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1.  Dysregulation of the intrinsic apoptotic pathway mediates megakaryocytic hyperplasia in myeloproliferative neoplasms 
Journal of Clinical Pathology  2016;69(11):1017-1024.
Aims
Megakaryocyte expansion in myeloproliferative neoplasms (MPNs) is due to uncontrolled proliferation accompanied by dysregulation of proapoptotic and antiapoptotic mechanisms. Here we have investigated the intrinsic and extrinsic apoptotic pathways of megakaryocytes in human MPNs to further define the mechanisms involved.
Methods
The megakaryocytic expression of proapoptotic caspase-8, caspase-9, Diablo, p53 and antiapoptotic survivin proteins was investigated in bone marrow specimens of the MPNs (n=145) and controls (n=15) using immunohistochemistry. The megakaryocyte percentage positivity was assessed by light microscopy and correlated with the MPN entity, JAK2V617F/CALR mutation status and platelet count.
Results
The proportion of megakaryocytes in the MPNs expressing caspase-8, caspase-9, Diablo, survivin and p53 was significantly greater than controls. A greater proportion of myeloproliferative megakaryocytes expressed survivin relative to its reciprocal inhibitor, Diablo. Differences were seen between myelofibrosis, polycythaemia vera and essential thrombocythaemia for caspase-9 and p53. CALR-mutated cases had greater megakaryocyte p53 positivity compared to those with the JAK2V617F mutation. Proapoptotic caspase-9 expression showed a positive correlation with platelet count, which was most marked in myelofibrosis and CALR-mutated cases.
Conclusions
Disruptions targeting the intrinsic apoptotic cascade promote megakaryocyte hyperplasia and thrombocytosis in the MPNs. There is progressive dysfunction of apoptosis as evidenced by the marked reduction in proapoptotic caspase-9 and accumulation of p53 in myelofibrosis. The dysfunction of caspase-9, which is necessary for proplatelet formation, may be the mechanism for the excess thrombocytosis associated with CALR mutations. Survivin seems to be the key protein mediating the megakaryocyte survival signature in the MPNs and is a potential therapeutic target.
doi:10.1136/jclinpath-2016-203625
PMCID: PMC5136711  PMID: 27060176
MYELOPROLIFERATIVE DISEASE; APOPTOSIS; IMMUNOHISTOCHEMISTRY
2.  Helicobacter pylori vacA transcription is genetically-determined and stratifies the level of human gastric inflammation and atrophy 
Journal of Clinical Pathology  2016;69(11):968-973.
Aims
Helicobacter pylori infection is the major cause of peptic ulceration and gastric cancer, and an important virulence determinant is its vacuolating cytotoxin vacA. Previously, we have described allelic variation in vacA which determines toxin activity and disease risk. Here we aimed to quantify vacA mRNA expression in the human stomach, define its genetic determinants and assess how well it predicts gastric pathology.
Methods
Gastric biopsies were donated by 39 patients with H. pylori infection attending for endoscopy at Queen's Medical Centre, Nottingham, UK. Total RNA was extracted, and vacA mRNA quantified by reverse transcriptase quantitative PCR. Separate biopsies were histologically scored for inflammation and atrophy using the updated Sydney system. H. pylori strains were isolated from further biopsies, and the nucleotide sequence upstream of vacA determined.
Results
vacA mRNA levels in human stomachs varied by two orders of magnitude independently of vacA allelic type. Among vacA i1-type (toxic) strains, increased vacA expression was strongly associated with higher grade gastric inflammation (p<0.02), neutrophil infiltration (p<0.005) and the presence of atrophy (p<0.01). A polymorphism at nucleotide +28 near the base of a potential stem-loop structure within the 5′ untranslated region was significantly associated with vacA transcript level and inflammation.
Conclusions
Increased gastric vacA expression during H. pylori infection is associated with inflammation and premalignant pathology. The +28 nucleotide within the vacA 5′ stem-loop stratifies disease risk among toxic vacA i1-type strains.
doi:10.1136/jclinpath-2016-203641
PMCID: PMC5136723  PMID: 27189958
TOXIN; MICROBIAL PATHOGENIC; HELICOBACTER PYLORI; INFLAMMATION; GASTRIC PATHOLOGY
3.  Kinetics of versican-expressing macrophages in bone marrow after cord blood stem cell transplantation for treatment of acute myelogenous leukaemia 
Journal of Clinical Pathology  2016;69(10):906-911.
Aims
To determine versican-producing cells in normocellular bone marrow and to evaluate chronological alteration in the number of versican-producing macrophages in bone marrow of patients with acute myelogenous leukaemia (AML) after cord blood stem cell transplantation (CBSCT) to gain insight in the significance of versican in recovery of haematopoiesis.
Methods
We enrolled seven age-matched unrelated patients with normocellular bone marrow for determining versican-producing cells in bone marrow, CBSCT-treated patients with AML, 18 with fine and other four with poor engraftment, for determining chronological alteration of versican-expressing and CD68-expressing cells in transplanted bone marrow in reference to the total cells. Clot samples of patients with AML were collected from the +16 to +55 day after transplantation and separated into four groups. We included an AML case whose specimen was obtained on the +9 day. Cells positive in immunohistochemistry using antibodies to versican and CD68 were counted to obtain the mean±SD in a unit area of the bone marrow, plotted chronologically and compared with the numbers from the age-matched normocellular group.
Results
We determined by a double immunohistochemistry that the versican-expressing cells in bone marrow are macrophages. The time-course curve demonstrated an inverse relationship between the versican-positive macrophages and the total cells in the transplanted bone marrow for over 55 days. In bone marrow of poor engraftment cases, versican-positive macrophages appeared to be decreased in comparison with age-matched and sampling day-matched patients.
Conclusions
These results suggest that versican and/or versican-expressing macrophages positively contribute to bone marrow regeneration of patients with AML after CBSCT.
doi:10.1136/jclinpath-2015-203496
PMCID: PMC5050288  PMID: 26951084
BONE MARROW; STEM CELL TRANSPLANTS; MACROPHAGES; IMMUNOHISTOCHEMISTRY
4.  Cancer stem cells in moderately differentiated oral tongue squamous cell carcinoma express components of the renin–angiotensin system 
Journal of Clinical Pathology  2016;69(10):942-945.
doi:10.1136/jclinpath-2016-203736
PMCID: PMC5050289  PMID: 27371611
CANCER RESEARCH; CANCER STEM CELLS; HEAD AND NECK CANCER; IMMUNOHISTOCHEMISTRY; TUMOUR BIOLOGY
5.  Less frequently mutated genes in colorectal cancer: evidences from next-generation sequencing of 653 routine cases 
Journal of Clinical Pathology  2016;69(9):767-771.
Aims
The incidence of RAS/RAF/PI3KA and TP53 gene mutations in colorectal cancer (CRC) is well established. Less information, however, is available on other components of the CRC genomic landscape, which are potential CRC prognostic/predictive markers.
Methods
Following a previous validation study, ion-semiconductor next-generation sequencing (NGS) was employed to process 653 routine CRC samples by a multiplex PCR targeting 91 hotspot regions in 22 CRC significant genes.
Results
A total of 796 somatic mutations in 499 (76.4%) tumours were detected. Besides RAS/RAF/PI3KA and TP53, other 12 genes showed at least one mutation including FBXW7 (6%), PTEN (2.8%), SMAD4 (2.1%), EGFR (1.2%), CTNNB1 (1.1%), AKT1 (0.9%), STK11 (0.8%), ERBB2 (0.6%), ERBB4 (0.6%), ALK (0.2%), MAP2K1 (0.2%) and NOTCH1 (0.2%).
Conclusions
In a routine diagnostic setting, NGS had the potential to generate robust and comprehensive genetic information also including less frequently mutated genes potentially relevant for prognostic assessments or for actionable treatments.
doi:10.1136/jclinpath-2015-203403
PMCID: PMC5036215  PMID: 26797410
COLON; MOLECULAR PATHOLOGY; MOLECULAR ONCOLOGY
6.  PERIVASCULAR EPITHELIOID TUMOURS (PEComas) OF THE GYNAECOLOGICAL TRACT 
Journal of clinical pathology  2015;68(6):418-426.
Perivascular epithelioid tumour (PEComas) of the gynaecological tract are rare tumours which were first recognised and diagnosed within the last twenty years. They represent a unique diagnostic challenge with regard to their accurate and reproducible distinction from more common entities such as smooth muscle tumours of the uterine corpus. In this review article we trace the development of the concept of the PEComa tumour family, highlight what is known about extra-gynaecological tract PEComa at an immunohistochemical, molecular and therapeutic level and then present a summary of all reported cases of gynaecological tract PEComa to date. In the summary, we highlight rare subtypes of gynaecological tract PEComa, and compare the performances of extant prognostic classification systems for malignancy in these tumours.
doi:10.1136/jclinpath-2015-202945
PMCID: PMC4984252  PMID: 25750268
7.  T-cell abnormalities in common variable immunodeficiency: the hidden defect 
Journal of Clinical Pathology  2016;69(8):672-676.
This review discusses how the T-cell compartment in common variable immunodeficiency is marked by the premature arrest in thymic output, leading to T-cell exhaustion and immune dysregulation. Although B cells have been the main focus of the disorder, ample experimental data suggest that T-cell abnormalities can be seen in a large proportion of Freiburg Group 1a patients and those suffering from inflammatory complications. The reductions in T-cell receptor excision circles, naïve T cells, invariant NKT cells and regulatory T cells suggest a diminished thymic output, while CD8 T cells are driven towards exhaustion either via an antigen-dependent or an antigen-independent manner. The theoretical risk of anti-T-cell therapies is discussed, highlighting the need for an international effort in generating longitudinal data in addition to better-defined underlying molecular characterisation.
doi:10.1136/jclinpath-2015-203351
PMCID: PMC4975840  PMID: 27153873
IMMUNODEFICIENCY; AUTOIMMUNITY; CMV; IMMUNOGLOBULIN; LYMPHOCYTES
8.  Cancer stem cells in moderately differentiated oral tongue squamous cell carcinoma 
Journal of Clinical Pathology  2016;69(8):742-744.
doi:10.1136/jclinpath-2015-203599
PMCID: PMC4975854  PMID: 27095085
CANCER; CANCER RESEARCH; CANCER STEM CELLS; CARCINOMA; HEAD AND NECK CANCER
9.  Loss of histone variant macroH2A2 expression associates with progression of anal neoplasm 
Journal of Clinical Pathology  2015;69(7):627-631.
Aims
The macroH2A histone variants are epigenetic marks for inactivated chromatin. In this study, we examined the expression of macroH2A2 in anal neoplasm from anal intraepithelial neoplasia (AIN) to anal squamous cell carcinoma (SCC).
Methods
AIN and anal SCC samples were analysed for macroH2A2 expression, HIV and human papilloma virus (HPV). The association of macroH2A2 expression with clinical grade, disease recurrence, overall survival and viral involvement was determined.
Results
macroH2A2 was expressed in normal squamous tissue and lower grade AIN (I and II). Expression was lost in 38% of high-grade AIN (III) and 71% of anal SCC (p=0.002). Patients with AIN with macroH2A2-negative lesions showed earlier recurrence than those with macroH2A2-positive neoplasm (p=0.017). With anal SCC, macroH2A2 loss was more prevalent in the HPV-negative tumours.
Conclusions
Loss of histone variant macroH2A2 expression is associated with the progression of anal neoplasm and can be used as a prognostic biomarker for high-grade AIN and SCC.
doi:10.1136/jclinpath-2015-203367
PMCID: PMC4941135  PMID: 26658220
ANUS; HPV; HIV; CANCER
10.  Gene of the month: SMARCB1 
Journal of Clinical Pathology  2016;69(6):484-489.
SMARCB1 is the core subunit of the SWI/sucrose non-fermenting ATP-dependent chromatin remodelling complex located on the long arm of chromosome 22 (22q11.2). Since discovering genetic alterations of the SMARCB1 gene in malignant rhabdoid tumours, the family of tumours harbouring loss of SMARCB1 expression has been steadily expanding. In this review, we give a general overview of SMARCB1, its role in various cancers including germline mutations, association with genetic syndromes and role in future targeted therapies.
doi:10.1136/jclinpath-2016-203650
PMCID: PMC4914571  PMID: 26941181
MALIGNANT TUMOURS; CARCINOMA; GENETICS
11.  TGFα expression in myeloid malignancies 
Journal of Clinical Pathology  2016;69(6):543-546.
Background
Transforming growth factor α (TGFα) is a peptide growth factor known to be expressed in normal haemopoiesis. It is also expressed in a range of epithelial neoplasms but has not been assessed in haemopoietic malignancies. We have performed an immunohistochemical evaluation of TGFα in acute and chronic myeloid malignancies.
Methods
TGFα expression was semiquantitatively assessed in 69 normal bone marrow trephines and 157 cases of myeloid malignancy using an immunohistochemical approach.
Results
Blast cells of myeloid origin in acute myeloid leukaemia (AML), myelodysplasia and accelerated and blast phases of chronic myeloid leukaemia (CML) were TGFα positive. In acute promyelocytic leukaemia the neoplastic cells had significantly weaker TGFα expression than seen in other forms of AML. The blast cells in CML-accelerated and blast phases were positive with similar expression to AML.
Conclusions
TGFα is expressed in neoplastic myeloblasts and could, therefore, be used as blast cell biomarker in diagnostic haematopathology. In addition, TGFα immunohistochemistry may be of use in identifying a therapeutic target.
doi:10.1136/jclinpath-2015-203526
PMCID: PMC4893134  PMID: 26984929
HAEMATOPATHOLOGY; IMMUNOHISTOCHEMISTRY; HAEMATO-ONCOLOGY
12.  IgG is involved in the migration and invasion of clear cell renal cell carcinoma 
Journal of Clinical Pathology  2015;69(6):497-504.
OBJECTIVE:
To investigate if IgG can be expressed in clear cell renal cell carcinoma (cRCC) , and the expression of IgG is involved in the cancer progression. If IgG expression can serve as a potential target in cancer therapies and be used for judging the prognosis.
MATERIALS AND METHODS:
By immunohistochemistry, we detected IgG in cRCC tissues(75 cRCC tissues and75 adjacent normal kidney tissues). Immunofluorescence and Western blot was used to detect the IgG in cRCC cell lines (786-0, ACHN and CAKI-I). By RT-PCR, the functional transcript of IgG heavy chain was detected. Knockdown of IgG was to analyze the proliferation, migration and invasion ability by CCK8, Transwell and Matrigel and apoptosis in cRCC cell lines.
RESULTS:
By immunohistochemistry, we found strong staining of IgG in 66 cases of 75 cRCC tissues and 63 cases of 75 adjacent normal kidney tissues. Immunofluorescence and Western blot was found IgG in cRCC cell lines. Knock-down IgG in cRCC cell lines resulted in significant inhibition of cell proliferation, migration and invasion, and the induction of apoptosis of the 786-0 cells. The immunohistochemistry analysis showed that high IgG expression significantly correlated with the poor differentiation and advanced stage of cRCC.
CONCLUSION:
IgG was over expressed in cRCC and was involved in the proliferation, migration and invasion of cancer cells. IgG expression may serve as a potential target in cancer therapies and could be used for judging the prognosis.
doi:10.1136/jclinpath-2015-202881
PMCID: PMC4893138  PMID: 26519488
RENAL CANCER; TUMOUR MARKERS; TUMOUR BIOLOGY
13.  Clinicopathological analysis of near-tetraploidy/tetraploidy acute myeloid leukaemia 
Journal of clinical pathology  2015;68(3):236-240.
Aims
Near-tetraploidy/tetraploidy (NT/T) is a rare cytogenetic alteration in acute myeloid leukaemia (AML). NT/T-AML is categorised as complex cytogenetics and therefore, presumed to have an unfavourable prognosis. Our aim is to further characterise the clinical, morphological, cytogenetic and prognostic features of NT/T-AML.
Methods
We searched our cytogenetic laboratory database from 1991 to 2012 to reveal 13 cases of NT/T-AML. Each case was evaluated with regard to its demographics, morphology, immunophenotype and prognosis. Specific morphological features included blast size, irregularity of nuclear contours, cytoplasmic vacuoles, and presence and lineage of dysplasia.
Results
Eleven men and two women had a median age of 68 years. Blasts were predominately large (11/13). Eight of 13 patients had AML with myelodysplasia-related changes. Sixty-nine per cent of patients achieved complete remission (CR). Median overall survival (OS) was 8.6 months. CR rate and median OS in cases with ≥5 cytogenetic abnormalities were 71% and 6 months, compared with 67% and 18.1 months in cases with <5 abnormalities.
Conclusions
NT/T-AML occurs in older males, exhibits large blast size and is associated with myelodysplasia. Unlike previously reported data, our study reveals an overall better prognosis in this older population with NT/T-AML than was expected for a complex karyotype AML. Cytogenetic complexity independent of ploidy status did not greatly affect the high CR rates, but did appear to be a better estimation of prognostic risk in terms of median OS.
doi:10.1136/jclinpath-2014-202697
PMCID: PMC4886850  PMID: 25563333
14.  The importance of tissue handling of surgically removed breast cancer for an accurate assessment of the Ki-67 index 
Journal of Clinical Pathology  2015;69(3):255-259.
Aim
Insufficient attention for the Ki-67 immunohistochemistry has been given to the importance of tissue handling for surgical breast cancer specimens. We sought to investigate the effect of fixation status on the Ki-67.
Methods
We examined the effect of fixative, time to and duration of fixation using surgical specimens, and finally, compared the paired Ki-67 index in the tumour between core needle and surgical specimen.
Results
The Ki-67 was significantly higher when 10% neutral buffered formalin was used (p=0.0276). Insufficient fixation caused a drastic reduction in the Ki-67 index (p=0.0177), but not significant in oestrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2). Sixteen hours delayed time to fixation also caused a reduction of the Ki-67 (p=0.0284), but not significant in ER. Prolonged fixation significantly led to a gradual reduction in the Ki-67 in a time-dependent manner, but not in both ER and HER2. Finally, cutting the tumour before fixation improved fixation status and consequently caused an increased level of the Ki-67 index (p=0.0181), which resulted in a strong correlation of the Ki-67 between core needle and surgical specimen (r=0.8595).
Conclusions
Tissue handling of surgical specimen is critical for assessing the Ki-67 compared with ER and HER2. We should pay more attention to tissue fixation status for the standard assessment of the Ki-67 index.
doi:10.1136/jclinpath-2015-203174
PMCID: PMC4789709  PMID: 26420767
TUMOUR BIOLOGY; IMMUNOHISTOCHEMISTRY; BREAST CANCER; PROLIFERATION
15.  Correction 
doi:10.1136/jclinpath-2015-203414corr1
PMCID: PMC4789710  PMID: 26893481
16.  Mechanisms and therapeutic effectiveness of lactobacilli 
Journal of Clinical Pathology  2015;69(3):187-203.
The gut microbiome is not a silent ecosystem but exerts several physiological and immunological functions. For many decades, lactobacilli have been used as an effective therapy for treatment of several pathological conditions displaying an overall positive safety profile. This review summarises the mechanisms and clinical evidence supporting therapeutic efficacy of lactobacilli. We searched Pubmed/Medline using the keyword ‘Lactobacillus’. Selected papers from 1950 to 2015 were chosen on the basis of their content. Relevant clinical and experimental articles using lactobacilli as therapeutic agents have been included. Applications of lactobacilli include kidney support for renal insufficiency, pancreas health, management of metabolic imbalance, and cancer treatment and prevention. In vitro and in vivo investigations have shown that prolonged lactobacilli administration induces qualitative and quantitative modifications in the human gastrointestinal microbial ecosystem with encouraging perspectives in counteracting pathology-associated physiological and immunological changes. Few studies have highlighted the risk of translocation with subsequent sepsis and bacteraemia following probiotic administration but there is still a lack of investigations on the dose effect of these compounds. Great care is thus required in the choice of the proper Lactobacillus species, their genetic stability and the translocation risk, mainly related to inflammatory disease-induced gut mucosa enhanced permeability. Finally, we need to determine the adequate amount of bacteria to be delivered in order to achieve the best clinical efficacy decreasing the risk of side effects.
doi:10.1136/jclinpath-2015-202976
PMCID: PMC4789713  PMID: 26578541
MICROBIOLOGY; MICROBIAL PATHOGENIC; GASTROINTESTINAL DISEASE; GENERAL
17.  An immunohistochemical comparison of two TTF-1 monoclonal antibodies in atypical squamous lesions and sarcomatoid carcinoma of the lung, and pleural malignant mesothelioma* 
Journal of Clinical Pathology  2015;69(2):136-141.
Immunohistochemical detection of thyroid transcription factor-1 (TTF-1) plays an important role in the diagnosis and subclassification of non-small cell carcinomas of the lung in biopsy and some cytology samples, specifically for identification of squamous cell carcinoma (classically negative) and non-mucinous adenocarcinoma (positive in most cases) and for discrimination between lung adenocarcinoma and pleural malignant mesothelioma (classically negative).
Aims and methods
We carried out a comparison of the widely used mouse monoclonal TTF-1 antibody based on the 8G7G3/1 clone versus the more recently introduced rabbit monoclonal antibody (MAb) based on the SP141 clone.
Results
Both antibodies labelled alveolar epithelium in normal lung parenchyma, but the SP141 antibody also labelled bronchial mucosal basal cells. All 13 cases of atypical squamous lesions (including one case of bronchial squamous dysplasia) were negative with the 8G7G3/1 antibody, but 6/13 cases of squamous carcinoma/dysplasia showed positive nuclear labelling with the SP141 antibody in the same tissue biopsy. All 35 cases of adenocarcinoma of the lung were positive with both antibodies. For 12 cases of sarcomatoid carcinoma of the lung, two cases were labelled with the 8G7G3/1 antibody, whereas positive labelling of 4/12 cases was observed with SP141. All 66 cases of epithelioid malignant mesothelioma were negative with both antibodies, but 8/19 cases of sarcomatoid mesothelioma showed positive nuclear labelling with the SP141 antibody (0/19 with 8G7G3/1).
Conclusions
Our findings indicate differences in the rates of positive and negative labelling with these two antibodies, and suggest the potential for misclassification of a proportion of squamous carcinomas of the lung as adenocarcinoma, and for misdiagnosis of some sarcomatoid mesotheliomas as sarcomatoid carcinoma of the lung. If the results of SP141 are assigned overriding significance, our findings further indicate that in isolation, neither negative labelling with either 8G7G3/1 or SP141 nor positive labelling with the SP141 MAb discriminates between sarcomatoid carcinoma and sarcomatoid mesothelioma, whereas positive labelling with the 8G7G3/1 MAb favours a diagnosis of sarcomatoid carcinoma. The literature suggests that these seemingly ‘aberrant’ results with the SP141 antibody are not ‘false’ positives, but rather real detection of low levels of TTF-1 protein in a broader range of tumours than is widely recognised.
doi:10.1136/jclinpath-2015-203184
PMCID: PMC4752635  PMID: 26281863
ANTIBODIES; DIAGNOSIS; LUNG CANCER; METHODOLOGY
18.  Serine-arginine protein kinase 1 (SRPK1), a determinant of angiogenesis, is upregulated in prostate cancer and correlates with disease stage and invasion 
Journal of Clinical Pathology  2015;69(2):171-175.
Vascular endothelial growth factor (VEGF) undergoes alternative splicing to produce both proangiogenic and antiangiogenic isoforms. Preferential splicing of proangiogenic VEGF is determined by serine-arginine protein kinase 1 (SRPK1), which is upregulated in a number of cancers. In the present study, we aimed to investigate SRPK1 expression in prostate cancer (PCa) and its association with cancer progression. SRPK1 expression was assessed using immunohistochemistry of PCa tissue extracted from radical prostatectomy specimens of 110 patients. SRPK1 expression was significantly higher in tumour compared with benign tissue (p<0.00001) and correlated with higher pT stage (p=0.004), extracapsular extension (p=0.003) and extracapsular perineural invasion (p=0.008). Interestingly, the expression did not correlate with Gleason grade (p=0.21), suggesting that SRPK1 facilitates the development of a tumour microenvironment that favours growth and invasion (possibly through stimulating angiogenesis) while having little bearing on the morphology or function of the tumour cells themselves.
doi:10.1136/jclinpath-2015-203125
PMCID: PMC4752636  PMID: 26500332
PROSTATE; CANCER; ANGIOGENESIS
19.  Molecular mechanisms in multiple myeloma drug resistance 
Journal of Clinical Pathology  2015;69(2):97-101.
Multiple myeloma (MM) is predominantly an incurable malignancy despite high-dose chemotherapy, autologous stem cell transplant and novel agents. MM is a genetically heterogeneous disease and the complexity increases as the disease progresses to a more aggressive stage. MM arises from a plasma cell, which produces and secretes non-functioning immunoglobulins. Most MM cells are sensitive to proteasome inhibitors (PIs), which have become the main drug in the treatment of newly diagnosed and relapsed MM. However, not all MM is sensitive to PIs. This review summarises the literature regarding molecular biology of MM with a focus on the unfolded protein response and explores how this could affect drug sensitivity and progression of disease.
doi:10.1136/jclinpath-2015-203414
PMCID: PMC4752637  PMID: 26598624
MYELOMA; CANCER; HAEMATOLOGY; MOLECULAR BIOLOGY
20.  Comparison of KRAS mutation analysis of colorectal cancer samples by standard testing and next-generation sequencing 
Journal of clinical pathology  2014;67(9):764-767.
Aims
Based on KRAS testing, the subset of patients with metastatic colorectal cancer (CRC) that could benefit from anti-EGFR therapy can be better delineated. Though KRAS testing has become significantly more prevalent over the last few years, methods for testing remain heterogeneous and discordance has been reported between methods.
Methods
In this study, we examined a CRC patient population and compared KRAS testing done in Clinical Laboratory Improvement Amendments (CLIA) approved laboratories as part of standard clinical care and by next-generation sequencing (NGS) using the Illumina platform. Discordances were further evaluated with manual review of the NGS testing.
Results
Out of 468 CRC patient samples, 77 had KRAS testing done by both CLIA assay and NGS. There were concordant results between testing methodologies in 74 out of 77 patients, or 96% (95% CI 89% to 99%). There were three patient samples that showed discordant results between the two methods of testing. Upon further investigation of the NGS results for the three discordant cases, one sample showed a low level of the mutation seen in the standard testing, one sample showed low tumour fraction and a third did not show any evidence of the mutation that was found with the standard assay. Five patients had KRAS mutations not typically tested with standard testing.
Conclusions
Overall there was a high concordance rate between NGS and standard testing for KRAS. However, NGS revealed mutations that are not tested for with standard KRAS assays that might have clinical impact with regards to the role for anti-EGFR therapy.
doi:10.1136/jclinpath-2014-202405
PMCID: PMC4743643  PMID: 25004944
22.  Extraneural hemangioblastoma of the kidney: the challenge for clinicopathological diagnosis 
Journal of Clinical Pathology  2015;68(12):1020-1025.
Background
Hemangioblastoma is a benign cerebellar tumour which may occur as a sporadic entity or in association with von Hippel-Lindau (VHL) disease in approximately 25% of cases. Renal hemangioblastoma (RH) is an extremely rare and newly recognised tumour. Here, we describe five cases of RH, one discovered by CT in an accident and the other four detected during routine examinations.
Methods
Five cases of renal hemangioblastoma retrieved from the Department of Pathology, Fudan University Shanghai Cancer Center were studied and the literatures were reviewed. Immunohistochemistry was used to differentiate and confirm this tumour.
Results
Pathological examination following tumour resection revealed RH in all cases, the first patient was also diagnosed with renal cell carcinoma (RCC), suggesting the possibility of VHL syndrome, but PCR sequencing analysis of the VHL gene confirmed no mutation in any of the three exons, implying sporadic disease .Histologically, the tumours were circumscribed, composed of sheets of oval or polygonal cells and a prominent vascular network. Tumour cells had pleomorphic nuclei, but mitotic figures were rare. The diagnosis of hemangioblastoma was confirmed by immunohistochemistry.
Conclusions
RH is very rare and is challenging to differentially diagnose. Distinguishing RCC and RH is difficult and each has a different prognosis, so differentiating between them is essential for avoiding over-diagnosis and unnecessary treatment.
doi:10.1136/jclinpath-2015-202900
PMCID: PMC4717387  PMID: 26201545
KIDNEY; RENAL CANCER; IMMUNOHISTOCHEMISTRY
23.  Decreased argyrophilic nucleolar organiser region (AgNOR) expression in Purkinje cells: first signal of neuronal damage in sudden fetal and infant death 
Journal of Clinical Pathology  2015;69(1):58-63.
Aims
The nucleolus is an important cellular component involved in the biogenesis of the ribosome. This study was performed in order to validate the introduction of the argyrophilic nucleolar organiser region (AgNOR) stain technique, specific for the nucleoli detection, in neuropathological studies on sudden fetal and infant death.
Methods
In a wide set of fetuses and infants, aged from 27 gestational weeks to eight postnatal months and dead from both known and unknown causes, an in-depth neuropathological study usually applied at the Lino Rossi Research Center of the Milan University was implemented by the AgNOR method.
Results
Peculiar abnormalities of the nucleoli, as partial or total disruption above all in Purkinje cells (PCs), were exclusively found in victims of sudden fetal and infant death, and not in controls. The observed nucleolar alterations were frequently related to nicotine absorption in pregnancy.
Conclusions
We conclude that these findings represent early hallmarks of PC degeneration, contributing to the pathophysiology of sudden perinatal death.
doi:10.1136/jclinpath-2015-202961
PMCID: PMC4717426  PMID: 26567317
AGNORS; NEUROPATHOLOGY; SIDS
24.  Pre-trial inter-laboratory analytical validation of the FOCUS4 personalised therapy trial 
Journal of Clinical Pathology  2015;69(1):35-41.
Introduction
Molecular characterisation of tumours is increasing personalisation of cancer therapy, tailored to an individual and their cancer. FOCUS4 is a molecularly stratified clinical trial for patients with advanced colorectal cancer. During an initial 16-week period of standard first-line chemotherapy, tumour tissue will undergo several molecular assays, with the results used for cohort allocation, then randomisation. Laboratories in Leeds and Cardiff will perform the molecular testing. The results of a rigorous pre-trial inter-laboratory analytical validation are presented and discussed.
Methods
Wales Cancer Bank supplied FFPE tumour blocks from 97 mCRC patients with consent for use in further research. Both laboratories processed each sample according to an agreed definitive FOCUS4 laboratory protocol, reporting results directly to the MRC Trial Management Group for independent cross-referencing.
Results
Pyrosequencing analysis of mutation status at KRAS codons12/13/61/146, NRAS codons12/13/61, BRAF codon600 and PIK3CA codons542/545/546/1047, generated highly concordant results. Two samples gave discrepant results; in one a PIK3CA mutation was detected only in Leeds, and in the other, a PIK3CA mutation was only detected in Cardiff. pTEN and mismatch repair (MMR) protein expression was assessed by immunohistochemistry (IHC) resulting in 6/97 discordant results for pTEN and 5/388 for MMR, resolved upon joint review. Tumour heterogeneity was likely responsible for pyrosequencing discrepancies. The presence of signet-ring cells, necrosis, mucin, edge-effects and over-counterstaining influenced IHC discrepancies.
Conclusions
Pre-trial assay analytical validation is essential to ensure appropriate selection of patients for targeted therapies. This is feasible for both mutation testing and immunohistochemical assays and must be built into the workup of such trials.
Trial registration number
ISRCTN90061564.
doi:10.1136/jclinpath-2015-203097
PMCID: PMC4717430  PMID: 26350752
COLORECTAL CANCER; ANTIBODIES; LABORATORY TESTS; MOLECULAR PATHOLOGY
25.  Clinicopathological characteristics of RHOA mutations in a Central European gastric cancer cohort 
Journal of Clinical Pathology  2015;69(1):70-75.
Genomically stable gastric cancers (GCs) are enriched for the diffuse phenotype and hotspot mutations of RHOA. Here we aimed to validate the occurrence, phenotype and clinicopathological characteristics of RHOA mutant GCs in an independent Central European GC cohort consisting of 415 patients. The RHOA genotype (exon 2 and 3) was correlated with various genotypic, phenotypic and clinicopathological patient characteristics. Sixteen (3.9%) tumours had a RHOA mutation including four hitherto unreported mutations, that is, p.G17Efs*24, p.V24F, p.T37A and p.L69R. RHOA mutation was more prevalent in women (5.4% vs 2.8%), distal GCs (4.5% vs 2.4%), in poorly differentiated GCs (G3/G4; 4.8% vs 1.1%), T1/T2 tumours (6.2% vs 3.1%) and lacked distant metastases. Nine RHOA mutant GCs had a diffuse, four an intestinal, two an unclassified and one a mixed Laurén phenotype. KRAS and RHOA mutations were mutually exclusive. A single case showed both a RHOA and a PIK3CA mutation. No significant difference was found in the overall survival between RHOA mutant and wildtype GCs. Our study confirms the occurrence and clinicopathological characteristics of RHOA hotspot mutations in an independent patient cohort. However, we found no evidence for a prognostic or growth advantageous effect of RHOA mutations in GC.
doi:10.1136/jclinpath-2015-202980
PMCID: PMC4717431  PMID: 26251521
GASTRIC CANCER; GASTRIC PATHOLOGY; GENETICS

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