Both the putative modifier gene ATP5a1 and the tumour suppressor gene TP53 are involved in the regulation of apoptosis and may be involved in the development of colorectal cancers.
To investigate the relationship between these genes in 16 colorectal cancer cell lines.
Each gene was screened for mutation using high resolution melting analysis and sequencing. Expression of ATP5a1 mRNA was tested by quantitative PCR.
Sequence changes in ATP5a1 were found in 9/16 (56%) cell lines and consisted of mainly novel single nucleotide polymorphisms (SNPs) found in the 5' UTR, introns 4/5/9 and exon 7. TP53 mutations were also found in 9/16 (56%) cell lines; these were consistent with previous reports. High levels of ATP5a1 expression were seen in cell lines with TP53 mutation compared with those with wild type TP53 (p = 0.02). Furthermore, an A->G change at the –18 position in intron 4 of ATP5a1 was significantly associated with increased gene expression (p = 0.0391). Comparison with genotype showed that cell lines with chromosomal instability (CIN) had significantly higher levels of ATP5a1 expression than those with microsatellite instability (MSI) (p = 0.02).
Higher levels of ATP5a1 expression are associated with certain SNPs and with TP53 mutation. High expression also occurs in CIN and may facilitate tumour development along this pathway. Conversely, low levels of ATP5a1 expression may facilitate development of tumours with MSI.
Secretory breast cancer (SBC) is a rare entity characterised by indolent clinical behaviour, distinctive histological features and the presence of a recurrent chromosomal translocation t(12;15)(p13;q25), leading to the formation of the ETV6–NTRK3 fusion gene.
To describe the molecular genetic features of a case of SBC which harbours a duplication of the t(12;15) translocation.
Tiling path array comparative genomic hybridisation (aCGH) analysis and fluorescence in situ hybridisation (FISH) using in-house-generated probes for ETV6, NTRK3 and the fusion genes, centromeric probes for chromosomes 12 and 15, and a commercially available split-apart ETV6/NTRK3 probe.
FISH revealed the presence of a duplication of the translocation t(12;15), which resulted from the gain of one copy of the derivative chromosome der(15)t(12;15), retention of one normal copy of both ETV6 and NTRK3 genes and deletion of the derivative chromosome der(12)t(12;15). Consistent with FISH findings, aCGH revealed copy number gains of ETV6 and NTRK3 and deletions encompassing the regions centromeric to ETV6 and telomeric to NTRK3. Additional regions of copy number changes included gains of 10q21, 10q26.3, 12p13.3–p13.31 15q11–q25.3 and 16pq and losses of 6q24.1–q27, 12p13.2–q12 and 15q25.3–q26.3.
To the best of our knowledge, this is the first time a carcinoma has been shown to harbour a duplication of the ETV6–NTRK3 translocation. The presence of an additional copy of the derivative chromosome der(15)t(12;15) coupled with deletion of the other derivative der(12)t(12;15) in the modal population of cancer cells suggests that this was either an early phenomenon or conferred additional growth advantage on neoplastic cells.
A small subset (10–15%) of gastrointestinal stromal tumours (GISTs) lack mutations in KIT and PDGFRA (wild-type GIST). Recently, a novel BRAF exon 15 mutation (V600E) was detected in imatinib-naive wild-type high-risk intestinal GISTs (4%). However, the frequency and distribution of BRAF mutations within the spectrum of GISTs, and whether they might represent secondary events acquired during tumour progression, remain unknown.
69 GISTs (39 KIT mutants, 2 PDGFRA mutants and 28 wild-type) were analysed for mutations in BRAF exon 15 and KRAS exon 2. To assess the stage at which these mutations might occur in GIST, a considerable number of incidental gastric (n = 23) and intestinal (n = 2) tumours were included.
BRAF mutations (V600E) were detected in 2 of 28 wild-type GISTs (7%), but in none of the 41 KIT/PDGFRA mutants. No KRAS mutation was detected. The two BRAF-mutated GISTs measured 4 mm in diameter and originated in the gastric body and the jejunum in two men (mean age, 76 years). Both tumours were mitotically inactive KIT-positive spindle-cell GISTs that were indistinguishable histologically from their more common KIT-mutated counterparts.
BRAF mutations represent an alternative molecular pathway in the early tumorigenesis of a subset of KIT/PDGFRA wild-type GISTs and are per se not associated with a high risk of malignancy. Mutations in KIT, PDGFRA and BRAF were mutually exclusive in this study. Results from this and a previous study indicate that BRAF-mutated GISTs show a predilection for the small bowel (four of five tumours), but this needs further evaluation in larger studies.
A better understanding of the biology of nodal metastatic disease is of indisputable value. Three-dimensional (3D) serial section alignment and reconstruction techniques can be used for visualisation of nodal metastasis and could provide better understanding of disease growth patterns.
19 tumour-involved sentinel nodes (SLNs) from breast cancer patients were serially sectioned, immunohistochemically stained, and digitally scanned. Digital image alignment and voxel-based rendering was used to construct informative 3D visual representations of metastatic tumour distribution within involved nodes.
The 3D reconstruction technique was successful and informative. The reconstructions of all 19 SLNs enabled the metastatic tumour cells to be viewed infiltrating normal SLN tissue from all angles. Metastases were present at the afferent lymphatic pole in 17/19 cases, confined to the afferent pole only in 7 cases, located at the efferent pole in 12/19 cases, and efferent pole only in just 2 cases. Finally, this study made the novel observation that metastatic growth occurs in three distinct patterns: sinusoidal, nodular and diffuse.
This methodology provides improved understanding of metastatic disease development and potentially could be used to develop strategies to improve techniques for its routine detection. Further studies are required in order to evaluate the prognostic and biological significance of the growth patterns identified.
Basal-like breast tumours, as defined by microarrays, carry a poor prognosis and therapeutic options are limited to date. Often, these tumours are defined as oestrogen receptor (ER) negative/progesterone receptor (PR) negative/human epidermal growth factor receptor 2 (HER-2) negative (triple negative) by immunohistochemistry (IHC), but a more complete definition should include expression of basal cytokeratins (CK5/6, CK14 or CK17) and/or human epidermal growth factor receptor 1 (HER-1). The aim of this study was to investigate to what extent CK5/6 and HER-1 characterise the group of triple negative breast cancers.
Expression of CK5/6 and HER-1 was studied by IHC in 25 triple negative breast carcinomas and 32 grade-matched, non-triple-negative controls. All 57 cases were further subjected to fluorescence in situ hybridisation to investigate HER-1 gene copy number.
CK5/6 and HER-1 expression was most frequent in triple negative tumours: 22 out of 25 cases (88.0%) expressed at least one of these markers (60.0% CK5/6 positive and 52.0% HER-1 positive). In the control group, CK5/6 and HER-1 expression was found in ER-negative but not in ER-positive tumours (ER negative/PR negative/HER-2 positive tumours: 20.0% CK5/6 positive and 46.7% HER-1 positive). HER-1 gene amplification was found in five cases only: four triple negative (16.0%) and one ER-negative control (ER negative/PR negative/HER-2 positive, 6.7%). Of interest, all five HER-1 amplified cases showed a remarkably homogeneous HER-1 expression pattern.
Expression of CK5/6 and HER-1 is frequent in ER-negative breast cancers, in triple negative and in non-triple negative tumours. In a minority of cases, HER-1 overexpression may be caused by HER-1 gene amplification. Further studies are needed to investigate whether such cases might benefit from anti-HER-1 therapy
It has been suggested that the donor tissue cores used in tissue microarrays (TMAs) may not be representative of the whole tissue section.
To validate the use of TMA technology in the study of malignant peripheral nerve sheath tumours (MPNSTs).
A TMA was constructed containing five independent core biopsy samples of 14 formalin-fixed, paraffin-embedded MPNSTs. The immunohistochemical (IHC) results of the five cores from the same tissue block on TMA were compared with readings from whole sections using two antibodies: anti-Ki-67 and anti-S-100. Digital image analysis was performed to calculate the percentage of positive stain areas. The agreement between IHC results obtained with TMA cores and whole sections was assessed using the statistic.
There was good to very good agreement between IHC results for whole and TMA sections from MPNSTs. In relation to S-100, very good agreement (92% agreement; = 0.77) was observed using a minimum of four TMA cores. Staining results for Ki-67 from at least four readable TMA cores were the same as those for the whole section in 86% of cases, with good agreement, using weighted statistics ( = 0.63).
This study indicates that the TMA technique can be used in the IHC study of MPNSTs, even with the use of heterogeneous markers such as S-100 protein.
Sinonasal intestinal-type adenocarcinomas (ITACs) are rare neoplasms resembling intestinal adenocarcinomas. Although several studies have documented neuroendocrine differentiation in ITACs, the combination of ITAC and small cell carcinoma has not been previously described in detail. The aim of this report is to detail the histopathological and immunohistochemical characteristics of two cases of composite ITAC with small cell carcinoma.
Two cases of composite ITAC with small cell carcinoma were routinely processed, and representative sections were stained with CAM5.2, AE1:AE3, keratin 7, keratin 20, keratin 19, CDX-2, p63, villin, chromogranin, synaptophysin and CD56.
One tumour consisted of a mixed-type ITAC showing colonic-type and poorly differentiated adenocarcinoma with foci of "signet-ring" cells combined with small cell carcinoma. Both components stained positively with CAM5.2, AE1:AE3, CK7, CK20 and CK19, whereas only the small cell carcinoma expressed synaptophysin and CD56. Both components stained negatively with CDX-2, villin, CD99 and p63. The "signet-ring" cells stained positively with chromogranin and synaptophysin. The second tumour showed a papillary-type ITAC combined with a small cell carcinoma. The adenocarcinoma and small cell carcinoma stained positively with CAM5.2, AE1:AE3, CK7, CK19 and CK20. Only the adenocarcinoma was CDX-2 positive, whereas the small cell carcinoma expressed CD56 and synaptophysin.
The two components of the combined ITACs and neuroendocrine small cell carcinoma show significant immunohistochemical overlap, supporting a common origin. The occurrence of a distinct neuroendocrine carcinoma combined with ITACs expands the histopathological spectrum of these tumours.
To describe the cytological appearances of melanoma in fluid specimens and potential diagnostic pitfalls in interpreting such specimens, which have been infrequently reported in the literature.
Cases of melanoma diagnosed at a single institution between 1993 and 2008 in cytology specimens of fluids (pleural, ascitic, cerebrospinal and other fluids), but excluding fine needle biopsy specimens, were identified and reviewed.
32 fluid specimens containing metastatic melanoma (from 26 patients) were identified. Most of the specimens were moderately cellular and showed moderate to marked nuclear pleomorphism. Mitotic figures and intranuclear cytoplasmic invaginations were identified in 11 (34.4%) and seven (21.9%) cases, respectively. Melanin pigment was seen in eight (25.0%) cases. Variable numbers of histiocytes were present, and mesothelial cells were present in body cavity fluid specimens.
In fluid specimens, reactive mesothelial cells and histiocytes may mimic epithelioid melanoma cells. Awareness of the morphological features and diagnostic pitfalls of melanoma in fluids is necessary to avoid the potentially serious consequences of misdiagnosis.
To determine whether measurement of soluble CD30 (sCD30) levels predicts for early rejection in a cohort of first deceased kidney transplant recipients.
Pre-transplant serum samples were analysed for sCD30 levels using a commercial ELISA kit (Biotest). A 100 U/ml cut-off for "high sCD30" was applied. Clinical outcome parameters were biopsy-proven rejection episodes, creatinine levels and glomerular filtration rate.
In the cohort of patients who experienced at least one episode of rejection in the first 6 months post-transplant, levels of pre-transplant sCD30 were significantly higher than in those who did not experience rejection. Despite this association, the occurrence of a high sCD30 level did not predict for rejection on an individual basis.
The prognostic value of pre-transplant sCD30 testing is diminished by the large number of patients with high sCD30 levels who do not develop rejection. Although this limits the utility of the test in informing clinical management of individual patients, a high pre-transplant sCD30 level should still be considered a risk factor for poorer outcome.
There has been a steady decline in the overt teaching of many basic and pathology sciences in the medical curriculum worldwide. As interns are the doctors most likely to request and act on tests, an assessment of their confidence in dealing with laboratory investigations was undertaken.
Interns at two hospitals in Cape Town, South Africa, were asked to complete a structured questionnaire designed to assess their confidence in ordering and interpreting a number of tests. The questionnaire also probed their desire for further teaching and the preferred delivery vehicle for such teaching.
61 out of 117 questionnaires were returned. Interns were confident in the use of common tests, but 23% were not confident in interpreting a test that they were confident in ordering. All respondents felt they would benefit from teaching in at least one area and lectures were the preferred method, although the majority felt it very likely that they would complete an online tutorial if available. The results suggest that institutions need to devise strategies to fulfil the learning needs of new graduates in the area of chemical pathology and clinical biochemistry.