Her-2/neu evaluation in Sister Mary Joseph's nodule from breast carcinoma: a case report and review of the literature.

Sister Mary Joseph's nodule (SMJN) involving the umbilicus can often be a clinical sign of metastatic cancer, but rarely cancer originating from the breast. We report a rare case of umbilical metastases from breast cancer and reviewed the literature. A 54-year-old woman was referred to a pre-surgery clinic for an examination of an umbilical nodule. The patient had a history of ductal breast carcinoma. Cytological smear from fine needle aspiration showed epithelial neoplastic cells resembling those of breast carcinoma. Neoplastic cells from tissue were positive for cytokeratin 8-18, estrogen and progesterone receptor and negative for E-cadherin and had a low proliferative index. Her-2/neu immunodetection showed a 2+ equivocal positive rate, but Her-2/neu gene amplification was found on the cytological smear by fluorescence in situ hybridization analysis. Similar results were obtained within a tissue section. Concordant findings have been obtained when comparing the recent American Society of Clinical Oncology/College of American Pathologists scoring system. Fine needle aspiration from the SMJN is a useful tool for the diagnosis of metastatic breast cancer. Furthermore, the predictive biomarkers for tumors of the breast, hormonal receptors and Her-2/neu not only assist with the identification of the source of the metastatic disease but also provide clinical information for patient management.

Subepithelial pelvic hematoma (Antopol--Goldman lesion) simulating renal neoplasm: report of a case and review of the literature.

The Antopol-Goldman lesion is a subepithelial pelvic hematoma simulating a renal neoplasm. We report the clinico-pathological features of a single case and a review of the literature. A 76-year-old man presented with flank pain and hematuria. Computed tomography showed a hypodense lesion of 6 cm at the left kidney with filling defect at pyelogram. The patient underwent nephroureterectomy for suspected neoplasm. Macroscopically, a mass of 6 cm was present impinging on the pelvi-caliceal system. Microscopically, the lesion was composed by hemorragic material with feature of an hematoma. A diffuse eosinophilic amorphous material suspicious for amyloid was observed among intra- and extraparenchymal vessels. The Congo-Red staining verified the presence of amyloid. The diagnosis was subepithelial pelvic hematoma with severe amyloidosis. Antopol-Goldman lesion should be kept in mind as a possible differential diagnosis of upper urinary tract lesion to avoid unnecessary nephrectomies. The anamnestic knowledge of amiloydosis may increase this diagnostic hypothesis.

Pulmonary glomus tumor.

Glomus tumor, also known as glomangioma, is a neoplasm derived from cells of the neuromyoarterial glomus or glomus body. We report a case of glomus tumor of the lung arising in the left lower lobe, incidentally found in a patient who underwent right bilobectomy for a carcinoma localized in the right upper lobe.

Periorbital subcutaneous tumor-like lesion due to Dirofilaria repens.

Dirofilariasis is a zoonotic infection, which is occasionally seen in humans and rarely found as a subcutaneous orbital swelling. The authors report a case of a 62-year-old woman presented with a 3-month history of a right periorbital subcutaneous nodule. Treatment with antibiotics and corticosteroids was not satisfactory. Magnetic resonance imaging analysis showed a nodule with a central colliquative area. The lesion displaced the eyeball superiorly but did not affect the intraorbital muscles. The patient was subjected to excisional biopsy and the nodule measured 15 mm. Histological findings showed microabscess reaction with heterogeneous lymphoid infiltration. Additional consecutive sections finally showed Dirofilaria repens, curled up in spirals with external cuticular ridges in an environment characterized by epithelioid cells. The lesion did not recur for 5 months. Periorbital swelling can be rarely caused by Dirofilaria repens; therefore, this diagnosis should be considered in all cases of subcutaneous inflammatory or tumor-like lesion of unknown etiology.

Primary pleomorphic rhabdomyosarcoma of the kidney in an adult.

Sarcoma of the kidney is uncommon and represents between 1% and 3% of all malignant renal tumors. Primary rhabdomyosarcoma of the kidney in adult age is unusual, and only sporadic cases have been reported. This is a very aggressive tumor with dismal prognosis. We report a new case of pleomorphic rhabdomyosarcoma of the kidney in an adult patient.

Microfluidic deletion/insertion analysis for rapid screening of KIT and PDGFRA mutations in CD117-positive gastrointestinal stromal tumors: diagnostic applications and report of a new KIT mutation.

Gastrointestinal stromal tumors (GISTs) frequently harbor mutations in the KIT and PDGFRA genes, the presence and type of which correlate with the response to the kinase inhibitor imatinib mesylate. Because most GIST mutations are deletions/insertions, we used a microfluidic apparatus to detect these size variations in polymerase chain reaction-amplified DNA. This approach, termed microfluidic deletion/insertion analysis (MIDIA), identified mutations in 30 of 50 DNA samples from paraffin-embedded CD117-positive GISTs (60%), comprising 25 deletions and five insertions. Sequencing of 14 MIDIA-positive samples confirmed the deletions/insertions, including two 3-bp alterations. Sequencing of all 20 MIDIA-negative samples also showed highly consistent results with MIDIA because 10 cases were wild type and eight displayed a single base substitution in which detection by MIDIA was not expected. Sequencing also revealed a 3-bp deletion undetected by MIDIA, thus establishing the resolution limit of MIDIA at deletions/insertions >or=3 bp. Denaturing high-pressure liquid chromatography analysis confirmed all mutations detected by MIDIA and sequencing. We pro-pose MIDIA as the first step in mutational screening of GIST because it allowed the detection of 75% of mutated cases (94% of deletions/insertions) in less than 30 minutes after polymerase chain reaction amplification and at a lower cost compared with denaturing high-pressure liquid chromatography and sequencing, which might then be used only for MIDIA-negative cases.

Establishment of the MAVER-1 cell line, a model for leukemic and aggressive mantle cell lymphoma.

BACKGROUND AND OBJECTIVES: Mantle cell lymphoma (MCL) cell lines are difficult to generate; only nine lines have been described so far and few of them have been thoroughly characterized. DESIGN AND METHODS: We established MAVER-1, a new MCL cell line, obtained from a leukemic MCL harboring both a t(11;14) translocation and a MYC rearrangement, and used immunohistochemistry, flow cytometry, molecular biology and cytogenetic techniques in order to characterize the cell line precisely. RESULTS: By immunohistochemistry and flow cytometry MAVER-1 displayed a classical MCL phenotype (IgM+, l+, CD5+, CD10-, CD19+, CD20+, CD23-, CD79a+, cyclin D1+) and genetic analysis showed a typical V/D/J rearrangement with naïve mutational status. According to both classic cytogenetic analysis and spectral karyotyping, MAVER-1 harbored the t(11;14) translocation associated with a complex karyotype. Molecular analysis by polymerase chain reactions showed that the t(11;14) breakpoint is within the major translocation cluster. Other important abnormalities of MAVER-1 include TP53 gene inactivation by a combined mutation of exon 8 and chromosome 17p13 deletion, ATM deletion, 8q24 (MYC) rearrangement and 8p22 deletion. INTERPRETATION AND CONCLUSIONS: The new cell line will be useful for in vitro studies regarding MCL pathogenesis and drug sensitivity, as well as a diagnostic control material.

Molecular characterization of composite mantle cell and follicular lymphoma.

Composite lymphomas are rare associations of two distinct lymphoma types at the same anatomical site. Reporting of such cases is important because they pose major biologic, diagnostic, and therapeutic dilemmas. In this study, we describe the third reported case of mantle cell and follicular lymphoma. We performed accurate immunohistochemical and molecular studies to define the mono- vs biclonal nature of this neoplasm. We used both manual and LASER-capture microdissection combined to multiple molecular approaches for clonality determination, including detection of heavy and light chain recombination, as well as presence of kappa/Kde recombination and sequence analysis. By immunohistochemistry and FISH, we confirmed the presence of two distinct lymphoma types characterized by specific translocations [namely, t(11;14) and t(14;18)], while we demonstrated two distinct and not clonally related cell populations by molecular techniques. The light chain approach, and particularly the kappa and kappa/Kde recombination detection, proved very useful for solving the clonality issue.

Primary mediastinal B-cell lymphoma: hypermutation of the BCL6 gene targets motifs different from those in diffuse large B-cell and follicular lymphomas.

BACKGROUND AND OBJECTIVES: Somatic hypermutation of the BCL6 gene and its expression in lymphoma represent specific markers for B-cell transit through the germinal center. Thus, analysis of BCL6 may aid in clarifying the relationship between primary mediastinal B-cell lymphoma (PMBL) and other non-thymic diffuse large cell lymphomas (DLCL). DESIGN AND METHODS: Twenty-four PMBL were analyzed for BCL6 status, including first intron mutations, by quantitative reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry. We also performed a meta-analysis of reported BCL6 mutations in PMBL (n=141), DLCL (n=233), and follicular lymphoma (n=120). RESULTS: Thirteen PMBL (54%) showed hypermutation of BCL6. All cases showed bcl6 mRNA and immunohistochemical expression. Meta-analysis demonstrated that the preferentially altered sequence motifs of BCL6 in PMBL were TA (p=0.002) and AT (p=0.0008) dinucleotides and TAT trinucleotides (p=0.001). GC and RGYW/WRCY motifs were a target in DLCL and FL but not in PMBL. Moreover, the DNA stretch spanning nucleotides 150-270 was highly targeted only in PMBL. INTERPRETATION AND CONCLUSIONS: The consistent expression of bcl6 protein and occurrence of hypermutation indicate that PMBL should be considered of germinal center origin. The fact that the hypermutation sites and mutational spectrum of BCL6 in PMBL differ from those found in FL and DLCL might suggest that the maturation block of the transforming cells differs among these tumor types, and that the characteristic mutational pattern is present before neoplastic transformation. Thus, our findings strengthen the hypothesis that PMBL originate from an already defined sub-population of B-cells, which are different from those leading to either DLCL or FL.

Constitutive expression of DeltaN-p63alpha isoform in human thymus and thymic epithelial tumours.

p63, a member of the p53 family, is involved in the survival and differentiation of reserve/stem cells in different epithelia. To unveil the possible role of p63 in thymic physiology and pathology, we investigated the expression of p63 isoforms in normal thymus, thymomas and other mediastinal tumours. All samples were analysed using immunohistochemistry with three different antibodies: 4A4 antibody recognising all p63 isoforms, p40 antibody reacting only with truncated dominant-negative isoforms (DeltaN-p63) and H-129 antibody recognising all alpha-isoforms. Reverse-transcription polymerase chain reaction (RT-PCR), and real-time PCR analyses were performed on RNA extracted from frozen samples of four thymomas and two primary-mediastinal large-B-cell lymphoma (PMLBCL). In normal thymus, DeltaN-p63alpha was expressed in all cortical and medullary epithelial cells, with decreasing intensity in Hassall's corpuscles. This phenotype was conserved in neoplastic transformation since all 54 investigated thymomas (World Health Organization types A, AB, B1, B2, B3, C) expressed DeltaN-p63alpha (virtually 100% cells). The predominance of DeltaN-p63alpha isoform mRNA was confirmed by real-time PCR. Among other mediastinal tumours, DeltaN-p63alpha was only expressed in those displaying either a stratified epithelial component (teratomas) or epidermoid differentiation (lung carcinoma). Among lymphomas, T-cell-precursor lymphomas did not express p63, whereas most PMLBCL expressed TA-p63alpha (7/8).

Runx2 mRNA expression in the tissue, serum, and circulating non-hematopoietic cells of patients with thyroid cancer.

CONTEXT: Runx2, a master gene of osteogenic differentiation, is also expressed in nonosseous cancer cells. Microcalcifications are characteristic of papillary thyroid carcinoma and represent a useful find for diagnosis. However, the molecular expression of osteogenic differentiation transcription factor Runx2 has been poorly investigated in this tumor. OBJECTIVE: The aim of this study was to investigate Runx2 mRNA expression in normal and pathological thyroid tissue, serum, and circulating non-hematopoietic cells. SETTING: The study was performed in the Endocrine Unit of Internal Medicine of "Azienda Ospedaliera Universitaria Integrata of Verona" (Verona, Italy). PATIENTS: We enrolled 12 patients with a papillary thyroid carcinoma (PTC), who had undergone total thyroidectomy performed by the same surgeon. The results, obtained by real-time RT-PCR, were compared with biological samples obtained from 13 sex- and age-matched normal donors. RESULTS: Our data demonstrated that Runx2 mRNA is overexpressed (7.81-fold expression) in pathological thyroid tissue than in normal tissue (P < 0.05). Runx2 mRNA overexpression was also observed in serum and circulating non-hematopoietic cells of PTC patients with respect to normal donors (5.91-fold expression, P < 0.001; 3.82-fold expression, P < 0.05, respectively). We also observed that patients with microcalcifications expressed significantly higher levels of Runx2 mRNA in serum with respect to patients without microcalcifications (P < 0.05). CONCLUSION: This study can open up new research perspectives in the diagnosis and follow-up of PTC, even if further and larger cohort studies will be necessary to validate the Runx2 expression as biomarkers in thyroid cancer.

Application of microfluidic technology to the BIOMED-2 protocol for detection of B-cell clonality.

The BIOMED-2 protocol is widely used for detecting clonality in lymphoproliferative disorders. The protocol requires multiple PCR reactions, which are analyzed by either capillary electrophoresis (GeneScan analysis) or heteroduplex PAGE analysis. We tested a microfluidic chip-based electrophoresis device (Agilent 2100 Bioanalyzer) for the analysis of B-cell clonality using PCR for the three framework subregions (FR) of the Ig heavy chain gene (IGH) and PCR for two rearrangements occurring in the Ig κ chain gene (IGK-VJ and IGK-DE). We analyzed 62 B-cell lymphomas (33 follicular and 29 nonfollicular) and 16 reactive lymph nodes. Chip-based electrophoresis was conclusive for monoclonality in 59/62 samples; for 20 samples, it was compared with GeneScan analysis. Concordant results were obtained in 45/55 IGH (FR1, FR2, and FR3) gene rearrangements, and in 34/37 IGK gene rearrangements. However, when the chip device was used to analyze selected IGK gene rearrangements (biallelic IGK rearrangements or IGK rearrangements in a polyclonal background), its performance was not completely accurate. We conclude, therefore, that this microfluidic chip-based electrophoresis device is reliable for testing cases with dominant PCR products but is less sensitive than GeneScan in detecting clonal peaks in a polyclonal background for IGH PCR, or with complex IGK rearrangement patterns.

Phospho-proteomic analysis of mantle cell lymphoma cells suggests a pro-survival role of B-cell receptor signaling.

BACKGROUND: Mantle cell lymphoma (MCL) is currently an incurable entity, and new therapeutic approaches are needed. We have applied a high-throughput phospho-proteomic technique to MCL cell lines to identify activated pathways and we have then validated our data in both cell lines and tumor tissues. METHODS: PhosphoScan analysis was performed on MCL cell lines. Results were validated by flow cytometry and western blotting. Functional validation was performed by blocking the most active pathway in MCL cell lines. RESULTS: PhosphoScan identified more than 300 tyrosine-phosporylated proteins, among which many protein kinases. The most abundant peptides belonged to proteins connected with B-cell receptor (BCR) signaling. Active BCR signaling was demonstrated by flow cytometry in MCL cells and by western blotting in MCL tumor tissues. Blocking BCR signaling by Syk inhibitor piceatannol induced dose/time-dependent apoptosis in MCL cell lines, as well as several modifications in the phosphorylation status of BCR pathway members and a collapse of cyclin D1 protein levels. CONCLUSION: Our data support a pro-survival role of BCR signaling in MCL and suggest that this pathway might be a candidate for therapy. Our findings also suggest that Syk activation patterns might be different in MCL compared to other lymphoma subtypes.

Utility of racemase and other immunomarkers in the detection of adenocarcinoma in prostatic tissue damaged by high intensity focused ultrasound therapy.

AIMS: High intensity focused ultrasound (HIFU) is an emerging alternative for the treatment of prostate adenocarcinoma. Alpha-methylacyl-CoA racemase (AMACR) has been shown to be a sensitive immunomarker for prostate cancer, however, there is no information available concerning its utility and that of other immunomarkers for the detection of malignancy after HIFU therapy. METHODS: AMACR expression was examined in 11 cases of prostatic carcinoma treated by HIFU, with histological evidence of residual carcinoma. In seven cases tumour was examined from thin core biopsies and in four cases from tissue fragments obtained by transurethral resection of prostate (TURP). In addition to AMACR, immunostaining was also undertaken for p63, cytokeratin 34betaE12, cytokeratin 5, cytokeratin 8-18, prostate specific alkaline phosphatase (PSAP), prostate specific antigen (PSA), chromogranin and CD56. RESULTS: In two of the cases foci of tumour were cut out in serial sections. AMACR was expressed in eight of nine evaluable cases (4/5 biopsies and 4/4 TURP specimens). Cytokeratin 8-18 and PSAP were positive in all cases, whereas PSA was positive in five of nine cases. Cytokeratin 34betaE12, cytokeratin 5, and p63 marked the basal layer in normal prostatic glands, but were negative in neoplastic glands. In four cases we found tumour cells with positive staining for CD56 and chromogranin. CONCLUSIONS: A panel with positive markers for AMACR, and negative markers for p63/cytokeratin 5/cytokeratin 34betaE12 confirms the neoplastic nature of the residual glands on biopsies or TURP fragments sampled after HIFU therapy.

Diagnostic usefulness of fluorescent cytogenetics in differentiating chromophobe renal cell carcinoma from renal oncocytoma: a validation study combining metaphase and interphase analyses.

We investigated the usefulness of interphase fluorescence in situ hybridization (FISH) analysis to differentiate between 11 chromophobe renal carcinomas and 12 renal oncocytomas, showing different clinical outcomes, when compared with conventional metaphase cytogenetics by karyotyping. Karyotypically, 3 chromophobe renal cell carcinomas showed losses of chromosomes, 3 were polyploid, 1 was normal, and 4 failed to grow. Of 12 oncocytomas, 5 showed a normal numeric karyotype and 6 additional structural rearrangements. FISH on chromophobe renal cell carcinomas showed a high percentage of cases (10/11 [91%]) with multiple numeric losses among chromosomes 1, 2, 6, 10, and 17; this interphase pattern was observed irrespective of the 3 different metaphase karyotypes. Of 12 oncocytomas, 11 (92%) revealed a normal numeric chromosomal status showing at least 2 chromosomes without aneusomy by interphase FISH. The study demonstrates that indeed FISH performed on formalin-fixed, paraffin-embedded tissue can provide clinically useful information more reliably than karyotyping of most of these tumors.

Analysis of colorectal cancers for human cytomegalovirus presence.

BACKGROUND: A possible association between human cytomegalovirus (HCMV) infection and colorectal cancer progression has been inferred by the identification in tumour tissues of HCMV antigens and specific viral DNA or RNA sequences. To further investigate the relationship between HCMV and colorectal cancers we developed qualitative and quantitative PCR assay to detect HCMV DNA in 56 formalin-fixed paraffin-embedded (FFPE) tissue samples from patients belonging to 4 different histological phenotypes: adenoma; poorly, moderately and well differentiated adenocarcinomas. RESULTS: Of the 56 FFPE tested tissue samples, 6 (11%) were positive for HCMV nested PCR amplification, and more precisely 1 (5%) of 20 cases of adenoma and 5 (21%) of 24 cases of moderately differentiated adenocarcinoma. No PCR positivity was obtained in samples from well and poorly differentiated adenocarcinomas. CONCLUSION: Our observations suggest that there is no evidence of a direct association between HCMV and colorectal cancer. Moreover, the results obtained are not supportive of a causal role of HCMV in the processes of carcinogenesis and/or progression of colorectal cancer. However, the fact that the virus may present a "hit and run" like-mechanism and HCMV can thus only be detectable at a particular stage of a processing adenocarcinoma, suggests that a significant number of colorectal cancers might have been the subject of HCMV infection that could contribute to trigger the oncogenic differentiation. Our analysis does not exclude the possibility of HCMV infection subsequent viral clearance.

Molecular diagnosis of renal cell neoplasms: the usefulness of immunohistochemistry and fluorescence in situ hybridization.

BACKGROUND: The classification of renal cell neoplasms includes different subtypes of tumors characterized by different outcome. Some overlapping morphological features and the increasing recognition of new entities are making the traditional histologic distinction of renal cell neoplasms difficult and more tools improving the specificity of the correct identification are needed. Among molecular analyses, immunohistochemistry and fluorescence in situ hybridization have become the most helpful procedures, solving many issues in the differential diagnosis of the renal cell neoplasms. OBJECTIVE: The aim of this review is to merge the large amount of recent knowledge regarding molecular markers of renal cell neoplasms into a helpful diagnostic algorithm. CONCLUSION: It is proposed that immunoreactions for CD10, Alpha-methylacyl-CoA racemase, cytokeratin 7, parvalbumin and S100A1, and the cytogenetical analysis of chromosomes 3p, 1, 2, 6, 7, 10, 17 and Y can now offer the most specific tools for the classification of renal cell tumors.