with multiple small nodules within fibrous septa; these nodules were composed of spindle cells and epithelioid cells and contained scattered multinucleated osteoclast-like cells. The tumor cells showed ultrastructural and immunohistochemical features of myofibroblasts and histiocyte-like cells. Thus, there was an abundance of lysosomes, prominent filopodia and bundles of thin cytofilaments along the cytoplasmic border, as well as immunoreactivity for alpha-smooth-muscle-specific actin, alpha-1-antitrypsin and alpha-1-antichymotrypsin. Ultrastructurally there were tumor cells exhibiting features of histiocytes which also contained bundles of actin of smooth muscle type. The presented case of plexiform fibrohistiocytic tumor appears to be composed of a rather peculiar cell form, somewhere between myofibroblasts and histiocytes.     

Currarino syndrome with pelvic neuroendocrine tumor diagnosed by post-mortem genetic analysis of tissue specimens

Currarino syndrome (CS) is an autosomal dominant disorder of embryonic development characterized by the triad of anorectal abnormalities, partial sacral agenesis, and presacral mass. Mutations of the HLXB9 gene have been identified in most CS cases, but a precise genotype-phenotype correlation has not been described so far. We report the clinical case of a 44-year-old Caucasian woman with malignant neuroendocrine transformation of a pre-sacrococcygeal mass combined with bicornuate uterus, dermoid cyst of the ovaries, and chronic constipation. After the patient died, a sacrococcygeal malformation and anterior meningocele were diagnosed in her 22-year-old son. CS diagnosis was then retrospectively confirmed by molecular analysis of normal and pathological tissue specimens of the mother, with identification of a HLXB9 mutation (c.727C>T; p.R243W). CS should be considered, and genetic counseling recommended, to all patients with presacral masses. Since malignant neuroendocrine transformation of presacral mass in CS is a possible complication, even thought rare, close follow up in these patients is advisable.     

Real-time quantitative analysis of telomerase activity in breast tumor specimens using a highly specific and sensitive fluorescent-based assay.

Telomerase activity has been associated with almost 90% of malignant human cancers from a variety of tissue sources, making it one of the most prominent molecular cancer markers known to date. As such, telomerase has become a very attractive diagnostic and therapeutic target. The advent of the telomeric repeat amplification protocol (TRAP) has allowed for the semiquantitative detection of telomerase from limiting sample amounts. Both the standard TRAP assay and a real-time assay using Amplifluor technology with primers designed specifically for telomerase activity amplification were used to quantitatively assess telomerase activity in primary tumors and tumor-derived cell lines. We have adapted the recently developed TRAPeze XL telomerase detection kit (Intergen, Gaithersburg, MD) for use with real-time polymerase chain reaction for more accurate quantification and high-throughput capabilities. In doing so, the reliability, assay time, and accuracy of quantitation have all been dramatically improved. A comparison of the quantitative analysis for the standard TRAP assay versus the real-time assay using 19 breast tumors revealed telomerase quantitation and standardization using the real-time assay was superior to the standard assay. Our data suggest that this assay will be useful for clinical and research studies involving detection of telomerase activity as it relates to cancer diagnosis.     

人源化肿瘤组织异种移植的进展

免疫缺陷动物模型用于对肿瘤机制和治疗的研究,并且普遍应用于临床前。但物种特异性差异阻碍了实验结果的可靠性,物种的特异性差异导致临床治疗失败率超过80%,在评估抗癌治疗的试验中成功率也低于10%。所以人源化肿瘤标本异种移植模型(PDX)能够尽可能的模拟人体内肿瘤生长的微环境,为肿瘤药物的安全性与有效性奠定基础。但PDX肿瘤模型也有自身的局限性,比如无法体现出肿瘤生长个体化的差异性,所以双人源化小鼠肿瘤模型(Hu-PDX模型)弥补了这项缺憾。本文总结了PDX模型发展史,PDX模型特征和优缺点,PDX模型的类型,以及最新进展(Hu-PDX模型)。     

Identification of tumor specimens by DNA analysis in a case of histocytological paraffin tissue block swapping

We report on a patient who was diagnosed with high-grade breast carcinoma by all the pre-surgery clinical evidence of malignancy, but histopathological reports did not reveal any such tumor residue in the post-surgical tissue block. This raised a suspicion that either exchange of block, labeling error, or a technical error took place during gross examination of the tissue. The mastectomy residue was unprocurable to sort out the problem. So, two doubtful paraffin blocks were sent for DNA fingerprinting analysis. The partial DNA profiles (8-9/15 loci) were obtained from histocytological blocks. The random matching probability for both the paraffin blocks and the patient's blood were found to be 1 in 4.43E4, 1.89E6, and 8.83E13, respectively for Asian population. Multiplex short tandem repeat analysis applied in this case determined that the cause of tumor absence was an error in gross examination of the post-surgical tissue. Moreover, the analysis helped in justifying the therapy given to the patient. Thus, with DNA fingerprinting technique, it was concluded that there was no exchange of the blocks between the two patients operated on the same day and the treatment given to the concerned patient was in the right direction.     

Fluorescent in situ hybridization on isolated tumor cell nuclei: a sensitive method for 1p and 19q deletion analysis in paraffin-embedded oligodendroglial tumor specimens.

In oligodendroglial neoplasms, losses of chromosomal material at 1p and 19q associate with chemosensitivity and prolonged survival. Thus, 1p/19q testing is increasingly proposed for use in brain tumor diagnosis and prognostic assessment. Fluorescent in situ hybridization (FISH) is a classic technique for investigation of 1p/19q status in paraffin-embedded tissues. A major limitation of this method is truncation of tumor cell nuclei complicating assessment of hybridization results. In our study, we analyzed 1p and 19q status in a series of 79 oligodendroglial neoplasms (49 oligodendrogliomas, 30 oligoastrocytomas, WHO: 57 Grade II, 22 Grade III tumors) and controls (gliotic brain tissue: n = 4, diffuse low-grade astrocytoma: n = 4) using FISH on isolated whole tumor cell nuclei, prepared as cytospin preparations, thus bypassing the problem of nuclear truncation. For interpretation of FISH results, we used consensus criteria as defined by the SIOP-Europe Neuroblastoma Study Group for analysis of peripheral neuroblastic tumors. FISH yielded interpretable results in 98.7% for 1p and 92.1% for 19q. Chromosome 1p/19q alterations comprised deletions (1p: 79.5%, 19q: 80%) and imbalances (1p: 11.5%, 19q: 12.9%). 1p aberrations were more frequent in oligodendroglioma than in oligoastrocytoma (100% versus 75.9%, P =.001). The frequency of 1p/19q alterations was not significantly different in WHO Grade II or Grade III tumors or in primary and recurrent tumors. We conclude that FISH on isolated cell nuclei, with application of the SIOP Europe Neuroblastoma consensus criteria, is a sensitive method for detection and interpretation of 1p and 19q aberrations in paraffin-embedded tissue specimens of oligodendroglial neoplasms.     

Routine analysis of p53 mutation in clinical breast tumor specimens using fluorescence-based polymerase chain reaction and single strand conformation polymorphism.

Improved prognostic and predictive markers in breast cancer management would help considerably in therapeutic decision making, particularly in patients with early-stage breast cancer. Tumor factors currently used for prognostication and management decisions are tumor size, histologic type and grade, axillary lymph node status, and estrogen receptor content. The discovery of various somatic genetic alterations in breast cancer has raised the possibility that these may provide additional and independent prognostic and predictive information. Alterations of the p53 tumor suppressor gene in particular have received the most attention as potential prognostic and predictive factors. In multivariate analysis, p53 gene mutation is consistently associated with a two- to threefold increased risk of relapse and death from breast cancer. One of the major reasons preventing the introduction of p53 mutation as a routine marker to assist in therapeutic decision making is the lack of a simple, reproducible, and inexpensive assay. In the present study the authors optimized a polymerase chain reaction-based mutation screening method, fluorescence-single strand conformation polymorphism (F-SSCP), that allows p53 status to be assessed accurately and reproducibly in routinely handled, formalin-fixed and paraffin-embedded tumor specimens. The frequency of p53 mutation observed using F-SSCP in a consecutive series of invasive ductal breast carcinomas was 17% (28/164). The authors propose that the prognostic and predictive values of p53 mutation in breast cancer should be further evaluated in prospective, randomized studies using this standardized technique.     

A new approach to enzyme histochemical analysis of biopsy specimens.

A novel technique combining the freeze drying and embedding in glycol methacrylate at low temperature of tissue permitted the histochemical demonstration of a variety of enzymes, showing maintenance of enzyme activity, accurate enzyme localisation without apparent diffusion, and excellent morphological detail. The results obtained with this new approach were superior to standard techniques used for both enzyme histochemical and morphological studies. Moreover, blocks of the embedded tissue were stored for at least one year at room temperature without loss of enzyme activity. This method should find a wide range of applications in histopathology.     

Molecular epidemiologic analysis of Staphylococcus aureus isolated from clinical specimens.

Nosocomial infections caused by Staphylococcus aureus are clinically serious and control of such infections requires strain typing to identify the source of contamination. Recently, pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) assay have been introduced and have provided a high level of strain discrimination of S. aureus isolated from clinical specimens. This study was performed to classify 82 strains of S. aureus isolated from 4 hospitals in the Kwangju-Chonnam area by PFGE and RAPD assay. Methicillin-resistant S. aureus (MRSA) was identified by disk diffusion method using the oxacillin disk and polymerase chain reaction of mecA gene was done in 69 strains. Eight-three strains including S. aureus ATCC 25923 were classified into 10 groups by RAPD assay, and into 8 groups by PFGE. Classified groups were not related to area or hospital. Classification was not characteristic between MRSA and methicillin-susceptible strains. Nosocomial infections due to outbreak were suggested because some strains disclosed identical band patterns by PFGE. These results indicate that medical personnels and instruments are routes of nosocomial infections caused by MRSA. PFGE and RAPD assay are powerful tools for the epidemiological study of S. aureus, but PFGE is more effective than RAPD assay. RAPD assay needs optimal combination of primers.     

Detection of loss of heterozygosity in formalin-fixed paraffin-embedded tumor specimens by the polymerase chain reaction.

A polymerase chain reaction-based procedure was used for the detection of DNA length polymorphisms generated by naturally occurring genetic deletions or insertions of known sequence. This method consists of a simple one-step assay that does not require any restriction enzyme analysis or Southern blot hybridization, allowing identification in ethidium bromide-stained gels. The procedure described here was used to detect loss of heterozygosity at various loci, including the Hbb beta-globin gene cluster, in chemically induced mouse skin tumors, using a variety of tissue preparations, including microdissection of formalin-fixed, paraffin-embedded specimens, short-term cultures, and fluorescence-activated cell sorting of epithelial populations. This approach may be useful in detecting tumor-specific reduction to homozygosity at polymorphic chromosomal loci, allowing the mapping of putative tumor-suppressor loci involved in carcinogenesis.     

Improved sensitivity and resolution in the flow cytometric DNA analysis of human solid tumor specimens. Use of in vitro fine-needle aspiration and uniform staining reagents.

Twenty-five unselected fresh colon or breast tumors were studied to identify specific components of sample preparation, sample staining, and flow cytometer operation affecting the sensitivity of DNA stemline analysis. Solid tumors were disaggregated using both a published method for mechanical/enzymatic whole cell (M/EWC) isolation and a fine-needle aspiration (FNA) technique. Staining FNA samples with CycleTEST propidium iodide reagents demonstrated improved sensitivity in the recognition of near diploid and near tetraploid aneuploid populations: 9 of 20 resolvable aneuploid DNA stemlines identified in FNA suspensions were not detected or clearly resolved in M/EWC preparations. These results suggest that previously reported discordances between flow and static image cytometry in the recognition of near tetraploid DNA stemlines may be related to inherent limitations of M/EWC. The in vitro FNA technique, when compared to M/EWC, may yield increased sensitivity and precision in clinical DNA stemline analysis when using fresh, unfixed, solid tumor specimens.     

Challenging testicular germ cell tumor diagnoses in post-chemotherapy resection specimens

Chemotherapy is often utilized in the treatment of patients with metastatic testicular germ cell tumors. However, assessment of post-chemotherapeutic specimens in this setting, mostly in the form of retroperitoneal lymph node dissection, can be challenging. This review will address common challenges with interpretation of post-chemotherapy resections, including florid reactions that mimic persistent tumor, the common over-interpretation of atypia in teratomatous elements, and almost unique neoplastic entities in this context (yolk sac tumor variants, rare trophoblastic tumors, and secondary somatic-type malignancies).     

Granular cell tumor. Immunohistochemical analysis of 21 benign tumors and one malignant tumor.

We examined the immunohistochemical profile of 21 granular cell tumors (GCTs) and a single clinically malignant GCT using a panel of commercially available antibodies. All cases showed diffuse cytoplasmic and nuclear staining for S100 protein. Fourteen cases stained for myelin basic protein, Leu-7, or both. Immunostains for neurofilament protein and glial fibrillary acidic protein were negative in all cases. Stains for cathepsin B and alpha 1-antichymotrypsin were positive in 21 and 15 cases, respectively. Cathepsin-B reactivity may reflect autodigestion of myelin, while the presence of alpha 1-antichymotrypsin is less specific and may be related to cellular production of this product or to nonspecific uptake of alpha 1-antichymotrypsin in serum during the formation of phagolysosomes. All tumors expressed vimentin, often in a distinctive peripheral cytoplasmic pattern. Focal desmin staining was seen in three separate specimens from the patients with the malignant GCT, but this tumor also expressed S100 protein, myelin basic protein, and Leu-7 and did not stain for muscle-specific actin. The desmin reactivity in this single case probably represents non-specific staining rather than myogenous differentiation, since the reactivity to other nerve sheath markers shows histogenetic similarity with the benign GCTs. These findings support a Schwann cell origin for nongingival GCTs and illustrate a useful panel of commercially available antibodies to diagnose these distinctive tumors.     

Giardiasis: analysis of histological changes in biopsy specimens of 80 patients.

Histological changes in duodenal biopsy specimens from 80 patients infected with Giardia lamblia were defined and compared with changes in duodenal biopsy specimens from 80 randomly chosen "healthy" patients. Villous architecture, the number of intraepithelial lymphocytes, the occurrence of lymphoid follicles and the density of colonisation by G lamblia trophozoites were examined. Grade 1 villous flattening was observed in 41% of cases positive for Giardia and in 37.5% of the control cases. The mean content of intraepithelial lymphocytes was 24.5 compared with 22.6 intraepithelial lymphocytes/100 epithelial cells in the positive and negative groups, respectively. Lymphoid follicles were more frequently observed in Giardia positive specimens, but their incidence was not significantly higher compared with that of controls. Twenty five per cent of patients positive for Giardia showed light, 41% intermediate, and 34% heavy colonisation by Giardia trophozoites. In 76% the parasites were observed in all specimens obtained. It is concluded that histological changes induced by G lamblia are not specific, and that two duodenal biopsy specimens might suffice to detect the trophozoites.     

A novel approach for reliable microarray analysis of microdissected tumor cells from formalin-fixed and paraffin-embedded colorectal cancer resection specimens

We present a novel approach for microarray analysis of RNA derived from microdissected cells of routinely formalin-fixed and paraffin-embedded (FFPE) cancer resection specimens. Subsequent to RNA sample preparation and hybridization to standard GeneChips (Affymetrix), RNA samples yielded 36.43 ± 9.60% (FFPE), 49.90 ± 4.43% (fresh-frozen), and 53.9% (cell line) present calls. Quality control parameters and Q-RT-PCR validation demonstrated reliability of results. Microarray datasets of FFPE samples were informative and comparable to those of fresh-frozen samples. A systematic measurement difference of differentially processed tissues was eliminated by a correction step for comparative unsupervised data analysis of fresh-frozen and FFPE samples. Within FFPE samples, unsupervised clustering analyses clearly distinguished between normal and malignant tissues as well as to further separate tumor samples according to histological World Health Organization (WHO) subtypes. In summary, our approach represents a major step towards integration of microarrays into retrospective studies and enables further investigation of the relevance of microarray analysis for clinico-pathological diagnostics. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Transport of viral specimens.

The diagnosis of viral infections by culture relies on the collection of proper specimens, proper care to protect the virus in the specimens from environmental damage, and use of an adequate transport system to maintain virus activity. Collection of specimens with swabs that are toxic to either virus or cell culture should be avoided. A variety of transport media have been formulated, beginning with early bacteriological transport media. Certain swab-tube combinations have proven to be both effective and convenient. Of the liquid transport media, sucrose-based and broth-based media appear to be the most widely accepted and used. Studies on virus stability show that most viruses tested are sufficiently stable in transport media to withstand a transport time of 1 to 3 days. Some viruses may withstand longer transport times. In many cases, it is not necessary to store virus specimens in a refrigerator or send them to the laboratory on wet ice or frozen on dry ice. However, the specimen should not be exposed to environmental extremes. Modern viral transport media allow for more effective use of viral culture and culture enhancement techniques for the diagnosis of human viral infections.     

Line-focusing x-ray monochromator for the analysis of trace elements in biological specimens.

A line-focusing x-ray monochromator has been designed and developed to be used with an energy dispersive detection system for the quantitative analysis of trace elements in biological specimens. This instrument uses a cylindrically curved Johansson-type crystal to monochromatize and focus the excitation radiation into a line 0.5 mm wide and 40 mm long. The excitation radiation chosen for these experiments was Mo Kalpha and the total intensity of the line-focused beam was estimated to be 2 X 10(7) cps. Because of the narrow width and high power density of the excitation between beam, small tissue specimens 1 X 1 X 12 mm in size along with a single strand of hair were analyzed for trace elements from Al through Sr. For the transition elements, concentrations of a few parts per million were easily detected with this analyzer.