Diagnosis of cystic lung diseases: a position statement from the UK Cystic Lung Disease Rare Disease Collaborative Network.

BACKGROUND: Rare cystic lung diseases are increasingly recognised due the wider application of CT scanning making cystic lung disease management a growing part of respiratory care. Cystic lung diseases tend to have extrapulmonary features that can both be diagnostic but also require surveillance and treatment in their own right. As some of these diseases now have specific treatments, making a precise diagnosis is crucial. While Langerhans cell histiocytosis, Birt-Hogg-Dubé syndrome, lymphoid interstitial pneumonia and lymphangioleiomyomatosis are becoming relatively well-known diseases to respiratory physicians, a targeted and thorough workup improves diagnostic accuracy and may suggest other ultrarare diseases such as light chain deposition disease, cystic pulmonary amyloidosis, low-grade metastatic neoplasms or infections. In many cases, diagnostic information is overlooked leaving uncertainty over the disease course and treatments. AIMS: This position statement from the Rare Disease Collaborative Network for cystic lung diseases will review how clinical, radiological and physiological features can be used to differentiate between these diseases. NARRATIVE: We highlight that in many cases a multidisciplinary diagnosis can be made without the need for lung biopsy and discuss where tissue sampling is necessary when non-invasive methods leave diagnostic doubt. We suggest an initial workup focusing on points in the history which identify key disease features, underlying systemic and familial diseases and a clinical examination to search for connective tissue disease and features of genetic causes of lung cysts. All patients should have a CT of the thorax and abdomen to characterise the pattern and burden of lung cysts and extrapulmonary features and also spirometry, gas transfer and a 6 min walk test. Discussion with a rare cystic lung disease centre is suggested before a surgical biopsy is undertaken. CONCLUSIONS: We suggest that this focused workup should be performed in all people with multiple lung cysts and would streamline referral pathways, help guide early treatment, management decisions, improve patient experience and reduce overall care costs. It could also potentially catalyse a national research database to describe these less well-understood and unidentified diseases, categorise disease phenotypes and outcomes, potentially leading to better prognostic data and generating a stronger platform to understand specific disease biology.

Histological clues to the diagnosis of metastasis to the breast from extramammary malignancies.

AIMS: The aims of this study were to review the histological features useful for the identification of metastases to the breast and to investigate the impression that this diagnosis has become more common. METHODS AND RESULTS: The histological features of metastases to the breast from 2008 to 2018 were reviewed. Seventy-four biopsies from 66 patients were identified: 1% compared with primary carcinoma of the breast. Non-haematological metastases comprised 0.75% compared with 0.3% in a series from 1996 to 2005. The most common tumour types were pulmonary carcinoma (22), lymphoma (15), melanoma (13), gastrointestinal carcinoma (eight) and serous papillary carcinoma (four). In 73% there were histological features that were not typical of primary mammary carcinoma. Some metastases were histologically similar to breast cancer and the history was essential to making the correct diagnosis. Useful histological clues included small-cell morphology for pulmonary carcinoma, glands containing necrosis for gastrointestinal carcinoma, intranuclear inclusions, marked pleomorphism and spindle cells for melanoma, clear cells for renal carcinoma, papillary architecture for serous papillary carcinoma and sheets of centroblasts or nodules of centroblasts and centrocytes for lymphoma. Useful immunohistochemical markers included TTF-1 for pulmonary carcinoma, S100, melan-A and HMB45 for melanoma, CK20 and CDX2 for colorectal carcinoma, PAX8 and WT1 for serous papillary carcinoma and lymphoid markers for lymphomas, in addition to the absence of expression of mammary markers ER, GATA3 and GCDFP-15. CONCLUSION: The majority of metastases to the breast have histological clues to the diagnosis. Immunohistochemistry is helpful. This diagnosis is being made more frequently.

Idiopathic chronic productive cough and response to open-label macrolide therapy: An observational study.

BACKGROUND AND OBJECTIVE: Adult patients with chronic productive cough of unknown cause are commonly seen in respiratory clinics. We have previously described a subgroup of these patients who have a short-lived response to standard antibiotic treatment but a prolonged response to 3 months of low-dose azithromycin therapy. METHODS: This observational study describes the physiological, radiological and pathological features of this patient cohort along with their response to a 12-week open-label trial of 250 mg azithromycin thrice weekly. RESULTS: A total of 30 subjects with a mean age of 57 were recruited. The majority demonstrated airway dilatation on high-resolution computed tomography (HRCT) scan without evidence of established bronchiectasis (n = 21) and non-specific chronic inflammatory changes on bronchial biopsy (n = 15/17). Twenty-nine subjects completed 3 months of azithromycin with a significant improvement in median Leicester Cough Questionnaire (LCQ) score (-6.3 points, P < 0.00001), reduction in median 24-h sputum volume (-5.8 mL, P = 0.0003) and improvement in sputum colour (P = 0.003). Patients responsive to azithromycin (n = 22) demonstrated neutrophilic or paucigranulocytic airway inflammation, whereas five subjects with eosinophilic airways inflammation did not respond symptomatically to azithromycin. CONCLUSION: We describe a cohort of patients with chronic productive cough not adequately described by existing disease labels whose symptoms responded well to low-dose azithromycin. Many of the features are similar to the paediatric condition protracted bacterial bronchitis.

Matrix Metalloproteinase-1 Activation Contributes to Airway Smooth Muscle Growth and Asthma Severity.

RATIONALE: Matrix metalloproteinase-1 (MMP-1) and mast cells are present in the airways of people with asthma. OBJECTIVES: To investigate whether MMP-1 could be activated by mast cells and increase asthma severity. METHODS: Patients with stable asthma and healthy control subjects underwent spirometry, methacholine challenge, and bronchoscopy, and their airway smooth muscle cells were grown in culture. A second asthma group and control subjects had symptom scores, spirometry, and bronchoalveolar lavage before and after rhinovirus-induced asthma exacerbations. Extracellular matrix was prepared from decellularized airway smooth muscle cultures. MMP-1 protein and activity were assessed. MEASUREMENTS AND MAIN RESULTS: Airway smooth muscle cells generated pro-MMP-1, which was proteolytically activated by mast cell tryptase. Airway smooth muscle treated with activated mast cell supernatants produced extracellular matrix, which enhanced subsequent airway smooth muscle growth by 1.5-fold (P < 0.05), which was dependent on MMP-1 activation. In asthma, airway pro-MMP-1 was 5.4-fold higher than control subjects (P = 0.002). Mast cell numbers were associated with airway smooth muscle proliferation and MMP-1 protein associated with bronchial hyperresponsiveness. During exacerbations, MMP-1 activity increased and was associated with fall in FEV1 and worsening asthma symptoms. CONCLUSIONS: MMP-1 is activated by mast cell tryptase resulting in a proproliferative extracellular matrix. In asthma, mast cells are associated with airway smooth muscle growth, MMP-1 levels are associated with bronchial hyperresponsiveness, and MMP-1 activation are associated with exacerbation severity. Our findings suggest that airway smooth muscle/mast cell interactions contribute to asthma severity by transiently increasing MMP activation, airway smooth muscle growth, and airway responsiveness.

The role of adjuvant platinum-based chemotherapy in esophagogastric cancer patients who received neoadjuvant chemotherapy prior to definitive surgery.

BACKGROUND AND OBJECTIVES: For patients with operable esophagogastric cancer, peri-operative chemotherapy confers a significant overall survival benefit compared to surgery alone, however only 30-40% of patients demonstrate histopathological response. It is unclear whether those with no neoadjuvant chemotherapy response should go onto receive adjuvant chemotherapy, as no further benefit may be conferred. METHODS: Esophagogastric cancers were prospectively captured with associated histopathological tumor regression grades following neoadjuvant chemotherapy. This cohort was then interrogated for clinico-pathological and survival outcomes. RESULTS: Following neoadjuvant chemotherapy and surgery, patients with chemotherapy responsive cancers, who were administered adjuvant chemotherapy gained a significant overall survival benefit. Multivariate Cox analysis, demonstrated a final adjusted hazard ratio for adjuvant therapy of 0.509; (95%CI 0.28-0.93); P = 0.028. In contrast, patients with non-responsive tumors, who underwent adjuvant chemotherapy, did not show any survival benefit. Chemotherapy toxicity was prevalent and contributed to only half of patients receiving adjuvant chemotherapy. CONCLUSIONS: These results suggest the benefit of the adjuvant portion of chemotherapy is limited to those who demonstrate a histopathological response to neoadjuvant chemotherapy. The administration of the adjuvant portion of chemotherapy to patients without a response to neoadjuvant chemotherapy may not provide any survival benefit, while potentially causing increased morbidity.

Dysplasia in Barrett's oesophagus: p53 immunostaining is more reproducible than haematoxylin and eosin diagnosis and improves overall reliability, while grading is poorly reproducible.

AIMS: p53 immunostaining in Barrett's oesophagus (BO) has been shown to be predictive of progression, but data regarding its generalizability to routine practice are lacking. This study compared the reliability of p53 and dysplasia interpretation and grading. METHODS AND RESULTS: Seventy-two cases encompassing the full spectrum of BO were circulated to 10 pathologists from four institutions after a brief training session in p53 interpretation. Each pathologist classified cases on haematoxylin and eosin (H&E) alone using the Vienna classification and assessed the p53 staining using a qualitative system. Agreement was assessed using kappa statistics. For the four-tier Vienna system, the average unweighted kappa was 0.30. Weighted kappa values varied from 0.27 to 0.69 with an average of 0.47. When grouped into definite dysplasia versus no definite dysplasia the average kappa was 0.55, but the kappa for low-grade dysplasia (LGD) versus high-grade dysplasia (HGD) was only 0.31. For p53, using the three recognized patterns, the unweighted kappa was 0.6 (confidence interval 0.58-0.63). When cases were evaluated with both H&E and p53 the average kappa was 0.61 for definite dysplasia versus the rest. CONCLUSIONS: p53 immunohistochemistry interpretation is more reliable than dysplasia diagnosis, even with limited training. As it is predictive of prognosis and improves diagnostic reproducibility, it is suitable for routine use by pathologists as an adjunct to dysplasia diagnosis. The distinction of LGD versus HGD was poor. This study supports simplifying dysplasia diagnosis into 'present', 'indefinite' or 'absent', and the use of p53 as an ancillary marker in difficult cases. This should help to prevent overdiagnosis of dysplasia and inappropriate treatment.

BAI3, CDX2 and VIL1: a panel of three antibodies to distinguish small cell from large cell neuroendocrine lung carcinomas.

AIMS: Discriminating small-cell lung carcinoma (SCLC) from large-cell neuroendocrine carcinoma (LCNEC) rests on morphological criteria, and reproducibility has been shown to be poor. We aimed to identify immunohistochemical markers to assist this diagnosis. METHODS AND RESULTS: Gene expression profiling on laser captured frozen tumour samples from eight SCLC and eight LCNEC tumours identified a total of 888 differentially expressed genes (DEGs), 23 of which were validated by qRT-PCR. Antibodies to four selected gene products were then evaluated as immunohistochemical markers on a cohort of 173 formalin-fixed paraffin-embedded (FFPE) SCLC/LCNEC tumour samples, including 26 indeterminate tumours without a consensus diagnosis. Three markers, CDX2, VIL1 and BAI3, gave significantly different results in the two tumour types (P < 0.0001): CDX2 and VIL1 in combination (either marker positive) showed sensitivity and specificity of 81% for LCNEC while BAI3 showed 89% sensitivity and 75% specificity for SCLC. Of the 26 indeterminate tumours 15 (58%) showed an immunophenotype suggesting either SCLC or LCNEC, eight (31%) showed staining of both tumour types, and three (11%) were negative for all markers. CONCLUSION: A panel of three markers, BAI3, CDX2 and VIL1, is a useful adjunct in the diagnosis of these tumour types.

Prevention is better than Cure - Healthcare Systems in United Kingdom and Pakistan.

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Landscape of cancer biomarker testing in England following genomic services reconfiguration: insights from a nationwide pathologist survey.

AIMS: Cancer diagnostics have been evolving rapidly. In England, the new National Health Service Genomic Medicine Service (GMS) provides centralised access to genomic testing via seven regional Genomic Laboratory Hubs. The PATHways survey aimed to capture pathologists' experience with current diagnostic pathways and opportunities for optimisation to ensure equitable and timely access to biomarker testing. METHODS: A nationwide survey was conducted with consultant pathologists from regional laboratories, via direct interviews based on a structured questionnaire. Descriptive analysis of responses was undertaken using quantitative and qualitative methods. RESULTS: Fifteen regional centres completed the survey covering a median population size of 2.5 (1.9-3.6) million (each for n=12). The median estimated turnaround time (calendar days) for standard molecular markers in melanoma, breast and lung cancers ranged from 2 to 3 days by immunohistochemistry (excluding NTRKfus in breast and lung cancers, and PD-L1 in melanoma) and 6-15 days by real-time-PCR (excluding KIT for melanoma), to 17.5-24.5 days by next-generation sequencing (excluding PIK3CA for breast cancer). Tests were mainly initiated by pathologists and oncologists. All respondents discussed the results at multidisciplinary team (MDT) meetings. The GMS roll-out was perceived to have high impact on services by 53% of respondents, citing logistical and technical issues. Enhanced education on new pathways, tissue requirements, report interpretation, providing patient information and best practice sharing was suggested for pathologists and other MDT members. CONCLUSION: Our survey highlighted the role of regional pathology within the evolving diagnostic landscape in England. Notable recommendations included improved communication and education, active stakeholder engagement, and tackling informatics barriers.

A comparative characterization of indigenous keratinase enzymes from district Khairpur, Sindh, Pakistan.

To isolate and characterize keratinolytic fungi and bacteria from indigenous soils, a total of 80 samples were collected from Ghari Mori District. Khairpur, and these organisms were isolated using standard microbiological technique. The isolated keratinolytic microorganisms comprised: Absidia sp., Chrysosporium asperatum, Chrysosporium keratinophilum, Entomophthora coronata, Bacillus subtilis and Staphylococcus aureus and their keratinolytic properties were distinguished from the production of keratinase by measurement of zone of hydrolysis on skimmed milk agar (p<0.05). C.keratinophylum and B. subtilis produced largest zone among all the isolated species. The crude keratinase revealed that the optimum time for production of the enzyme was seven days, optimum temperature 30°C and optimum pH 9 for C.keratinophylum but for B. subtilis, the optimum time was three days, optimum temperature 37°C and optimum pH 7. The enzyme activity of C. keratinophylum and B. subtilis were determined to be 220 U/ml and 260 U/ml respectively (P<0.05).

Pathology of thymoma-where are we today?

Thymoma is the commonest epithelial neoplasm arising from thymus gland. Tumour is slow growing and in the absence of metastasis, surgery is the treatment of choice. Complete resection and bland morphology are important prognostic features. However, a significant proportion of these tumours tend to recur. These recurrent tumours, advanced thymomas and thymic carcinomas require platinum-based combination chemotherapy and radiotherapy. Efforts are being made to explore additional treatment modalities to control disease with the aim of improving survival. Number of thymoma cases worldwide is small in comparison to lung cancers. As a result, fewer studies have been carried out to enhance our understanding of molecular events responsible for the initiation, maintenance, and progression of thymomas. Inspite of this there are advances in understanding the pathology of thymic epithelial neoplasms including genetics, PD-L1 and molecular testing which has bearing on the prognosis, post-surgical management, and testing algorithm. Similar to pulmonary pathology, thymic epithelial tumours will require adequate tumour sampling to carry out ancillary testing. Mutational analytical tests include EGFR, RAS, BRAF, RET, AKT1, PIK3CA and T53 genes. If adequate sample is available (upto100 cells), PD-L1 testing should be considered for immunotherapy in recurrent/ advanced thymomas and thymic carcinomas. This list is likely to expand in future with increasing emphasis on molecular testing to support treatment with newer therapies.

Evolution of lung pathology in lymphangioleiomyomatosis: associations with disease course and treatment response.

Lymphangioleiomyomatosis (LAM) is a rare multisystem disease with a variable clinical course. The lungs are infiltrated by nodules of LAM cells, stromal cells and inflammatory cells, causing lung cysts and respiratory failure. We used immunohistochemical markers in lung biopsy and transplant samples from a national cohort of women with LAM with linked clinical data to understand how LAM nodule cell populations changed with disease progression. Marker distribution was examined qualitatively by dual immunohistochemistry, and markers for LAM cells, fibroblasts, lymphatics, mast cells, proliferation, cathepsin K and mTOR pathway activity were quantitated in LAM nodules and compared with clinical features and prospective lung function loss. The LAM cell marker PNL2 was more extensively expressed in those with higher forced expiratory volume in one second (FEV1 ), higher diffusion in the lung for carbon monoxide (DLCO ) and less extensive disease involvement whilst the converse was true for the protease cathepsin K. Each percentage increase in cathepsin K reactivity was associated with a 0.65% decrease in FEV1 (95% CI -1.11 to -0.18) and a 0.50% decrease in DLCO (95% CI -0.96 to -0.05). Higher reactivity to the mTOR complex 1 activation marker, phospho-ribosomal protein S6, was associated with a better lung function response to rapamycin (p = 0.0001). We conclude that LAM nodules evolve with disease progression, with LAM cells becoming outnumbered by fibroblasts. Increasing cathepsin K expression is associated with more severe disease and lung function loss. Markers of mTOR activation predict the response to rapamycin, suggesting that more advanced LAM may be less mTOR responsive and treatments specifically targeted towards LAM associated fibroblasts may have roles as adjuncts to mTOR inhibition.

Tumour regression grade (TRG) analyses in patients with resectable gastro-oesophageal adenocarcinomas treated with platinum-based neoadjuvant chemotherapy.

AIMS: Neoadjuvant chemotherapy followed by surgery is the standard of care for patients with gastro-oesophageal adenocarcinoma. The aims were to validate the utility of the tumour regression grade (TRG) in patients who have received chemotherapy and to investigate if (i) TRG correlates with tumour downstaging and (ii) TRG could provide a comparative platform for future predictive biomarker investigations. METHODS AND RESULTS: Three pathologists were blinded to the treatment approaches. Review included diagnosis, tumour grade, TNM staging, vascular invasion, perineural invasion, resection margin involvement and histopathological response to chemotherapy, as measured by TRG. In the neoadjuvant chemotherapy (CS) group (n = 84), 46.7% of gastric/gastro-oesophageal junction adenocarcinomas, and 45.5% of lower third oesophageal adenocarcinomas had TRG 1, 2 or 3 compared with 13.7% in the primary surgery group (n = 124) (P < 0.001 and P = 0.006, respectively). In the CS group, responders (TRG 1, 2 or 3) showed significant tumour downstaging [early ypT-stage disease (P = 0.002)]. In gastric cancers specifically, additional associations were seen with negative nodal disease (P = 0.044) and absence of vascular invasion (P = 0.027). CONCLUSIONS: TRG may reflect response to chemotherapy. In addition, positive correlations between TRG and ypTNM staging were demonstrated that would suggest tumour downstaging.

Barrett's dysplasia and the Vienna classification: reproducibility, prediction of progression and impact of consensus reporting and p53 immunohistochemistry.

AIMS: The Vienna classification is used to classify dysplasia in Barrett's oesophagus (BO), but reproducibility and value of diagnosis of lower grades in particular are often questioned. The aim was to test the diagnostic variability and correlation with patient outcome and to attempt to define histological features causing discrepant diagnoses, as well as to test the impact of adding p53 immunohistochemistry on reproducibility and prediction of outcome. METHODS AND RESULTS: One hundred and forty-three patients with 154 sets of biopsy specimens originally diagnosed with Barrett's dysplasia were retrieved from the pathology records of Nottingham University Hospital. Thirty-two Barrett's patients without dysplasia were added. Anonymized slides were graded independently by five pathologists without and with p53-stained slides. Interobserver variation, correlation with outcome and diagnostic accuracy were determined. Weighted kappa scores between pairs of pathologists showed substantial agreement and improved after p53 immunohistochemistry. Agreement with the original diagnosis was substantially lower. Fourteen of 34 low-grade dysplasias (LGD) and 27 of 30 high-grade dysplasias on consensus progressed within 10 years compared with 18/94 and 28/39 of original diagnoses. Progression correlated with p53 positivity. CONCLUSION: The Vienna classification is useful and reproducible in BO. Consensus diagnosis by gastrointestinal pathologists produces high specificity and predictive value, even for LGD. p53 immunohistochemistry assists in diagnosis in difficult cases and predicts progression.

Ex vivo Raman spectroscopy mapping of lung tissue: label-free molecular characterization of nontumorous and cancerous tissues.

Raman spectroscopy mapping was used to study ex vivo fresh lung tissues and compare to histology sections. The Raman mapping measurements revealed differences in the molecular composition of normal lung tissue, adenocarcinoma, and squamous cell carcinoma (SCC). Molecular heterogeneity of the tissue samples was well captured by the k -means clustering analysis of the Raman datasets, as confirmed by the correlation with the adjacent haematoxylin and eosin (H&E) stained tissue sections. The results indicate that the fluorescence background varies considerably even in samples that appear structurally uniform in the H&E images, both for normal and tumor tissue. The results show that characteristic Raman bands can be used to discriminate between tumorous and nontumorous lung tissues and between adenocarcinoma and SCC tissues. These results indicate the potential to develop Raman classifications models for lung tissues based on the Raman spectral differences at the microscopic level, which can be used for tissue diagnosis or treatment stratification.

Stratification of resectable lung adenocarcinoma by molecular and pathological risk estimators.

BACKGROUND: Mortality in early stage, resectable lung cancer is sufficiently high to warrant consideration of post-surgical treatment. Novel markers to stratify resectable lung cancer patients may help with the selection of treatment to improve outcome. METHODS: Primary tumour tissue from 485 patients, surgically treated for stage I-II lung adenocarcinoma, was analysed for the RNA expression of 31 cell cycle progression (CCP) genes by quantitative polymerase chain reaction (PCR). The expression average, the CCP score, was combined with pathological stage into a prognostic score (PS). Cox proportional hazards regression assessed prediction of 5-year lung cancer mortality above clinical variables. The PS threshold was tested for risk discrimination by the Mantel-Cox log-rank test. RESULTS: The CCP score added significant information above clinical markers (all patients, P=0.0029; stage I patients, P=0.013). The prognostic score was a superior predictor of outcome compared to pathological stage alone (PS, P=0.00084; stage, P=0.24). Five-year lung cancer mortality was significantly different between the low-risk (90%, 95% confidence interval (CI) 81-95%), and high-risk groups (65%, 95% CI 57-72%), P=4.2×10(-6)). CONCLUSIONS: The CCP score is an independent prognostic marker in early stage lung adenocarcinoma. The prognostic score provides superior risk estimates than stage alone. The threefold higher risk in the high-risk group defines a subset of patients that should consider therapeutic choices to improve outcome.

Histological determinants of survival in completely resected T1-2N1M0 nonsmall cell cancer of the lung.

BACKGROUND: The histologic determinants of survival after surgical resection of stage II nonsmall cell lung cancer are poorly understood. We analyzed the prognostic significance of a number of histologic features after complete resection of T1-2N1M0 nonsmall cell cancer of the lung. METHODS: The case notes and histology of all patients who underwent a potentially curative surgical resection for T1-2N1M0 nonsmall cell carcinoma of the lung between 1991 and 1997 were reviewed retrospectively. The following histologic factors were recorded: histologic type of tumor; number of nodes with metastatic deposits together with their nodal station; the presence of vascular invasion, visceral pleural involvement, and cellular necrosis; and grade of tumor. The results from 98 patients were analyzed. Univariate and multivariate analyses were performed to identify prognostic factors. RESULTS: Univariate analysis showed that only three factors had a statistically significant correlation with a poor prognosis: vascular invasion (p = 0.002), nonsquamous histology (p = 0.005), and visceral pleural involvement (p = 0.002). Multivariate analysis revealed that all three factors were significant independent adverse prognostic indicators. CONCLUSIONS: Visceral pleural involvement, nonsquamous histology, and vascular invasion are all significant adverse prognostic factors after surgical resection of T1-2N1M0 nonsmall cell cancer of the lung. These findings conflict with previously published reports, and we advocate a prospective, large-scale study in order to clarify the prognostic significance of histologic characteristics in stage II disease.

Characterization of T-cell non-Hodgkin's lymphoma and its association with Epstein-Barr virus in Pakistani patients.

This study analyzes the prevalence of T-cell non-Hodgkin's lymphoma (T-NHL) in a major referral center of Pakistan and its association with Epstein-Barr virus (EBV). Ninety-two cases of T-NHL were characterized on the basis of morphology, immunohistochemistry and genetic features. The prevalence of T-NHL was 22.2% of the total NHLs diagnosed during the eight years period (1992-1999). Polymerase chain reaction (PCR) technique was used to assess T-cell clonality in paraffin-embedded tissues of known T-NHL. Amplifiable DNA was isolated from all the cases, which were further studied for T-cell receptor (TcR)-beta, gamma, delta, and IgH chain gene rearrangements. Out of 92 cases 51 cases showed clonal product and 33 demonstrated polyclonal smear for beta, gamma, or delta chain genes, respectively, whereas 8 cases exhibited IgH chain gene rearrangement for FR2 region. This study demonstrated frequent presence of EBV in T-NHL (55.4%) by PCR, which were further tested for the localization of the virus by in situ hybridization (ISH). The extent of polymorphism in EBV genome was studied by single stranded conformation polymorphism (SSCP) technique for Bam HI E, K, N and Z regions. Hypervariability in Bam HI K, and N regions was noticeably higher compared to E or Z regions. In conclusion, our study demonstrated that the prevalence of T-NHL in Pakistan is slightly higher to that reported for Western communities. In addition, the frequency of EBV genome in T-NHL is intermediate as compared to other studies. No association was established between EBV variants differentiated on the basis of sequence heterogeneity in Bam HI K, N, E and Z regions with the manifestation of different subsets of T-NHL.