Assessment of a rapid liquid-based cytology method for measuring sputum cell counts.

Differential sputum cell counting is not widely available despite proven clinical utility in the management of asthma. We compared eosinophil counts obtained using liquid-based cytology (LBC), a routine histopathological processing method, and the current standard method. Eosinophil counts obtained using LBC were a strong predictor of sputum eosinophilia (≥3%) determined by the standard method suggesting LBC could be used in the management of asthma.

Analysis of lymphatic and blood vessel invasion biomarkers in T1 esophagogastric adenocarcinomas for improved patient prognostication.

Lymphovascular invasion (LVI) in T1 esophagogastric adenocarcinoma may predict risk of recurrence despite definitive treatment with surgery or endoscopic resection. Podoplanin and CD34 are emerging biomarkers of lymphatic and blood vessel invasion, respectively, and could be adopted to refine LVI assessment. A consecutive series of 65 patients with T1 adenocarcinomas diagnosed at Nottingham University Hospitals were investigated. T1 tumors from 43/65 patients who received primary surgery only were suitable for LVI evaluation by hematoxylin and eosin (H&E) staining as well as by CD34 and Podoplanin immunohistochemistry. LVI was correlated to clinicopathological features and recurrence free survival. H&E staining detected LVI in 11.6% (5/43) of T1 tumors. CD34 and Podoplanin immunohistochemistry significantly improved LVI detection to 25.6% (11/43). Compared with LVI by H&E, immunohistochemical evaluation of blood vessel invasion (CD34) or lymphatic vessel invasion (Podoplanin) was significantly associated with higher grade (P = 0.005), submucosal invasion (T1b) (P = 0.018), lymph node positivity (N1) (P = 0.029) and poor recurrence free survival (P = 0.0003). Our study provides evidence that CD34 and Podoplanin immunohistochemistry could improve LVI detection and allow better prognostication of patients and optimum selection of definitive treatment. Larger multicenter studies are required for further validation that could have significant clinical implications.

Tumour regression and ERCC1 nuclear protein expression predict clinical outcome in patients with gastro-oesophageal cancer treated with neoadjuvant chemotherapy.

AIMS: Neoadjuvant chemotherapy followed by surgery is the standard of care for patients with gastro-oesophageal adenocarcinoma. Previously, we validated the utility of the tumour regression grade (TRG) as a histopathological marker of tumour downstaging in patients receiving platinum-based neoadjuvant chemotherapy. In this study we profiled key DNA repair and damage signalling factors and correlated them with clinicopathological outcomes, including TRG response. METHODS AND RESULTS: Formalin-fixed human gastro-oesophageal cancers were constructed into tissue microarrays (TMAs). The first set consisted of 142 gastric/gastro-oesophageal cancer cases not exposed to neoadjuvant chemotherapy and the second set consisted of 103 gastric/gastro-oesophageal cancer cases exposed to preoperative platinum-based chemotherapy. Expressions of ERCC1, XPF, FANCD2, APE1 and p53 were investigated using immunohistochemistry. In patients who received neoadjuvant chemotherapy, favourable TRG response (TRG 1, 2 or 3) was associated with improvement in disease-specific survival (P=0.038). ERCC1 nuclear expression correlated with lack of histopathological response (TRG 4 or 5) to neoadjuvant chemotherapy (P=0.006) and was associated with poor disease-specific (P=0.020) and overall survival (P=0.040). CONCLUSIONS: We provide evidence that tumour regression and ERCC1 nuclear protein expression evaluated by immunohistochemistry are promising predictive markers in gastro-oesophageal cancer patients receiving neoadjuvant platinum-based chemotherapy.

Human apurinic/apyrimidinic endonuclease (APE1) is a prognostic factor in ovarian, gastro-oesophageal and pancreatico-biliary cancers.

BACKGROUND: Altered DNA repair may be associated with aggressive tumour biology and impact upon response to chemotherapy and radiotherapy. We investigated whether expression of human AP endonuclease (APE1), a key multifunctional protein involved in DNA BER, would impact on clinicopathological outcomes in ovarian, gastro-oesophageal, and pancreatico-biliary cancer. METHODS: Formalin-fixed human ovarian, gastro-oesophageal, and pancreatico-biliary cancers were constructed into TMAs. Expression of APE1 was analysed by IHC and correlated to clinicopathological variables. RESULTS: In ovarian cancer, nuclear APE1 expression was seen in 71.9% (97 out of 135) of tumours and correlated with tumour type (P=0.006), optimal debulking (P=0.009), and overall survival (P=0.05). In gastro-oesophageal cancers previously exposed to neoadjuvant chemotherapy, 34.8% (16 out of 46) of tumours were positive in the nucleus and this correlated with shorter overall survival (P=0.005), whereas cytoplasmic localisation correlated with tumour dedifferentiation (P=0.034). In pancreatico-biliary cancer, nuclear staining was seen in 44% (32 out of 72) of tumours. Absence of cytoplasmic staining was associated with perineural invasion (P=0.007), vascular invasion (P=0.05), and poorly differentiated tumours (P=0.068). A trend was noticed with advanced stage (P=0.077). CONCLUSIONS: Positive clinicopathological correlations of APE1 expression suggest that APE1 is a potential drug target in ovarian, gastro-oesophageal, and pancreatico-biliary cancers.

Cytological assessment of conventional transbronchial fine needle aspiration of lymph nodes.

OBJECTIVES: Transbronchial fine needle aspiration (TBNA) is a minimally invasive bronchoscopic technique that allows pathological examination of mediastinal and hilar lymph nodes. The aim of this study was to assess the cytopathological outcome of TBNA. METHODS: One hundred and eighty-seven patients who underwent TBNA of mediastinal and hilar lesions from May 2000 to June 2007 were reviewed. RESULTS: TBNA results were considered to be adequate if the cytological material revealed a malignant lesion or sufficient number of benign lymphoid cells. In the current study, 40 cases (21.9%) were reported as inadequate. When inadequate tests were excluded, the overall sensitivity and accuracy of TBNA in the diagnosis of malignant lesions were 83.5% and 88.0% respectively. The lowest sensitivity was noted in lymph node involvement by lymphoma. Regarding the workload associated with TBNA cytology, we found that the average number of conventionally prepared cytological slides per case was high (17 slides per case). CONCLUSION: Our results confirm that conventional TBNA is a sensitive and useful technique but it is relatively expensive and the protocols should be adapted to allow appropriate material to be collected for ancillary diagnostic tests.

Biomarkers of response to therapy in oesophago-gastric cancer.

Cancer of the oesophagus, gastro-oesophageal junction (GOJ) and stomach remains a major health problem worldwide. The evidence base for the optimal management of patients with operable oesophago-gastric cancer is evolving. Accepted approaches include preoperative chemotherapy followed by surgery (oesophageal cancer), chemo-radiotherapy alone (oesophageal cancer) and perioperative chemotherapy (gastric and gastro-oesophageal adenocarcinomas). The underlying principles behind neoadjuvant therapy are to improve resectability of the tumour by tumour shrinkage/downstaging and to treat occult metastatic disease as early as possible. The response rate to cytotoxic therapy is about 40% in oesophago-gastric cancer. Available evidence suggests that a favourable histopathological response to cytotoxic therapy may be a useful positive predictive marker in oesophago-gastric cancer. However, the ability to predict tumour response in routine clinical practice is difficult and is an area of intense investigation. There is evolving evidence for the role of predictive biomarkers in cancer in general and oesophago-gastric cancer in particular. We provide an overview on the current status of radiological and biological predictive biomarkers. We have focussed on clinical translational investigations and, where appropriate, provided pre-clinical insights. Whether predictive markers will be routinely incorporated in clinical practice remains to be seen as biomarker research is expensive and the data generated from these investigations are complex. It is clear that a concerted international effort between academia and industry is critical if personalised medicine as a practical reality for our cancer patients is to be realised.

A rare case of follicular thyroid carcinoma in ovarian struma.

Struma ovarii is a form of specialised mature teratoma, with predominantly mature thyroid tissue in an ovarian teratoma as seen in 2% of cases. Its malignant transformation is even rarer and is seen in only 5% of those cases. This 40-year-old female patient had an incidental finding of a pelvic mass during investigation of secondary amenorrhoea. She underwent a staging laparotomy and pelvic clearance. The histopathology revealed a bilateral mature teratoma of the ovary with follicular thyroid carcinoma in the right ovarian struma (malignant struma). A total thyroidectomy was performed followed by a whole body 31I scintigraphy which did not reveal any abnormal uptake of isotope. The patient remains well after four years and is being followed-up with serial serum thyroglobulin surveillance.

Postoperative survival following perioperative MAGIC versus neoadjuvant OE02-type chemotherapy in oesophageal adenocarcinoma.

The optimal management of resectable oesophageal adenocarcinoma is controversial, with many centres using neoadjuvant chemotherapy following the Medical Research Council (MRC) oesophageal working group (OE02) trial and the MRC Adjuvant Gastric Infusional Chemotherapy (MAGIC) trial. The more intensive MAGIC regimen is used primarily in gastric cancer but some also use it for oesophageal cancer. A database of cancer resections (2001-2013) provided information on survival of patients following either OE02 or MAGIC-type treatment. The data were compared using Kaplan-Meier analysis. Straight-to-surgery patients were also reviewed and divided into an 'early' cohort (2001-2006, OE02 era) and a 'late' cohort (2006-2013, MAGIC era) to estimate changes in survival over time. Subgroup analysis was performed for responders (tumour regression grade [TRG] 1-3) versus non-responders (TRG 4 and 5) and for anatomical site (gastro-oesophageal junction [GOJ] vs oesophagus). An OE02 regimen was used for 97 patients and 275 received a MAGIC regimen. Those in the MAGIC group were of a similar age to those undergoing OE02 chemotherapy but the proportion of oesophageal cancers was higher among MAGIC patients than among those receiving OE02 treatment. MAGIC patients had a significantly lower stage following chemotherapy than OE02 patients and a higher median overall survival although TRG was similar. On subgroup analysis, this survival benefit was maintained for GOJ and oesophageal cancer patients as well as non-responders. Analysis of responders showed no difference between regimens. 'Late' group straight-to-surgery patients were significantly older than those in the 'early' group. Survival, however, was not significantly different for these two cohorts. Although the original MAGIC trial comprised few oesophageal cancer cases, our patients had better survival with MAGIC than with OE02 chemotherapy in all anatomical subgroups, even though there was no significant change in operative survival over the time period in which these patients were treated. The use of the MAGIC regimen should therefore be encouraged in cases of operable oesophagogastric adenocarcinoma.

Expression of mesothelial markers in malignant mesotheliomas: an immunohistochemical evaluation of 173 cases.

OBJECTIVE: To see the distribution of Calretinin, thrombomodulin, CK5/6 and HBME-1 markers in various subtypes of mesotheliomas and extend the published data on this topic. The positivity of adenocarcinoma specific markers (CEA and BerEP4) in malignant mesotheliomas have also been evaluated. METHODS: Various markers in 173 cases of malignant mesotheliomas received over a period of 8 years were evaluated by immunohistochemistry. RESULTS: In majority of malignant mesotheliomas i.e., epithelioid and biphasic types, the positive staining patterns complement the gold standard histologic diagnosis. However, in a small minority mainly sarcomatoid variant, heavy reliance cannot be placed on these markers. CEA and BerEP4 are useful negative markers of mesotheliomas, although occasionally these are positive in clear cut mesotheliomas. CONCLUSIONS: Specificity of various markers in malignant mesotheliomas should be assessed according to histologic subtypes. The existing generation of markers is not reliable in diagnosis of sarcomatoid mesotheliomas. Fortunately this forms only a small group of mesothelial malignancy. In common epithelioid and biphasic variants calretinin, thrombomodulin, CK5/6, HBME-1 are sensitive positive markers whereas CEA and BerEP4 are negative markers of malignant mesotheliomas.

Vascular amyloidosis causing spontaneous mediastinal haemorrhage with haemothorax.

Bleeding diathesis is a recognised complication of amyloid disease. Localised and generalised bleeding manifestations are usually associated with intravascular coagulopathy related to isolated or multiple coagulation factor deficiencies. Recently, there have been reports of haemorrhage due to amyloid deposition in blood vessel walls and in the perivascular region leading to increased fragility and poor haemostasis. We report a case of spontaneous mediastinal haemorrhage due to amyloid involvement of vascular tissue in the absence of coagulopathy.

Distribution and estimation of nucleolar organizer regions in various human lung tumours.

A quantitative study of nucleolar organizer regions in human lung carcinomas was carried out on routinely processed paraffin embedded tissue sections. We examined 104 lung carcinomas including 38 squamous cell carcinomas, 36 adenocarcinomas, 18 large cell anaplastic carcinomas, 6 small cell carcinomas and 6 carcinoids. No significant differences were found in mean number of NORs between squamous, adenocarcinoma and undifferentiated carcinomas including large cell and small cell carcinomas. Carcinoids had comparatively lower means except for one typical carcinoid. Considering the high incidence of overlap between ranges of NOR counts in these groups of tumours and in agreement with the only other study of lung tumours (which comprised only carcinoids and small cell carcinomas), we conclude that this technique cannot be reliably used to discriminate between various histologic types of lung cancers. However, long term follow up of these patients is needed to establish the value of the AgNOR technique for prognostic guidance.

Assessment of nucleolar organizer regions (NORs) in proliferative conditions of the liver.

To overcome the diagnostic dilemma in proliferative conditions of the liver which sometimes pose a problem to the working pathologist especially when the material is inadequate, a special staining technique (AgNOR) has been applied. By using this technique, nucleolar organizer regions were counted which determine the proliferative status of the cells. This prospective study included 65 cases of randomly selected liver core and fine needle aspiration biopsies. AgNOR staining was performed on formalin-fixed, paraffin-embedded tissue sections NOR dots were counted in 100 randomly selected hepatocytes at x100 oil immersion objective, and the mean count per cell was calculated for each case. Statistical analysis was done by using the Mann Whitney U test. AgNOR count results were later compared with the histologic diagnosis. The study revealed a gradual increase in mean AgNOR counts from normal liver through cirrhosis to hepatocellular carcinoma. The difference in NOR counts was significant in these three groups. The hepatocellular carcinomas were graded according to the Edmondson-Steiner histological grading system. The Grade I hepatocellular carcinomas show AgNOR counts ranging between 5-6/cell, a score which is much higher than in the normal liver, where it ranges between 1.2-2.0/cell. This technique can be used to assess the lesions where the distinction between normal liver and Grade I hepatocellular carcinoma is difficult with the use of routine methods. AgNOR counts in normal liver and chronic hepatitis cases were insignificant, but there was an appreciable difference between cases of chronic hepatitis, cirrhosis and hepatocellular carcinoma. In view of the results of this study, the AgNOR staining method is found to be a useful diagnostic tool to differentiate between normal liver, cirrhosis and hepatocellular carcinoma and also to precisely discriminate between cases of normal liver and Grade I hepatocellular carcinoma.

Clinicopathological profile of Wilms' tumor.

The profile of renal tumors in children less than 15 years of age during the period 1991-1997 is presented. Among the 37 children with kidney tumors, 29 (78.4%) had Wilms' tumor. There was also a 20-year-old female with Wilms' tumor. The median age at presentation was 2.6 years (range 2.5 months to 20 years). 66.7% of the cases diagnosed were < or = 3 years and 90% were < or = 6 years. Five cases were under one year of age. The male to female ratio was 2:1. Twenty-two cases (73.3%) were triphasic and 7 (23.3%) were biphasic. Only one case was monophasic with blastemal component. Five cases (16.7%) showed nephrogenic rests in the uninvolved renal parenchyma and one case had nephroblastomatosis. The tumor was favorable in 26 cases (86.7%) and unfavorable in 4. Fourteen cases were in-patients while 16 were outside referrals. The pathological (10 cases whose specimens were sent from other centers) and clinicopathological (13 hospitalized patients) staging showed 10 cases (43.5%) with stage 1, 4 cases (17.4%) with stage 2, and 7 cases (30.4%) with stage 3. In two cases (8.7%), there was stage 4 disease. The length of the follow-up period in the 13 hospitalized patients ranged from 7 days to 5 years 5 months (median 14 months). There was one recurrence and one death after 2 years of diagnosis.

Predicting prognosis in lung cancer: use of proliferation marker, Ki67 monoclonal antibody.

An investigation was carried out to assess the prognostic significance of proliferation marker Ki67 in a group of lung cancer patients treated by surgery (limited disease). Tissue was not available for Ki67 immunostaining in inoperable group. The diagnosis is established by bronchial biopsy which does not carry enough tissue for frozen section and counting. This study is supplemented by estimating the prognostic significance of histological sub-types in the operable group and in a group of inoperable patients with extensive disease. These are usually treated by radiotherapy and/or chemotherapy. In all, 267 patients were studied including 105 treated by surgery. These patients attended King's College and Brompton Hospital, UK, between 1986 and 1989. With regard to proliferation marker Ki67 done for the surgical group, only patients with Ki67 scores of less than 5% did survive significantly longer than the rest. Histology did not make any significant contribution in determining prognosis in both operable and inoperable groups. Although follow-up is limited (mean 20 months), Ki67 antibody seems promising in identifying low and high grade disease in the initial stage of lung cancer. It may prove useful for category of patients with high scores to be placed on chemotherapy/radiotherapy. Results suggest that in the case of lung tumour, proliferative activity is a better prognostic indicator than histological type.

Association of Epstein Barr Virus (EBV) with nasopharyngeal carcinoma (NPC).

AIM: To observe the frequency of nasopharyngeal carcinoma (NPC) and its association with Epstein Barr Virus (EBV) infection. SETTING: This study included consecutive cases of nasopharyngeal carcinoma, which were diagnosed in the Department of Pathology at the Aga Khan University Hospital, Karachi in the period of two years (1996-97). METHODS: These tumors were initially evaluated on H&E stained sections. The tumors showing evidence of keratinization were excluded from the study. The Epstein Barr Virus was detected with the help of Polymerase chain reaction in formalin fixed, paraffin embedded tissue sections. RESULTS: During the study period, seventeen cases of nasopharyngeal carcinoma were diagnosed which comprised 0.3% of all malignant tumors. The age ranged from 5 years to 70 years with male to female ratio of 2.4:1. The NPC was more prevalent in adults (71%) as compared to children (29%) under 15 years. Six cases (35%) exhibited positive signal for Epstein Barr Virus. CONCLUSION: Nasopharyngeal carcinoma is an infrequent tumor. The prevalence of Epstein Barr virus infection in nasopharyngeal carcinoma is quite low as compared to other regions of the world.

Detection of Mycobacterium tuberculosis in paraffin embedded intestinal tissue specimens by polymerase chain reaction: characterization of IS6110 element negative strains.

Tuberculosis is still one of the most widespread infection known to mankind. Although lung is the predominant site of disease, a sizeable population in Pakistan gets intestinal disease. Clinical presentation, radiologic and endoscopic examination provide clues to the diagnosis. However, a definitive diagnosis requires biopsy material with granulomas and/or caseation complemented by acid fast staining and culture. There are many occasions when biopsy material is scanty and even in some intestinal resection cases histologic evaluation fails to confirm or rule out tuberculosis. Therefore, an investigation was conducted to assess the efficacy of PCR in the detection of mycobacterial DNA in paraffin embedded intestinal tissue. In this study 12 histologically confirmed cases of intestinal tuberculosis and 2 cases with non specific inflammation but clinically suspected for abdominal tuberculosis were selected. One case of rectal polyp was included to serve as a negative control. M. tuberculosis DNA was amplified in 8 out of 12 histologically confirmed cases and in 2 cases diagnosed with non specific inflammation. Amplified products were obtained in 6 out of 10 PCR positive specimens with IS6110 region specific primers while 4 samples were negative, suggesting the absence of insertion sequence 6110 in these strains. However, amplification was obtained in these negative specimens with a second primer pair confirming them as M. tuberculosis complex species. On the basis of this study we conclude that; (1) Processed and paraffin embedded tissue material is suitable for PCR analysis, (2) PCR assay can be used to complement the diagnosis of intestinal tuberculosis especially in situations where a definite conclusion can not be drawn by conventional methods, (3) M. tuberculosis species lacking insertion sequence 6110 element are present in our population. Therefore, several primer pair sets should be included when applying PCR for the detection of mycobacterial DNA.

Morphological pattern and frequency of thyroid tumors.

OBJECTIVE: The present study was done to evaluate the frequency of thyroid cancer and to find out the prevalence of histological types of thyroid tumor with respect to age and sex group. SETTING: This study included consecutive cases of malignant tumors of thyroid gland, which were diagnosed in the Department of Pathology at the Aga Khan University Hospital, Karachi during the period of three years (1995-1997). METHODS: These cases were evaluated on H & E stained sections from paraffin embedded 10% buffered formalin fixed tissue blocks. Special stains and immunohistochemical analysis were performed whenever required. RESULTS: A total of 8541 malignant tumors were diagnosed in a period of 3 years which included 103 (1.2%) cases of thyroid cancer. Thyroid tumors were more prevalent in females with female to male ratio of 2.6:1. Papillary carcinoma (69%) was the most common histological type of thyroid tumors, followed by follicular carcinoma (11.6%), medullary carcinoma (9.7%), anaplastic carcinoma (5.9%), non-Hodgkin's lymphoma (2.9%) and unclassified tumors (0.9%) in order of frequency. CONCLUSION: Thyroid cancer was more common in females. Papillary carcinoma was the most common histological type of thyroid tumors in females as well as in males. Papillary carcinoma was more prevalent in third, fourth and fifth decades of life while follicular and anaplastic carcinomas were more frequent after the fourth decade of life.

p53 and PCNA expression in benign, atypical and malignant meningiomas.

OBJECTIVE: Alterations: p53 genes are turning out to be the most common genetic alterations in human cancers. Due to long half-life of mutated p53, its detection is possible by immunohistochemistry. Proliferating cell nuclear antigen (PCNA) is expressed by dividing cells, hence has been shown to correlate with prognosis. We have used monoclonal antibodies protein DO-7 (p53) and PC10 (PCNA) to see whether their expression correlates with histological grading in meningethelial tumour. MATERIAL AND METHODS: Twenty nine meningiomas (20 benign, 7 atypical and 2 malignant) were selected from the records of our laboratory. p53 and PCNA expression was sought by immunohistochemistry using Peroxidase Anti Peroxidase (PAP) technique. RESULTS: Four benign and 2 atypical meningiomas showed weak staining for p53. Both malignant meningiomas showed strong positivity for p53. Six benign meningiomas had less than 5% PCNA positivity, one 10% positivity and three showed 20% positivity. PCNA positivity ranged for 10-80% in atypical meningiomas. In two malignant meningiomas PCNA positivity was 70% and 90%. CONCLUSION: It is worthwhile to include p53 and PCNA expression along with histologic assessment in predicting outcome of meningiomas. A larger series with complete follow-up is essential in assessing value of these markers which unfortunately remains a dream in our country.

Spectrum of cutaneous appendage tumors at Aga Khan University Hospital.

OBJECTIVE: To determine the frequency of various types of cutaneous appendage tumors in our practice. METHOD: This is a partly retrospective and partly prospective study conducted at the Department of Pathology, Histopathology Section, The Aga Khan University Hospital, Karachi between 1st January 1997 and 31st December 2001. RESULTS: One hundred sixty six skin appendage tumors were diagnosed during the study period. 87.3% were benign, while 12.6% were malignant. Male female ratio was almost equal. Mean age was 41.72 years. 37.34% showed eccrine differentiation, 14.45% showed apocrine differentiation and 41.56% showed pilosebaceous differentiation, 6.62% exhibited mixed differentiation. The 5 commonest tumors were pilomatricoma, nodular hidradenoma (eccrine acrospiroma), syringocystadenoma papilleferum, eccrine poroma and eccrine spiradenoma. The commonest malignant tumors were porocarcinoma and sebaceous carcinoma. Pilomatricoma were common in children. CONCLUSION: Most of our findings roughly correlate with the western published data. However, commonest site for eccrine poromas in our study was head and neck. Also, not a single case of eccrine spiradenoma was seen in the first two decades of life. These findings differ significantly from western data.