In the frame of translational breast cancer research, eligibility criteria for formalin-fixed paraffin-embedded tissue (FFPE) material processing for gene expression studies include tumor cell content (TCC) and sample site (primary vs metastatic tumors). Herein we asked whether the observed differences in gene expression between paired samples with respect to TCC and sample site also have different clinical significance. We assessed ESR1, ERBB2, MAPT, MMP7, and RACGAP1 mRNA expression with real time PCR in paired samples before (NMD) and after macrodissection (MD) from 98 primary tumors (PMD, PNMD) and 72 metastatic lymph nodes (LNMD, LNNMD), as well as from 93 matched P (mP) and LN (mLN). TCC range was 2.5–75 % in the NMD series and 28–98 % in the MD and in the mP/mLN series. The prognostic effect of these markers, individually or in clusters, remained stable between paired PMD/NMD. In comparison, cluster classification failed in the LNNMD group with lower TCC. In the mP/mLN cohort, RACGAP1 mRNA expression was of prognostic significance when tested in mLN samples (p < 0.001). Similarly, luminal B, HER2, and triple negative tumors were of dismal prognosis when classified in the LN component of the same series (mLN, overall survival: p = 0.013, p = 0.034, and p = 0.007, respectively). In conclusion, the clinical relevance of the RNA markers examined may be affected by TCC in metastatic LN samples but not in primary tumors, while it differs between primary tumors and matched metastases. These data will facilitate the design of translational studies involving FFPE sample series.
Electronic supplementary material
The online version of this article (doi:10.1007/s00428-012-1357-1) contains supplementary material, which is available to authorized users.
Macrodissection; Tumor cell content; Gene expression; FFPE; Primary tumor; Metastatic lymph node; Breast cancer; Translational study
Splenic marginal zone lymphoma (SMZL) is a low-grade lymphoma showing a rather nonspecific immunophenotype. Gene expression profiling studies suggested that TCL1A could be a marker of SMZL, but reported data are conflicting. We evaluated TCL1A expression in a series of spleen and bone marrow samples involved by SMZL and correlated the findings with other immunophenotypical, morphological, and clinical data. In addition, we evaluated the expression of TCL1A in a series of spleens and lymph nodes involved by lymphomas that might mimic SMZL (13 nodal marginal zone lymphomas (NMZL), 39 follicular lymphomas (FL), 30 B-cell chronic lymphocytic leukemias (B-CLL), 31 mantle cell lymphomas (MCL), 1 lymphoplasmacytic lymphoma) and 15 bone marrow specimens involving hairy cell leukemia (HCL). TCL1A staining was negative in 24/31 cases of SMZL (77 %); 27/31 MCL and all B-CLL were positive for TCL1A; 32/34 cases of nodal FL (96 %) and all five splenic FL were positive for TCL1A, although at a lower intensity. Eight of 13 NMZL were positive for TCL1A, often showing a heterogeneous staining pattern. All HCL samples were strongly positive for TCL1A. No correlation was found between the pattern of splenic infiltration, TCL1A expression, and the clinical course. TCL1A-positive SMZL showed a higher rate of DBA44 staining compared to the negative ones, and this difference was statistically significant (Fisher test, single-tailed, p = 0.0397). Our data support the use of TCL1A in the panel of diagnostic markers used in the differential diagnosis of splenic low-grade B-cell lymphoma; a possible prognostic value, however, needs a larger series to be established.
Splenic marginal zone lymphoma; TCL1A; Immunohistochemistry; Differential diagnosis
In patients with serous adenocarcinoma (SAC) of the endometrium, we evaluated the prognostic importance of clinicopathological parameters, DNA ploidy, and immunoexpression of p53, estrogen receptor (ER), progesterone receptor (PR), and Ki-67. In a series of 73 stage I and II SAC, DNA ploidy analysis was performed on hysterectomy specimens using DNA image cytometry. Immunohistochemical analysis of p53, ER, PR, and Ki-67 expression was additionally performed. In the review of the histological slides by three gynecologic pathologists, the presence of a serous component was not agreed upon in 17 (23 %) cases. The remaining 56 cases, consisting of pure SAC or SAC mixed with endometrioid adenocarcinoma, were further analyzed. Tumor recurrence was observed in 14 patients, and 28 patients died during the follow-up period. Patients with diploid (n = 19), aneuploid (n = 29), and tetraploid (n = 8) tumor had 5-year recurrence rates of 10, 38, and 53 %, respectively (p = 0.09). A DNA ploidy parameter, 5c exceeding rate, was found to be a prognostic marker for recurrence (p = 0.03), progression-free survival (p < 0.01), and overall survival (p = 0.02). Immunoexpression of p53, ER, PR, and Ki-67 did not have prognostic value, and the same was true for FIGO stage, lymphovascular invasion, the extent of myometrial invasion, and lymphadenectomy. The histological diagnosis of SAC may be difficult in some cases. Established clinicopathological parameters do not seem to be strong prognosticators in stage I and II disease. A DNA ploidy parameter, 5c exceeding rate, may be a prognostic marker in this patient group and should be further validated in larger series.
Serous adenocarcinoma; Endometrial carcinoma; DNA ploidy; p53; Estrogen receptor; Progesterone receptor
The enzyme alpha-methylacyl-coenzyme A racemase plays an important role in the beta-oxidation of branched-chain fatty acid and its derivatives. It has been used to detect prostatic adenocarcinoma and high-grade intraepithelial neoplasia, and recently also as a marker for other neoplasms, including those of the genitourinary system, breast, upper and lower gastrointestinal tract and their precursor lesions. We assessed expression of alpha-methylacyl-coenzyme A racemase by immunohistochemistry in neuroendocrine tumours of the stomach to determine differences in the incidence and pattern of expression among different types of gastric neuroendocrine tumours. While none of the grade 1 neuroendocrine tumours were immunoreactive, 67 % of grade 2 neuroendocrine tumours and 90 % of neuroendocrine carcinomas were positive for alpha-methylacyl-coenzyme A racemase. Furthermore, an adenocarcinoma component was found in 72.5 % (37 of 51) of neuroendocrine carcinomas, whereas none of the grade 1 and 2 neuroendocrine tumours contained an adenocarcinoma component. In 83 % of neuroendocrine carcinomas, the adenocarcinoma component was positive for alpha-methylacyl-coenzyme A racemase, and both adenocarcinoma and neuroendocrine carcinoma components stained positively in 78 % of these cases. Our results indicate that alpha-methylacyl-coenzyme A racemase is a useful marker for distinguishing between grade 1 (negative) and grade 2 neuroendocrine tumours, and neuroendocrine carcinoma of the stomach (frequently positive). Different patterns of alpha-methylacyl-coenzyme A racemase expression between gastric neuroendocrine tumours and neuroendocrine carcinoma suggest that these might develop via different tumourigenic pathways.
Alpha-methylacyl-coenzyme A racemase; Stomach; Neuroendocrine tumour; Neuroendocrine carcinoma
Urothelial cell carcinoma (UCC) with musculus detrusor (MD) invasion is treated by cystectomy. Subsequent pathologic evaluation of cystectomies does not reveal MD invasion (
Cystectomy; Urothelial cell carcinoma; Down-staging; pT0
For tularemia, a zoonosis caused by the gram-negative coccobacillus Francisella tularensis, research of the relation between skin lesions and lymph node lesions has not been reported in the literature. This report describes skin lesions and lymph node lesions and their mutual relation over time for tularemia in Japan. Around the second day after infection (DAI), a subcutaneous abscess was observed (abscess form). Hand and finger skin ulcers formed during the second to the fourth week. Subcutaneous and dermal granulomas were observed with adjacent monocytoid B lymphocytes (MBLs) (abscess–granulomatous form). From the sixth week, large granulomas with central homogeneous lesions emerged diffusely (granulomatous form). On 2–14 DAI, F. tularensis antigen in skin lesions was detected in abscesses. During 7–12 DAI, abscesses with adjacent MBLs appeared without epithelioid granuloma (abscess form) in regional lymph nodes. During the second to fifth week, granulomas appeared with necrosis (abscess–granulomatous form). After the sixth week, large granulomas with a central homogeneous lesion (granulomatous form) appeared. F. tularensis antigen in lymph node lesions was observed in the abscess on 7–92 DAI. Apparently, F. tularensis penetrates the finger skin immediately after contact with infected hares. Subsequently, the primary lesion gradually transfers from skin to regional lymph nodes. The regional lymph node lesions induced by skin lesion are designated as dermatopathic lymphadenopathy. This study revealed temporal differences of onset among the skin and lymph node lesions.
Tularemia; Primary skin lesions; Regional lymph node lesions; Temporal differences of onset
Myoepithelial carcinoma of soft tissue (MEC) and cellular extraskeletal myxoid chondrosarcoma (cEMC) share striking similarities. In this paper, we compare ten MECs with five cEMCs. MEC patients had an equal gender distribution. The age range was 15–76 years (mean, 42 years). Tumours were located on extremities, pelvic girdle, vulva and neck. Follow-up, available for nine patients, ranged from 4 to 85 months (mean, 35 months). Five patients were alive without evidence of disease, two were alive with disease and two died 8 months after the initial diagnosis. cEMCs were from three males and two females with an age range of 37–82 years (mean, 57 years); they presented in extremities, shoulder and paravertebral/cervical. Follow-up, available for four patients, ranged from 6 to 220 months (mean, 61 months). All patients were alive, two with recurrences and/or metastases and two without evidence of disease. Morphologically, the distinction between these two entities was difficult since all cases exhibited features typically seen in myoepithelial tumours. Immunohistochemically, MECs expressed pan-keratin (80 %), epithelial membrane antigen (EMA; 57 %), S100 (50 %), alpha-smooth muscle actin (ASMA; 75 %), calponin (67 %) and p63 (25 %). S100 and EMA were expressed in 40 % of cEMC cases respectively with additional immunoreactivity for p63, ASMA and glial fibrillary acidic protein in one case. Pan-keratin was negative in all neoplasms. NR4A3 rearrangement was present in four of four cEMCs and in none of the MECs. In contrast, three of nine (33 %) MECs and four of five (80 %) cEMCs showed an EWSR1 rearrangement. In summary, MECs and cEMCs share clinical, morphological, immunohistochemical and genetic characteristics. The pathognomic rearrangement of NR4A3 is a useful diagnostic feature in identifying cEMCs.
Myoepithelial carcinoma of soft tissue; Extraskeletal myxoid chondrosarcoma; Soft tissue tumours; EWSR1 rearrangement; NR4A3 rearrangement
Analysis of sentinel lymph node (SLN) by means of One-Step Nucleic Acid Amplification (OSNA) is being used increasingly as a very sensitive and quick method for intraoperative axillary staging in patients with breast cancer. This molecular diagnostic assay detects the expression level of cytokeratin 19 (CK19), a luminal epithelial cell marker broadly expressed in most breast carcinomas and not normally found in lymph nodes. Almost all breast cancers express this cytoskeleton protein, but some breast tumors have been found to lose the expression of CK19. CK19 immunostaining in core biopsies has been recommended in selecting patients eligible for OSNA analysis because SLNs with metastatic involvement by CK19-negative breast cancers may result in a false negative result by OSNA. However, the real frequency of CK19-negative breast cancer has to be elucidated. In this study, we have assessed the frequency and molecular profile of CK19-negative breast carcinomas in three series of cases. The first is a prospective series of 197 breast carcinomas, 111 of which were subjected to SLN evaluation by OSNA. The second is a retrospective series of 41 triple-negative (TN) breast carcinomas, and the third is a retrospective series of 68 breast cancer patients (matched core biopsies and metastatic lymph nodes) that had been evaluated by conventional procedures before the OSNA methodology was adopted in our institution. Our results not only demonstrate that lack of expression of CK19 is infrequent in breast cancers but also that performing CK19 immunohistochemical staining is important on diagnostic core biopsies in taking the decision of using OSNA methodology in the evaluation of sentinel nodes in breast cancer patients.
Breast carcinoma; OSNA; CK19; Luminal; “Basal-like”
Vestibular schwannomas show a large variation in growth rate, making prediction and anticipation of tumor growth difficult. More accurate prediction of clinical behavior requires better understanding of tumor biological factors influencing tumor progression. Biological processes like intratumoral hemorrhage, cell proliferation, microvessel density, and inflammation were analyzed in order to determine their role in vestibular schwannoma development. Tumor specimens of 67 patients surgically treated for a histologically proven unilateral vestibular schwannoma were studied. Preoperative magnetic resonance imaging (MRI) scans were used to determine tumor size and to classify tumors as homogeneous, inhomogeneous, and cystic. Immunohistochemical studies evaluated cell proliferation (histone H3 and Ki-67), microvessel density (CD31), and inflammation (CD45 and CD68). Intratumoral hemorrhage was assessed by hemosiderin deposition. The expression patterns of these markers were compared with tumor size, tumor growth index, MRI appearance, patients’ age, and duration of symptoms. No relation between cell proliferation and clinical signs of tumor volume increase or MRI appearance was found. Intratumoral hemosiderin, microvessel density, and inflammation were significantly positively correlated with tumor size and the tumor growth index. Cystic and inhomogeneous tumors showed significantly more hemosiderin deposition than homogeneous tumors. The microvessel density was significantly higher in tumors with a high number of CD68-positive cells. The volume increase of vestibular schwannomas is not based on cell proliferation alone. Factors like intratumoral bleeding, (neo)vascularization, and intensity of the inflammatory reaction also influence tumor volume.
Electronic supplementary material
The online version of this article (doi:10.1007/s00428-012-1236-9) contains supplementary material, which is available to authorized users.
Vestibular schwannoma; Neuropathology; Tumor biology
Sixty-four cases of malignant lymphoma involving the liver were examined. Of these, 20 cases were histologically confirmed to be primary hepatic B-cell lymphoma. Twelve of these 20 cases were diffuse large B-cell lymphoma (DLBCL) and eight cases were mucosa-associated lymphoid tissue (MALT) lymphoma. Of the 12 cases of DLBCL, six were immunohistologically positive for CD10 and/or Bcl6 (indicating a germinal center phenotype), six were positive for Bcl2, and five were positive for CD25. Eight of the 12 DLBCL cases (66.7%) and two of the eight MALT lymphoma cases (25%) had serum anti-hepatitis C virus (HCV) antibodies and HCV RNA. The incidence of HCV infection was significantly higher in the hepatic DLBCL cases than in systemic intravascular large B-cell cases with liver involvement (one of 11 cases, 9.1%) and T/NK-cell lymphoma cases (one of 19 cases, 5.3%) (p < 0.01 for both). Two hepatic DLBCL cases (16.7%) had rheumatoid arthritis treated with methotrexate, and four MALT lymphoma cases (50%) had Sjögren’s syndrome, primary biliary cirrhosis, or autoimmune hepatitis; one case in each of these two groups was complicated by chronic HCV-seropositive hepatitis. Although primary hepatic lymphoma is rare, persistent inflammatory processes associated with HCV infection or autoimmune disease may play independent roles in the lymphomagenesis of hepatic B cells.
Liver; Malignant lymphoma; HCV; Autoimmune disease
Gene expression profiles provide important information about the biology of breast tumors and can be used to develop prognostic tests. However, the implementation of quantitative RNA-based testing in routine molecular pathology has not been accomplished, so far. The EndoPredict assay has recently been described as a quantitative RT-PCR-based multigene expression test to identify a subgroup of hormone–receptor-positive tumors that have an excellent prognosis with endocrine therapy only. To transfer this test from bench to bedside, it is essential to evaluate the test–performance in a multicenter setting in different molecular pathology laboratories. In this study, we have evaluated the EndoPredict (EP) assay in seven different molecular pathology laboratories in Germany, Austria, and Switzerland. A set of ten formalin-fixed paraffin-embedded tumors was tested in the different labs, and the variance and accuracy of the EndoPredict assays were determined using predefined reference values. Extraction of a sufficient amount of RNA and generation of a valid EP score was possible for all 70 study samples (100%). The EP scores measured by the individual participants showed an excellent correlation with the reference values, respectively, as reflected by Pearson correlation coefficients ranging from 0.987 to 0.999. The Pearson correlation coefficient of all values compared to the reference value was 0.994. All laboratories determined EP scores for all samples differing not more than 1.0 score units from the pre-defined references. All samples were assigned to the correct EP risk group, resulting in a sensitivity and specificity of 100%, a concordance of 100%, and a kappa of 1.0. Taken together, the EndoPredict test could be successfully implemented in all seven participating laboratories and is feasible for reliable decentralized assessment of gene expression in luminal breast cancer.
Breast cancer; Prognosis; mRNA; Quality control
KRAS mutation testing is mandatory before prescribing anti-epidermal growth factor monoclonal antibodies in the treatment of advanced colorectal cancer. We describe the performance of a TaqMelt polymerase chain reaction (PCR) assay—the cobas® KRAS Mutation Test—designed to detect 19 mutations in codons 12, 13, and 61. The limit of detection was determined using DNA blends from cell lines, plasmids, and formalin-fixed paraffin-embedded tissue specimens. Assay performance was compared to Sanger sequencing using a panel of 188 specimens. Discordant specimens were subjected to next generation pyrosequencing (454). Assay repeatability was assessed using a panel of six specimens. A >95% correct mutation call rate was obtained in all specimen types with ~5% mutant alleles at DNA inputs of 0.8–6.3 ng per PCR reaction; 100% detection rate was observed at the recommended DNA input of 50 ng. The positive percent agreement with Sanger was 97.5% (79/81) for codons 12/13 and 85.7% (6/7) for codon 61. Negative percent agreement was 94.4% (101/107) for codon 12/13 and 99.4% (180/181) for codon 61. Nine of 10 discordant specimens yielded 454 results consistent with the cobas® results. With repeated testing, the assay showed a correct call rate of 100% (192/192) for all operators, instruments, reagent lots, and days tested. The cobas® test detects KRAS mutations in codons 12, 13, and 61 at a limit of detection of <5%. The PCR assay was more sensitive and specific than Sanger sequencing, and performance was highly reproducible. Test performance was not influenced by various endogenous interfering substances or common gut microbes.
KRAS mutations; Molecular diagnostics; Colorectal cancer; Polymerase chain reaction (PCR)
Intra-operative frozen section analysis (FS analysis) of sentinel lymph nodes (SLNs) in patients with breast cancer can prevent a second operation for axillary lymph node dissection. In contrast, loss of tissue during FS analysis may impair the probability to detect lymph node metastases. To determine the effect of tissue loss on the probability of detection of metastases, dimensions and tissue loss resulting from intra-operative frozen section analysis were measured for 21 SLNs. In a mathematical model, the influence of tissue loss on the probability to detect metastases was calculated in relation to SLN size for various pathology protocols: an American, a widely used European, the extensive ‘Milan’ and the Dutch protocol. For median-sized SLN 11 × 8 × 5 mm (length × width × height), FS analysis led to a median loss of 680 μm (13.6%) of the height of the SLN. Irrespective of SLN size or used pathology protocol, the probability of detecting 2 mm metastases remained unchanged or even increased (0–12.8%). Moreover, the probability to detect 0.2 mm metastases increased for the majority of tested combinations of SLN size, tissue loss and used protocol. Only when combining maximum tissue loss and smallest SLN size in the Dutch protocol, or when applying the extensive Milan protocol on a median-sized SLN, the probability to detect 0.2 mm metastases decreased by 2.7% and 14.3%, respectively. Contrary to ‘common knowledge’, doing FS analysis of SLNs does not impair the probability to detect lymph node metastases.
Frozen section; Sentinel lymph node; Lymphatic metastasis; Breast cancer; Mathematical model
Endochondral bone formation requires a cartilage template, known as the growth plate, and vascular invasion, bringing osteoblasts and osteoclasts. Endochondral chondrocytes undergo sequences of cell division, matrix secretion, cell hypertrophy, apoptosis, and matrix calcification/mineralisation. In this study, two critical steps of endochondral bone formation, the deposition of collagen X-rich matrix and blood vessel attraction/invasion, were investigated by immunohistochemistry. Fourteen multiple osteochondromas and six secondary peripheral chondrosarcomas occurring in patients with multiple osteochondromas were studied and compared to epiphyseal growth plate samples. Mutation analysis showed all studied patients (expect one) to harbour a germ-line mutations in either EXT1 or EXT2. Here, we described that homozygous mutations in EXT1/EXT2, which are causative for osteochondroma formation, are likely to affect terminal chondrocyte differentiation and vascularisation in the osteocartilaginous interface. Contrastingly, terminal chondrocyte differentiation and vascularisation seem to be unaffected in secondary peripheral chondrosarcoma. In addition, osteochondromas with high vascular density displayed a higher proliferation rate. A similar apoptotic rate was observed in osteochondromas and secondary peripheral chondrosarcomas. Recently, it has been shown that cells with functional EXT1 and EXT2 are outnumbering EXT1/EXT2 mutated cells in secondary peripheral chondrosarcomas. This might explain the increased type X collagen production and blood vessel attraction in these malignant tumours.
Growth plate; Osteochondroma; Peripheral chondrosarcoma; Type X collagen; Angiogenesis; Bone tumour
Adrenocortical carcinoma (ACC) is a rare, heterogeneous malignancy with a poor prognosis. According to WHO classification 2004, ACC variants include oncocytic ACCs, myxoid ACCs and ACCs with sarcomatous areas. Herein, we provide a comprehensive review of these rare subtypes of adrenocortical malignancy and emphasize their clinicopathological features with the aim of elucidating aspects of diagnostic categorization, differential diagnostics and biological behavior. The issue of current terminology, applied to biphasic tumors with pleomorphic, sarcomatous or sarcomatoid elements arising in adrenal cortex, is also discussed. We additionally present emerging evidence concerning the adrenal cortical tumorigenesis and the putative adenoma–carcinoma sequence as well.
Adrenal cortical neoplasia; Adenoma; Carcinoma; ACC variants; Oncocytic; Myxoid; Sarcomatoid; Carcinosarcoma
Periostin, also called osteoblast-specific factor 2, is a secreted cell adhesion protein, which shares a homology with the insect cell adhesion molecule fasciclin I. It has been shown to be an important regulator of bone and tooth formation and maintenance, and of cardiac development and healing. Recent studies revealed that periostin plays an important role in tumor development and is upregulated in a wide variety of cancers such as colon, pancreatic, ovarian, breast, head and neck, thyroid, and gastric cancer as well as in neuroblastoma. Periostin binding to the integrins activates the Akt/PKB- and FAK-mediated signaling pathways which lead to increased cell survival, angiogenesis, invasion, metastasis, and importantly, epithelial-mesenchymal transition of carcinoma cells. In this review we summarize recent clinicopathological studies that have investigated periostin expression in lung, kidney, prostate, liver cancer, and malignant pleural mesothelioma and discuss the role of periostin isoforms in tumorigenesis and their potential as targets for stroma-targeted anticancer therapy.
Periostin; EMT; Isoform; Stroma; Target
Epithelial barrier function is contingent on appropriate polarization of key protein components. Work in intestinal epithelial cell cultures and animal models of bowel inflammation suggested that atypical PKC (aPKC), the kinase component of the Par3–Par6 polarity complex, is downregulated by pro-inflammatory signaling. Data from other laboratories showed the participation of myosin light chain kinase in intestinal inflammation, but there is paucity of evidence for assembly of its major target, non-muscle myosin II, in inflammatory bowel disease (IBD). In addition, we showed before that non-muscle myosin IIA (nmMyoIIA) is upregulated in intestinal inflammation in mice and TNFα-treated Caco-2 cells. Thus far, it is unknown if a similar phenomena occur in patients with IBD. Moreover, it is unclear whether aPKC downregulation is directly correlated with local mucosal inflammation or occurs in uninvolved areas. Frozen sections from colonoscopy material were stained for immunofluorescence with extensively validated specific antibodies against phosphorylated aPKC turn motif (active form) and nmMyoIIA. Inflammation was scored for the local area from where the material was obtained. We found a significant negative correlation between the expression of active aPKC and local inflammation, and a significant increase in the apical expression of nmMyoIIA in surface colon epithelia in inflamed areas, but not in non-inflamed mucosa even in the same patients. Changes in aPKC and nmMyoIIA expression are likely to participate in the pathogenesis of epithelial barrier function in response to local pro-inflammatory signals. These results provide a rationale for pursuing mechanistic studies on the regulation of these proteins.
PKCι; PKCζ; Epithelial polarity; Crohn’s disease; Ulcerative colitis; Non-muscle myosin IIA