MD Beate Haugk
email: beate.haugk@nuth.nhs.uk

Pancreatic intraepithelial neoplasia -can we detect early pancreatic cancer?

Keywords: Carcinoma, pancreatic ductal, intraepithelial neoplasms, Pancreatic neoplasms, Carcinoma in-situ, Neoplasms, cystic, mucinous and serous Carcinoma, pancreatic ductal, intraepithelial neoplasms, Pancreatic neoplasms, Carcinoma in-situ, Neoplasms, cystic, mucinous and serous PDAC, pancreatic ductal adenocarcinoma, PanIN, pancreatic intraepithelial neoplasia, MCN, mucinous cystic neoplasm, IPMN, intraductal mucinous neoplasm, MUC, mucin, EUS, endoscopic ultrasound, COX, cycloxygenase

Pancreatic cancer is one of the most lethal cancers with an incidence equalling mortality. Pancreatic cancer is a heterogeneous group in which pancreatic ductal adenocarcinoma (PDAC) is the commonest. It is now established that PDAC develops through stepwise progression from precursor lesions. Detection and treatment of these precursor lesions would allow curative treatment. Three precursor lesions for PDAC have been identified. Two of these -mucinous cystic neoplasms (MCNs) and intraductal papillary mucinous neoplasms (IPMNs) -are rare, radiologically detectable, cystic precursor lesions which can be cured if treated at the pre-invasive stage. The third and commonest precursor lesion has recently been defined as pancreatic intraepithelial neoplasia (PanIN). PanINs are microscopic lesions with no clinical correlate. They display a spectrum of cyto-architectural changes (PanIN-1, PanIN-2 and PanIN-3) mirrored in an increasing accumulation of molecular genetic changes with PanIN-3 sharing many of the alterations with PDAC. The great advances in the understanding of pancreatic carcinogenesis have opened avenues for diagnosis and chemoprevention. However, access to the pancreas is limited, molecular tests are at the early stages and too little is known about the natural history of early PanINs to justify resection. Currently screening focuses on high risk individuals only.

Introduction

More than 230 000 new cases of pancreatic cancer were diagnosed worldwide in 2002. [START_REF] Parkin | Global Cancer Statistics[END_REF] In the UK it is the 11 th most common cancer with an annual incidence of approximately 7600 cases. [START_REF]UK Pancreatic Cancer incidence statistics[END_REF] Pancreatic cancer is the fifth most common cause of cancer related death in the UK. With a five year survival rate of only 2-3% it presents one of the most lethal cancers with a mortality which essentially equals incidence. 3 Despite many advances in modern medicine the prognosis of pancreatic cancer has remained very poor over the last two decades. [START_REF] Walsh | Chapter 19[END_REF] Approximately 85% of cases present at an advanced stage where curative surgical treatment is no longer possible. 15 -20% of cases are treated surgically with a curative intent, [START_REF] Garcea | Survival curative resection for pancreatic ductal adenocarcinoma. A systematic review of the literature[END_REF] however disease recurrence is frequent and 5 year survival low at 7-25%. [START_REF] Alexakis | Current standards of surgery for pancreatic cancer[END_REF] Surgery is currently the only hope for cure. In many solid cancers detection and treatment of early or preinvasive stages of the disease have significantly improved survival as has been well demonstrated with colorectal cancer, breast cancer and cervical cancer. 7,[START_REF] Berry | Effect of screening and adjuvant therapy on mortality from breast cancer[END_REF]9 Pancreatic cancer presents a particular challenge with respect to detection and treatment of early stage disease or pre-malignant conditions. First of all, the disease commonly presents with symptoms only at an advanced stage. [START_REF] Hariharan | Analaysis of mortality rates for pancreatic cancer across the world[END_REF] Secondly, the pancreas is a difficult organ to access. Visualisation is often limited to crosssectional imaging and ultrasound. Most pancreatic cancers show a highly infiltrative and partly discontinuous growth pattern rendering early radiological recognition difficult. Tissue biopsy sampling is restricted due to small size of the organ, its retroperitoneal location and risk of complications such as pancreatitis, fistula or metastatic implantation. The term pancreatic cancer is often used synonymously with pancreatic ductal adenocarcinoma (PDAC), which is by far the commonest primary malignant tumour of the pancreas. [START_REF] Lüttges | Gibt es Perspektiven zur Früherkennung des Pankreaskarzinoms?[END_REF][START_REF] Lüttges | Pancreatic ductal adenocarcinoma and its precursors[END_REF][START_REF] Verma | Pancreatic cancer epidemiology[END_REF] However, most cases which are diagnosed as pancreatic cancer are advanced adenocarcinomas with a tubular (pancreatico-biliary) growth pattern arising in the head of the pancreas. They may not just have arisen from the pancreatic parenchyma but in a significant proportion also the distal common bile duct and in some cases the ampulla of Vater. [START_REF] Carter | Surgery for pancreatic cancer[END_REF][START_REF] Verbeke | Resection and R1 rates in pancreatic cancer--are we there yet?[END_REF] They may therefore represent a heterogenous group of tumours with potentially different carcinogenetic pathways and pre-malignant conditions.

Pre-malignant epithelial lesions in the pancreas

Of all primary pancreatic carcinomas PDAC and its variants account for approximately 90%. [START_REF] Klöppel | Histological typing of Tumours of the Exocrine Pancreas[END_REF] Rare carcinomas such as pancreatic acinar cell carcinoma and its potential precursor lesions shall not be considered here. It is now established that PDAC develops through stepwise progression from pre-invasive lesions. [START_REF] Koorstra | Morphogenesis of pancreatic cancer: role of pancreatic intraepithelial neoplasia (PanINs)[END_REF][START_REF] Koorstra | Pancreatic carcinogenesis[END_REF][START_REF] Feldmann | Molecular genetics of pancreatic intraepithelial neoplasia[END_REF] These precursor or "precancer" lesions provide a potential substrate for curative intervention. Evidence underscoring their nature as precursor lesions stems from several sources. Morphological studies have laid the foundation for our current understanding of carcinogenesis. Molecular genetic analyses confirm the close relationship between pre-invasive lesions and invasive carcinoma. [START_REF] Hruban | Progression model for pancreatic cancer[END_REF] Genetically engineered mouse models allow recapitulation of stepwise progression from preinvasive to invasive neoplasms. 22 We currently recognise three precursor lesions for PDAC (Table 1). [START_REF] Hruban | Precursors to pancreatic cancer[END_REF] Pancreatic intraepithelial neoplasia (PanIN) represents the most important and most common lesion. It is the main subject of this review and will be discussed in detail in subsequent sections. Intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs) can also be associated with an invasive ductal type adenocarcinoma. [START_REF] Klöppel | Intraductal neoplasms of the pancreas: cystic and common[END_REF][START_REF] Kosmahl | Cystic pancreas tumors and their classification: features old and new[END_REF] IPMNs and MCNs belong to the group of cystic tumours of the pancreas which account for only 5% of all primary tumours of the pancreas. [START_REF] Visser | Diagnostic evaluation of cystic pancreatic lesions[END_REF] They are macroscopically cystic and display a continuous morphological spectrum which includes lesions with bland lining epithelium with little atypia (adenoma), lesions with moderate dysplasia (borderline), lesions with severe/high grade dysplasia (carcinoma in-situ) and invasive carcinoma. [START_REF] Klöppel | Intraductal neoplasms of the pancreas: cystic and common[END_REF][START_REF] Kosmahl | Cystic pancreas tumors and their classification: features old and new[END_REF] The prognosis for noninvasive IPMNs and MCNs is excellent and detection and surgical intervention at the pre-invasive stage is curative. [START_REF] Andrejevic-Blant | Pancreatic intraductal papillary mucinous neoplasms: a new and evolving entity[END_REF][START_REF] Crippa | Mucinous cystic neoplasm of the pancreas is not an aggressive entity: lessons from 163 resected patients[END_REF] The prognosis is poor for tumours with stromal invasion. 29,30 Due to their grossly cystic nature and mucinous cyst contents early detection is now facilitated by improved radiological techniques and cyst fluid examination including cytology. 31,32,33 It is vitally important to examine surgically resected mucin producing cystic tumours very thoroughly, in their entirety if possible, to exclude an invasive carcinoma which would require close follow-up and discussion of adjuvant therapy. [START_REF] Hruban | Precursors to pancreatic cancer[END_REF] Although IPMNs and MCNs share a macroscopically cystic nature and a similar spectrum of morphological changes of the epithelium they are two distinctly different entities. MCNs are very rare and in the Kiel series account only for 8% of all pancreatic cystic neoplasms reviewed. 34 MCNs occur almost exclusively in women.

The mean age at diagnosis lies between 40 and 50 years; patients with non-invasive lesions tend to be younger by 5-10 years compared to patients with an invasive carcinoma. 90% of tumours arise in the body and tail of the pancreas and do not show any connection with the pancreatic duct system. [START_REF] Hruban | Precursors to pancreatic cancer[END_REF] The histological identification of specialised ovarian type stroma is the defining feature. 35 The lining IPMNs in contrast are the most common cystic tumour of the pancreas accounting for 24% in the Kiel series. 34 By definition, IPMNs involve the main pancreatic duct and/or its branches (main duct type and/or branch duct type). They show a slight male predominance and occur usually between the age of 60 and 70. 80% occur in the head of the pancreas. [START_REF] Klöppel | Intraductal neoplasms of the pancreas: cystic and common[END_REF] We now distinguish four histological subtypes by the characteristics of the lining epithelium: a gastric foveolar type (MUC1-, MUC2-), an intestinal type (MUC1-, MUC2+ and CDX2+), a pancreaticobiliary type (MUC1+, MUC2-, CDX2-), and an oncocytic type (variable expression of MUC1 and MUC2). [START_REF] Adsay | Pathologically and biologically distinct types of epithelium in intraductal papillary mucinous neoplasms: delineation of an "intestinal" pathway of carcinogenesis in the pancreas[END_REF][START_REF] Campbell | Cystic neoplasms of the exocrine pancreas[END_REF] The intestinal type is the commonest. It usually affects the main pancreatic duct and an associated invasive carcinoma would commonly be of mucinous/colloid type with a more favourable prognosis than conventional PDAC. [START_REF] Adsay | Pathologically and biologically distinct types of epithelium in intraductal papillary mucinous neoplasms: delineation of an "intestinal" pathway of carcinogenesis in the pancreas[END_REF][START_REF] Seidel | Almost all infiltrating colloid carcinomas of the pancreas and periampullary region arise from in situ papillary neoplasms: a study of 39 cases[END_REF] The pancreatico-biliary type is rarer and when invasive usually associated with conventional ductal type adenocarcinoma. [START_REF] Adsay | Pathologically and biologically distinct types of epithelium in intraductal papillary mucinous neoplasms: delineation of an "intestinal" pathway of carcinogenesis in the pancreas[END_REF] The gastric foveolar type often affects branch ducts and appears less commonly associated with an invasive tumour. [START_REF] Adsay | Pathologically and biologically distinct types of epithelium in intraductal papillary mucinous neoplasms: delineation of an "intestinal" pathway of carcinogenesis in the pancreas[END_REF] The oncocytic type is exceptionally rare. [START_REF] Adsay | Intraductal oncocytic papillary neoplasms of the pancreas[END_REF] Other very rare neoplasms which enter the spectrum of intraductal lesions with potential of invasive carcinoma have also been described such as intraductal tubular neoplasms which lack the mucin production typically observed with IPMNs. [START_REF] Albores-Saavedra | Intraductal tubular adenoma, pyloric type, of the pancreas: additional observations on a new type of pancreatic neoplasm[END_REF][START_REF] Itatsu | Intraductal tubular carcinoma in an adenoma of the main pancreatic duct of the pancreas head[END_REF] Like IPMN, pancreatic intraepithelial neoplasia is also an intraductal lesion but in contrast to IPMNs and MCNs PanINs are a microscopic finding with no clinical correlate. Their relationship with IPMNs will be discussed later.

Pancreatic intraepithelial neoplasia -terminology and classification

Microscopic pancreatic duct lesions occurring in association with invasive ductal adenocarcinoma have been recognised and described for many decades. [START_REF] Sommers | Pancreatic duct hyperplasia and cancer[END_REF] Detailed studies examining surgically resected pancreases as well as autopsy material revealed a significantly increased incidence of atypical duct lesions in association with pancreatic carcinoma. [START_REF] Cubilla | Morphological lesions associated with human primary invasive nonendocrine pancreas cancer[END_REF][START_REF] Klöppel | Intraductal proliferation in the pancreas and its relationship to human and experimental carcinogenesis[END_REF][START_REF] Andea | Clinicopathological correlates of pancreatic neoplasia: a comparative analysis of 82 cases with and 152 cases without pancreatic ductal adenocarcinoma[END_REF] Atypical duct lesions were found more commonly in the head of pancreas and appeared to increase with age. [START_REF] Kozuka | of pancreatic duct hyperplasia to carcinoma[END_REF] The development of invasive ductal adenocarcinoma in pancreatic remnants months and years following incompletely resected incidental atypical duct lesions in the context of other benign pathology provided further evidence supporting a progression model from duct precursor lesions to adenocarcinoma. [START_REF] Brockie | Progression of atypical ductal hyperplasia/carcinoma in situ of the pancreas to invasive adenocarcinoma[END_REF][START_REF] Brat | Progression of pancreatic intraductal neoplasias to infiltrating adenocarcinoma of the pancreas[END_REF] However, systematic evaluation of these lesions was hindered by a lack of a unifying terminology. 70 different terminologies were used including duct lesions, flat or papillary hyperplasia or metaplasia with and without atypia, dysplasia and neoplasia. [START_REF] Hruban | Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions[END_REF] The need for an international nomenclature and classification was addressed at the Pancreas Cancer Think Tank meeting in Utah, US, in 1999. At this meeting there was agreement to adopt the new terminology of pancreatic intraepithelial neoplasia (PanIN). [START_REF] Hruban | Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions[END_REF] A 3 tier system was established: PanIN-1, PanIN-2 and PanIN-3 (Table 2).

Classification criteria were agreed and photographic and diagrammatic illustrations compiled. PanINs were distinguished from normal ductal epithelium which is cuboidal to low columnar with amphophilic cytoplasm and no evidence of mucinous cytoplasm, nuclear crowding or atypia (Figure 1). PanINs were also separated from PanIN-2 may be flat but is mostly papillary. These lesions have nuclear abnormalities by definition which may include nuclear enlargement, crowding, pseudostratification, hyperchromasia and some loss of polarity. Mitoses are infrequent, non-luminal and not atypical. No cribriform areas or necrosis is seen (Figure 5). PanIN -3 is usually papillary or micropapillary but could rarely be flat.

Cribriform changes, "budding off" of small clusters of epithelial cells into the lumen and necrosis may be seen. Cytologically these lesions resemble the cellular features of carcinoma with loss of nuclear polarity, nuclear irregularity, prominent (macro) nucleoli and mitoses which may be abnormal. Dystrophic goblet cells may also be seen (Figure 6). [START_REF] Hruban | Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions[END_REF] The new standardised classification of pancreatic ductal lesions has facilitated comparison of morphological and molecular genetic studies enhancing the evidence for the role of PanINs as precursors in the stepwise progression to ductal adenocarcinoma.

Molecular background

Cancer is caused by progressive accumulation of cellular genetic changes which will eventually lead to uncontrolled growth. 22 protein expression) level. [START_REF] Feldmann | Molecular genetics of pancreatic intraepithelial neoplasia[END_REF] Molecular changes can occur during the early phase of carcinogenesis (initiation) or late (progression). Changes at the genomic level can be chromosomal or affect cancer related genes directly. 22 Cancer related genes are divided in functionally different classes including oncogenes, tumour suppressor genes and caretaker genes.

It is thought that genomic instability caused by telomere shortening may be one of the earliest, initiating events occurring in pancreatic carcinogenesis. [START_REF] Van Heek | Telomere shortening is nearly universal in pancreatic intraepithelial neoplasia[END_REF] Telomeres, short DNA sequence repeats present at the end of chromosomes, protect them from fusion during cell division. Extreme shortening or "telomere erosion" can cause chromosomal bridging during the mitotic anaphase leading to significant chromosomal rearrangement precipitating further genetic disarray. 22 Telomere shortening is found in over 90% of PanIN-1A and over 95% of all PanIN lesions and likely presents one of the major causes for gain of oncogenes and loss of tumour suppressor genes in the progression to ductal adenocarcinoma. [START_REF] Van Heek | Telomere shortening is nearly universal in pancreatic intraepithelial neoplasia[END_REF] Loss of heterozygosity (LOH) presents genomic instability caused by loss of chromosomal material compared to normal. LOH of chromosome 9p and 17p occurs at high frequency in PDAC and pre-invasive atypical intraductal lesions (PanIN-3) with loss of 9p being an early event in the progression of PanINs. [START_REF] Yamano | Genetic and divergence in pancreatic carcinoma[END_REF] Oncogenes are genes which promote growth when activated. They act in a dominant fashion and can be activated by a variety of mechanisms including intragenic mutation and gene amplification. [START_REF] Koorstra | Pancreatic carcinogenesis[END_REF] The most important oncogene implicated in PDAC is KRAS. Its gene product is involved in cell cycle progression. [START_REF] Almoguera | Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes[END_REF] In one study over 80% of PDACs showed an activitating point mutation of codon 12 of the KRAS2 oncogene on chromosome 12p. [START_REF] Rh | K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and alleleoligonucleotide hybridization[END_REF] A meta-analysis of mutations in the KRAS oncogene in PanINs observed in the context of PDAC found KRAS mutations in just of PanIN-2 and PanIN-3 lesions which were combined due to their overall small number. [START_REF] Löhr | Frequency of K-ras in pancreatic intraductal neoplasias associated with pancreatic ductal adenocarcinoma and chronic pancreatitis: a meta-analysis[END_REF] These findings suggest that KRAS mutation is one of the earliest genetic abnormalities in pancreatic carcinogenesis. While testing of the KRAS mutation by PCR can be performed on small samples, the clinical relevance as a diagnostic marker is limited as the mutation affects the earliest precursor lesions of PDAC and it also has been demonstrated in a significant proportion of duct lesions in normal pancreases (19-38%) [START_REF] Lüttges | Duct changes and K-ras mutations in the disease-free pancreas: analysis of type, age relation and spatial distribution[END_REF] and chronic pancreatitis (26%). [START_REF] Lüttges | Ductal lesions in patients with chronic pancreatitis show K-ras mutations in a frequency similar to that in the normal pancreas and lack nuclear immunoreactivity for p53[END_REF] KRAS mutation appears to be associated with smoking. [START_REF] Lüttges | Ductal lesions in patients with chronic pancreatitis show K-ras mutations in a frequency similar to that in the normal pancreas and lack nuclear immunoreactivity for p53[END_REF] It does not appear to be present in pancreatic acinar cells and is only present in acinar-ductal metaplasia lesions in association with PanINs. [START_REF] Shi | KRAS2 mutations in human pancreatic acinar-ductal metaplastic lesions are limited to those with PanIN: implications for the human pancreatic cancer cell of origin[END_REF] Tumour suppressor genes contribute to tumour growth when inactivated. They are recessive and can be inactivated by intragenic mutation of one allele and loss of the other, homozygous deletion or epigenetic gene silencing by promoter methylation. [START_REF] Koorstra | Pancreatic carcinogenesis[END_REF] The commonest inactivated tumour suppressor genes implicated in pancreatic cancer are p16/CDKN2A/INK4A, TP53 and SMAD4/DPC4/MADH4. 22 Inactivation of the p16 gene has been demonstrated in up to 98% of pancreatic carcinomas. [START_REF] Schutte | Abrogation of the Rb/p16 tumorsuppressive pathway in virtually all pancreatic carcinomas[END_REF] Lack of the p16 protein, detected by immunohistochemistry, has been shown in 30% of flat duct lesions (PanIN-1A), 27% of papillary lesions (PanIN-1B), 55% of papillary duct lesions with atypia (PanIN-2) and 71% of carcinoma in-situ (PanIN-3) which were associated with PDAC indicating that loss of function of p16 is a relatively early event in pancreatic carcinogenesis. 59 Patients with the familial atypical multiple mole melanoma syndrome have a germline mutation in the p16/CDKN2A gene with an up to 22 fold risk of pancreatic cancer [START_REF] Shi | Familial pancreatic cancer[END_REF] with a strong association with the p16-Leiden deletion. [START_REF] De Vos Tot Nederveen | carcinoma in carriers of a specific 19 base pair deletion of CDKN2A/p16 (p16-leiden)[END_REF] 62 Concordant loss of MTAP and p16 has also been demonstrated in 8% of PanINs. [START_REF] Hustinx | Concordant loss of MTAP and p16/CDKN2A expression in pancreatic intraepithelial neoplasia: evidence of homozygous deletion in a noninvasive precursor lesion[END_REF] The MTAP gene product methylthioadenosine is involved in the synthesis of adenosine and chemotherapeutic agents targeting the loss of MTAP protein might be effective in a proportion of pancreatic carcinomas. [START_REF] Koorstra | Pancreatic carcinogenesis[END_REF] TP53 is a frequently inactivated tumour suppressor gene in many solid cancers and is inactivated in 50-75% of pancreatic carcinomas, usually by an intragenic mutation and loss of the second wild type allele. [START_REF] Feldmann | Molecular genetics of pancreatic intraepithelial neoplasia[END_REF][START_REF] Redston | p53 mutations in pancreatic carcinoma and evidence of common involvement of homocopolymer tracts in DNA microdeletions[END_REF] TP53 protein is a critical checkpoint in the repair of DNA damage inducing cell apoptosis in cases of irreparability. 22 When the gene is mutated the resulting protein tends to be stable and positive immunohistochemical staining indicates a mutated protein. TP53 staining is observed in varying proportions of PanIN-3 lesions suggesting that loss of TP53 function is a late event in carcinogenesis. [START_REF] Maitra | Multicomponent analysis of the pancreatic adenocarcinoma progression model using a pancreatic intraepithelial neoplasia tissue microarray[END_REF] DPC4/SMAD4/ MADH4 (DPC = deleted in pancreatic cancer) is a tumour suppressor gene which is affected in approximately 50% of PDACs and is rarely observed in other cancer types. [START_REF] Schutte | DPC4 gene in various tumor types[END_REF] The DPC4 gene product, SMAD4, is involved in the transforming growth factor beta pathway.

Immunostaining for SMAD4 protein expression is a sensitive method which will usually demonstrate loss of the protein if both copies of the gene are lost. 67 Positive SMAD4 staining is observed in PanIN-1and PanIN-2 but its absence has been demonstrated in 30% of PanIN-3 lesions indicating a late event in pancreatic carcinogenesis. [START_REF] Wilentz | Loss of expression of Dpc4 in pancreatic intraepithelial neoplasia: evidence that DPC4 inactivation occurs late in neoplastic progression[END_REF][START_REF] Maitra | Multicomponent analysis of the pancreatic adenocarcinoma progression model using a pancreatic intraepithelial neoplasia tissue microarray[END_REF] One of the care-taker/genome maintenance genes implicated in PDAC is BRCA2 which is associated with hereditary breast and ovarian cancer syndrome. An inactivating germline mutation with secondary loss of the second copy of the gene is seen in <10% of PDAC. [START_REF] Goggins | Germline BRCA2 gene mutations in patients with apparently sporadic pancreatic carcinomas[END_REF] BRCA2 inactivation has only been demonstrated in a single PanIN-3 lesion which may suggest that this is a late event in carcinogenesis. [START_REF] Goggins | BRCA2 is inactivated late in the development of pancreatic intraepithelial neoplasia: evidence and implications[END_REF] With regards to alterations on the transcriptomic level, small non-coding RNA molecules, micro-RNAs (mi-RNAs) have recently gained interest as potential markers and therapeutic targets in pancreatic cancer. 71 There are early indications that miRNA deregulation may be an early event in the development of pancreatic intraepithelial neoplasia. 72 In addition to the described altered genes and their products changes in the expression levels of many other proteins and apomucins have been observed in PanINs and PDAC using immunohistochemistry and tissue micro-arrays. Cyclin D1, a cell cycle regulating protein, has been shown to be over-expressed in over 60% of PDAC [START_REF] Poch | Epidermal growth factor induces cyclin D1 in human pancreatic carcinoma: evidence for a cyclin D1-dependent cell cycle progression[END_REF][START_REF] Gansauge | Overexpression of cyclin D1 in human pancreatic carcinoma is associated with poor prognosis[END_REF] and has also been demonstrated in 27% of PanIN-2 and 57% of PanIN-3 lesions as an intermediate step in the carcinogenetic process. [START_REF] Maitra | Multicomponent analysis of the pancreatic adenocarcinoma progression model using a pancreatic intraepithelial neoplasia tissue microarray[END_REF] The proliferation antigen Topoisomerase II alpha have been demonstrated in PanIN-3 lesions mainly as a late event in carcinogenesis. [START_REF] Maitra | Multicomponent analysis of the pancreatic adenocarcinoma progression model using a pancreatic intraepithelial neoplasia tissue microarray[END_REF] Ki67 expression appears to increase with the degree of PanINs with over 20% labelling found in PanIN-3. [START_REF] Maitra | Multicomponent analysis of the pancreatic adenocarcinoma progression model using a pancreatic intraepithelial neoplasia tissue microarray[END_REF][START_REF] Klein | Direct correlation between proliferative activity and dysplasia in pancreatic intraepithelial neoplasia (PanIN): additional evidence for a recently proposed model of progression[END_REF] The tumour marker prostate stem cell antigen (PSCA) is not present in normal ducts but expressed in all PanINs and ductal adenocarcinoma. Mesothelin appears to be expressed mainly in invasive ductal adenocarcinoma and is only found in a smaller percentage of PanINs.

Fascin is expressed in all PanINs but at a higher level in PanIN-2 and PanIN-3. 14-3-3σ is expressed in 85% of PanIN-3 lesions and 90% of ductal adenocarcinoma. [START_REF] Maitra | Multicomponent analysis of the pancreatic adenocarcinoma progression model using a pancreatic intraepithelial neoplasia tissue microarray[END_REF] Cyclo-oxygenase 2 (COX-2), implicated in activating signalling pathways, appears to be progressively overexpressed in PanIN lesions and invasive carcinoma making it a potential target for chemopreventative therapy. [START_REF] Albazaz | Cyclooxygenase-2 expression associated with severity of PanIN lesions: a possible link between chronic pancreatitis and pancreatic cancer[END_REF][START_REF] Maitra | Cyclooxygenase 2 expression in pancreatic adenocarcinoma and pancreatic intraepithelial neoplasia: an immunohistochemical analysis with automated cellular imaging[END_REF] Matrix metalloproteinase-7 is over-expressed in all PanIN lesions suggesting this is an early event in pancreatic carcinogenesis. [START_REF] Crawford | Matrix metalloproteinase-7 is expressed by pancreatic cancer precursors and regulates metaplasia in exocrine pancreas[END_REF] The epithelial apomucin MUC1 is expressed in normal ductal epithelium, less seen in PanIN-1 lesions but is re-expressed in PanIN-2 (43%) and PanIN-3 lesions (85%).

The gastric foveolar mucin MUC5 is not seen in normal ductal epithelium but its expression is seen in the earliest PanIN lesions and persists through all stages. [START_REF] Maitra | Multicomponent analysis of the pancreatic adenocarcinoma progression model using a pancreatic intraepithelial neoplasia tissue microarray[END_REF] MUC2 is not seen in PanIN lesions but is expressed in intestinal type IPMNs and could be used as a potential tool in the differential diagnosis. [START_REF] Maitra | Multicomponent analysis of the pancreatic adenocarcinoma progression model using a pancreatic intraepithelial neoplasia tissue microarray[END_REF] MUC4 is increasingly detected from lower PanIN lesions to invasive adenocarcinoma. 79,[START_REF] Ringel | The MUC gene family: their role in diagnosis and early detection of pancreatic cancer[END_REF] Abnormal transcriptional activation of the embryonic signalling pathways Notch and Hedgehog have been reported in PanIN lesions and the altered gastric epithelial differentiation in early PanINs may be related to activation of Hedgehog signalling. [START_REF] Feldmann | Molecular genetics of pancreatic intraepithelial neoplasia[END_REF]81 Further evidence for the progression model of pancreatic cancer comes from genetically engineered mouse models with a mutant KRAS which show the spectrum of PanIN lesions as in the human with development of invasive carcinoma in <10% of cases. Additional TP53 mutation results in PanIN lesions and highly aggressive, lethal invasive adenocarcinoma. 82,83 The described molecular genetic abnormalities are summarised in Table 3.

Differential diagnoses

PanIN lesions need to be distinguished from other duct lesions including IPMNs, reactive ductal changes and cancerisation of ducts.

IPMN and PanINs

The current definition of an IPMN is a grossly visible, mucin producing, predominantly papillary or, rarely flat, epithelial neoplasm arising from the main pancreatic duct or branch ducts with varying degree of duct dilatation. These lesions are usually >10mm in diameter and include a variety of cell types with a spectrum of cytological and architectural atypia. 84 share more molecular alterations with PanINs than intestinal type IPMNs. [START_REF] Chadwick | immunohistochemical, and molecular classification of 52 IPMNs of the pancreas[END_REF] There is also increasing evidence that intestinal and non-intestinal IPMNs follow different biological pathways. [START_REF] Adsay | Pathologically and biologically distinct types of epithelium in intraductal papillary mucinous neoplasms: delineation of an "intestinal" pathway of carcinogenesis in the pancreas[END_REF][START_REF] Ban | Intraductal papillary mucinous neoplasm (IPMN) of the pancreas: its histopathologic difference between 2 major types[END_REF][START_REF] Chadwick | immunohistochemical, and molecular classification of 52 IPMNs of the pancreas[END_REF][START_REF] Sadakari | Invasive carcinoma derived from the type intraductal papillary mucinous neoplasm of the pancreas has a poorer prognosis than that derived from the intestinal type[END_REF] These findings support suggestions that gastric IPMNs may in fact be large PanIN lesions and focal accentuation of a diffuse duct disease. [START_REF] Andrejevic-Blant | Pancreatic intraductal papillary mucinous neoplasms: a new and evolving entity[END_REF] This would explain the difficulties frequently encountered in trying to distinguish PanINs from small gastric IPMNs. Until the relationship of non-intestinal IPMNs with PanINs is fully characterised the two lesions have to be distinguished using the above guidelines as best as possible. Furthermore, many IPMNs show several epithelial types and prediction of biological behaviour may be difficult.

Reactive changes and cancerisation of ducts

The presence of numerous neutrophils in duct epithelium should alert to the possibility of reactive changes. In reactive duct epithelium the architectural changes of PanIN are usually absent. While enlarged the nuclei usually have smooth contours and finely dispersed chromatin. [START_REF] Hruban | Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions[END_REF] Cancerisation of pancreatic ducts in PDAC may be difficult to distinguish from PanIN-3 lesions. The close proximity of carcinoma to the duct and the abrupt transition from a highly atypical epithelium to normal duct epithelium favour cancerisation. Deeper sections may highlight the relationship of the duct with the carcinoma. [START_REF] Hruban | Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions[END_REF] Where do we stand in the quest for early detection and prevention of pancreatic cancer Not all "pancreatic cancers" are pancreatic ductal adenocarcinomas (PDACs).

Pancreatic head cancer is a heterogeneous group which may encompass a not insignificant proportion of carcinomas arising from the distal common bile duct and the ampulla. [START_REF] Verbeke | Resection and R1 rates in pancreatic cancer--are we there yet?[END_REF][START_REF] Nitecki | Long-term survival after resection for ductal adenocarcinoma of the pancreas. Is it really improving?[END_REF] A progression model for the development of PDAC from well defined precursor lesions has been established, supported by morphological, molecular and mouse model experimental studies. However, there may well be other carcinogenetic pathways to PDAC. [START_REF] Hruban | Progression model for pancreatic cancer[END_REF][START_REF] Di Magliano | Hedgehog/Ras interactions regulate early stages of pancreatic cancer[END_REF] We now recognise that the commonest precursor to PDAC is a microscopic lesion, termed pancreatic intraepithelial neoplasia (PanIN). The other two lesionsmucinous cystic tumours (MCNs) and intraductal papillary mucinous tumours (IPMNs) -are rare, macroscopically cystic lesions. Cysts are increasingly detected due to improving radiological techniques and a multimodal approach including cyst fluid analysis and cytology may correctly identify mucin producing cystic tumours.

Mucinous cystic tumours should always be removed. The management of IPMNs is more controversial. They can often be multifocal rendering a curative surgical approach more difficult. In general, they should be removed when >3cm, when they display mural nodules or show connection to the main pancreatic duct. However insitu and invasive carcinomas have been detected with IPMNs <1cm. [START_REF] Hruban | Precursors to pancreatic cancer[END_REF] As illustrated, the detection of specific molecular abnormalities in pancreatic samples could potentially help to identify otherwise undetectable microscopic precursor lesions. Unfortunately, the pancreas is rather difficult to access for any samples. In addition, detection of genetic abnormalities of early carcinogenesis, such as KRAS, leaves the clinician with the dilemma that the currently poorly understood natural history of the earliest precursor lesions may not justify a pancreatic resection. However there is evidence that high grade PanINs may progress to carcinoma and surgery may be indicated. 90 Detection of molecular abnormalities of early precursor lesions may also open avenues for chemoprevention of early pancreatic cancer. Currently, chemoprevention, such as COX-2 inhibitors, is studied in mouse models only. [START_REF] Feldmann | Molecular genetics of pancreatic intraepithelial neoplasia[END_REF][START_REF] Funahashi | Delayed progression of pancreatic intraepithelial neoplasia in a conditional Kras(G12D) mouse model by a selective cyclooxygenase-2 inhibitor[END_REF] Furthermore, pancreatic cancer is too uncommon for a general population screening be practical. Screening should focus on high risk individuals with a strong family history or a known germline mutation associated with an increased risk of pancreatic carcinoma. At least five genetic syndromes are currently known. [START_REF] Hruban | Precursors to pancreatic cancer[END_REF] Several of the syndromes are associated with a specific histomorphology which, when encountered, should raise the index of suspicion of a genetic basis. [START_REF] Shi | Familial pancreatic cancer[END_REF] It has also been observed that the patients with a strong family history of pancreatic cancer show multifocal PanINs with a characteristic lobulocentric atrophy which can be detected with EUS. 92 There are still enormous barriers to overcome before PanINs, the commonest precursor lesions for the most common pancreatic carcinoma, can be clinically

Conclusions for the practising pathologist

The vast majority of precursor lesions for PDAC will be encountered in pancreatic resection specimens. MCNs and IPMNs should be embedded entirely if possible to exclude any foci of invasive carcinoma. [START_REF] Hruban | Precursors to pancreatic cancer[END_REF] PanINs will usually be an incidental finding in a pancreatectomy specimen, mainly in the context of neoplastic disease. Their detection has currently no clinical implication except for the presence of PanIN-3 at the pancreatic neck resection margin. As there is evidence that PanIN-3 can develop into invasive carcinoma the possibility of further surgery should be considered and discussed, particularly if PanIN-3 is found during frozen section assessment of the pancreatic neck resection margin. 90 It is also important to realise that PanINs, including PanIN-3, are not only present in relation to PDAC but have also been encountered adjacent to distal common bile duct adenocarcinomas and ampullary carcinomas. Their presence can therefore not be used reliably to type a tumour as PDAC. 93 Diagnostic biopsies from the pancreas are infrequently obtained. EUS guided aspiration cytology is more commonly used in the assessment of pancreatic head lesions which does not allow a reliable distinction between PanIN-3 and invasive ductal adenocarcinoma. If a pancreatic biopsy is performed it will usually be in the context of a radiological lesion. Identification of isolated PanIN-3 in this context may also raise the question of surgery.

Although currently of limited clinical relevance, the accurate and consistent identification of PanINs in resection specimens and their distinction from cancerisation of ducts and IPMNs will facilitate systematic molecular and morphological studies. These will allow characterisation of the group of ductal Normal (Figure 1)

Cuboidal to low columnar epithelium with amphophilic cytoplasm.

No mucinous cytoplasm, nuclear crowding or atypia.

Squamous (transitional) metaplasia (Figure 2)

Mature stratified squamous or pseudostratified transitional epithelium without atypia.

PanIN-1A (Figure 3) Flat lesion with tall columnar cells with basally located nuclei and abundant supranuclear mucin.

Small round to oval nuclei.

Oval nuclei situated perpendicular to basement membrane.

PanIN-1B (Figure 4) Papillary, micropapillary or basally pseudostratified architecture.

Otherwise identical to PanIN-1A.

PanIN-2 (Figure 5) Mostly papillary but can be flat.

Nuclear abnormalities must be present and may include some loss of polarity, nuclear crowding, enlarged nuclei, pseudostratification, nuclear hyperchromatism.

Mitoses rare, non-apical and not atypical.

True cribriform structures with luminal necrosis and marked cytological atypia generally not seen.

PanIN-3 (Figure 6) Usually papillary or micropapillary architecture; rarely flat.

True cribriforming, "budding off" of small clusters of epithelial cells into lumen and luminal necrosis suggestive of PanIN-3.

Loss of polarity, dystrophic goblet cells, mitoses including occasional abnormal forms, nuclear irregularities and prominent (macro) nucleoli.

Resembles carcinoma without invasion.

11

 11

  in type and these lesions resemble mucinous cystic tumours encountered in the ovary and liver.

  is replacement of the ductal epithelium by squamous epithelium without any atypia (Figure2). PanIN-1 was divided into PanIN-1A and PanIN-1B. PanIN-1A is defined as a flat lesion composed of tall columnar cells with basally located nuclei and abundant supranuclear mucin. The nuclei are round to oval (Figure3). PanIN-1B lesions show a papillary, micropapillary or basally pseudostratified architecture but are otherwise identical to PanIN IA (Figure4).

  Molecular alterations can be observed at the genomic (DNA), transcriptomic (RNA) and the proteomic (post-translational

  one third of PanIN-1A lesions, over 40% of PanIN-1B lesions and almost 90%

  Features supportive of an IPMN are macroscopically visible papillae, macroscopically visible luminal mucin and tall papillae with stromal cores. In lesions <10mm deeper sections may reveal a connection to a larger duct lesion. Correlation with radiology may further help. PanINs are defined as microscopic duct lesions, usually involving ducts < 5mm in diameter, with flat or papillary cuboidal to columnar mucinous lining epithelium with varying cytological and architectural atypia. The duct diameter should be measured from basement membrane to basement membrane not including any periductal structures. In ducts only partially involved by PanIN the duct diameter should be used. PanINs can affect ducts >5mm, usually related to duct dilatation due to proximal obstruction. Careful sectioning of the duct system is recommended to assess lesions >5mm. PanINs affecting the main pancreatic duct will usually have less complex and tall papillary structures than main duct IPMNs. PanINs should be graded according to the highest grade in any given lesion. These guidelines will allow distinction of PanINs from the most common IPMN of intestinal type in most cases. Positive MUC2 staining in intestinal type IPMNs can further support the diagnosis. In contrast undoubted morphological overlap exists between the gastric foveolar type IPMN and PanINs which share a morphologically identical epithelium and MUC5 positivity.[START_REF] Adsay | Pathologically and biologically distinct types of epithelium in intraductal papillary mucinous neoplasms: delineation of an "intestinal" pathway of carcinogenesis in the pancreas[END_REF]84 Gastric IPMNs involve branch ducts, are smaller and are often associated with adjacent PanIN like complexes.[START_REF] Ban | Intraductal papillary mucinous neoplasm (IPMN) of the pancreas: its histopathologic difference between 2 major types[END_REF] They

  22

  detected and appropriately treated. Reliable molecular screening tests and chemoprevention are a long time away. Nevertheless over the last decades there have been phenomenal advances in the understanding of pancreatic carcinogenesis.Screening of high risk individuals using high resolution imaging and moleculartechniques may bring us one step closer to fighting one of the most lethal cancers of mankind.

  PanINs and their distinction from pancreatic carcinomas that may follow different carcinogenetic pathways. This will open avenues for early detection and chemoprevention in the future.

F

Figure 1 .

 1 Figure Legend

Figure 2 .

 2 Figure 2. Mature squamous metaplasia replacing part of duct epithelium with

Figure 3 .

 3 Figure 3. PanIN-1A: flat lesion with tall columnar, mucinous epithelium.

Figure 4 .

 4 Figure 4. PanIN-1B: papillary architecture with tall columnar, mucinous epithelium.

Figure 5 .

 5 Figure 5. PanIN-2: papillary architecture with pseudostratified tall columnar

Figure 6 .

 6 Figure 6. PanIN-3: Papillary architecture with cribriform areas and "budding off" of a

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .

 123456 Figure 1. Normal low columnar pancreatic ductal epithelium without atypia.

  Homozygous deletion of the

	p16/CDKN2A/INK4A gene occurs in 40% of PDAC and in 30% the neighbouring
	gene MTAP is also affected.
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Table 1 :

 1 29 Zamboni G, Scarpa A, Bogina G et al. Mucinous cystic tumors of the pancreas: clinicopathological features, prognosis, and relationship to other mucinous cystic tumors. Am. J. Surg. Pathol. 1999;23:410-22. 30 Woo SM, Ryu JK, Lee SH et al. Survival and prognosis of invasive intraductal papillary mucinous neoplasms of the pancreas: comparison with pancreatic ductal adenocarcinoma. Pancreas. 2008;36:50-5. 31 Linder JD, Geenen JE, Catalano MF. Cyst fluid analysis obtained by EUS-guided FNA in the evaluation of discrete cystic neoplasms of the pancreas: a prospective single-center experience. Gastrointest. Endosc. 2006;64:697-702. Ryu JK, Woo SM, Hwang JH et al. Cyst fluid analysis for the differential diagnosis of pancreatic cysts. Diagn. Cytopathol. 2004;31:100-5. Prasad NB, Biankin AV, Fukushima N et al. Gene expression profiles in pancreatic intraepithelial neoplasia reflect the effects of Hedgehog signaling on pancreatic ductal epithelial cells. Cancer Res. 2005;65:1619-26. Hingorani SR, Petricoin EF, Maitra A et al. and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell. 2003;4:437-50. 84 Hruban RH, Takaori K, Klimstra DS et al. An illustrated consensus on the classification of pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasms. Am. J. Surg. Pathol. 2004;28:977-87. Agoff SN, Crispin DA, Bronner MP, Dail DH, Hawes SE, Haggitt RC. Neoplasms of the ampulla of vater with concurrent pancreatic intraductal neoplasia: a histological and molecular study. Mod. Pathol. 2001;14:139-46. Precursor lesions of pancreatic ductal adenocarcinoma

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Table 2 :

 2 Pancreatic intraepithelial neoplasia (modified from Hruban 2001 49 )

Published on behalf of the British Division of the International Academy of Pathology Histopathology

Chaudhari VV, Raman SS, Vuong NL et al. Pancreatic cystic lesions: discrimination accuracy based on clinical data and high-resolution computed tomographic features. J. Comput. Assist. Tomogr. 2008;32:757-63.

Kosmahl M, Pauser U, Peters K et al. Cystic neoplasms of the pancreas and tumor-like lesions with cystic features: a review of 418 cases and a classification proposal. Virchows Arch. 2004;445:168-78.

Goh BK, Tan YM, Chung YF et al. review of mucinous cystic neoplasms of the pancreas defined by ovarian-type stroma: clinicopathological features of 344 patients. Surgery. 2007;141:834-5.

Acknowledgement

The author would like to express her gratitude to Prof AD Burt for critically reviewing the manuscript.


Eszter Turányi

Katalin Dezső

Judit Csomor

Zsuzsa Schaff

Sándor Paku

Peter Nagy
email: nagy@korb1.sote.hu

Katalin Dezsı

Péter Nagy

Peter Nagy

Immunohistochemical classification of ductular reactions in human liver

Keywords: progenitor cell CD 10, CD 56, CK 7, EMA, progenitor cell EMA epithelial membrane antigene, FHF: fulminant hepatic failure, FNH: focal nodular hyperplasia, PBC: primary biliary cirrhosis, PSC: primary sclerosing cholangitis

come

INTRODUCTION

Ductular reactions in human liver were described by Popper et al. [START_REF] Popper | Ductular reaction in the liver in hepatic injury[END_REF] fifty years ago. It had been a relatively neglected chapter of liver pathology until potential progenitor/stem cell origin of these structures was suggested. [START_REF] Aterman | The stem cells of the liver -a selective review[END_REF] Since then, the number of related publications has gradually been increasing and ductular reactions have been observed in a wide variety of liver diseases. [START_REF] Desmet | Ductular reaction in the liver[END_REF][START_REF] Burt | Bile duct proliferation-its true significance?[END_REF][START_REF] Demetris | Ductular reaction after submassive necrosis in humans[END_REF][START_REF] Vandersteenhoven | Characterization of ductular hepatocytes in end-stage cirrhosis[END_REF][START_REF] Fiel | Characterization of ductular hepatocytes in primar liver allograft failure[END_REF] It is clear from the beginning that, beside the presence of the ductular arrangement, there are obvious morphological differences among them. This is reflected by the tremendous confusion in the nomenclature. Several names are used (oval cells, oval like cells, neocholangioles, cholangiolar like structures, metaplastic ductules, pseudotubules etc.) without any consistency, obscuring the orientation in related publications. The most widely used distinction is between "typical" and "atypical" ductular proliferations. [START_REF] Desmet | Ductular reaction in the liver[END_REF] Typical ductules are similar to normal interlobular bile ducts, have a recognizable lumen and most frequently occur in connection with biliary obstruction. Atypical ductules are thin, elongated structures, lined by flattened cells, and they are likely the progenies of the hepatic progenitor/stem cell compartment. The confusion in terminology has lead a panel of experts to publish a consensus paper recommending to call all of these morphological reactions uniformly as "ductular reaction". [START_REF] Roskams | Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductulr reactions in human livers[END_REF] Although it is acknowledged in this review that ductular reactions may derive from several sources, no further classification is proposed, moreover, the distinction between typical and atypical reactions is also discouraged. In the meantime, it has become obvious that ductular reactions may have not only different origins but various functions [START_REF] Fujita | Sequential observation of liver cell regeneration after massive hepatic necrosis in auxiliary partial orthotopic liver transplantation[END_REF][START_REF] Clouston | Fibrosis correlates with a ductular reaction in hepatitis C: roles of impaired replication, progenitor cells and steatosis[END_REF] as well, suggesting the diversity of these histological reactions. There are several immunohistochemical studies on hepatic ductular reactions [START_REF] Thung | The development of proliferating ductular structures in liver disease[END_REF][START_REF] Crosby | Immunolocalization of putative human liver progenitor cells in livers from patients with end-stage primary biliary cirrhosis and sclerosing cholangitis using the monoclonal antibody OV-6[END_REF][START_REF] Craig | The histopathology of regeneration in massive hepatic necrosis[END_REF][START_REF] Kiss | Immunohistochemical analysis of atypical ductular reaction in the human liver, with special emphasis on the presence of growth factors and their receptors[END_REF] but as far as we know this characterization has not been applied for their classification. We collected paraffin embedded liver samples from various liver diseases with ductular reactions and stained them with a

Patients

Four normal and 69 liver specimens with ductular reactions were recovered from our archives, basic clinical data are given in Supplemental Table 1. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval of the ethical committee of Semmelweis University.

The statistical analysis was performed by Fisher's exact test.

RESULTS

The purpose of this study was the classification of ductular reactions occurring in various liver diseases. Several antibodies which have been described to stain hepatic ductules (AFP, Chromogranin, CEA, DLK-1, DMBT-1) did not yield any positive staining. Conversely, CK 19, EpCAM, E-cadherin, claudin-2 decorated all the ductules and biliary structures.

Therefore, these antibodies could not be used for any kind of sorting and were not considered further (data not shown). The CK7 reaction is positive on every ductule, in addition its pale staining shows excellently the intermediate hepatobiliary cells, so this antibody was used to visualize ductular reactions. Three immunohistochemical reactions (CD56, EMA, CD10) showed a diversified staining pattern in the different samples. In fact, the majority of the investigated liver samples fit in one of three different staining combinations (of the ductules) we called arbitrarily Type (P)rimitive (CD56+/EMA-/CD10-), Type (D)ifferentiating (CD56+/EMA+/CD10+) and Type (O)bstuctive (CD56-/EMA+/CD10-) ductular reactions.

Normal human liver

The immunohistochemical profile of 4 histologically normal liver samples was absolutely concordant.

The CD 56 reaction was completely negative in normal samples except the scarce nerve branches on the sections. The CD10 antibody did not react with interlobular bile ducts but showed a characteristic canalicular staining on hepatocytes in the liver parenchyma. [START_REF] Xiao | cDNA arrays and immunohistochemistry identification of CD10/CALLA expression on hepatocellular carcinoma[END_REF][START_REF] Shousha | CD-10 immunostaining of bile canaliculi in liver biopsies: change of staining pattern with the development of cirrhosis[END_REF] We were unable to identify unambiguously the intraparenchymal ductules [START_REF] Theise | The canals of Hering and hepatic stem cells in humans[END_REF] on immunostained sections so we could not make a firm statement about their phenotype. The most frequent immunophenotype among the investigated ductular reactions was CD56 positivity but no staining with the other two markers (Figure 1.). The HE morphology of these ductules was similar to the so called atypical ductular proliferation: they were composed by flat ductular cells mostly without obvious lumen formation. Intermediate hepatobiliary cells were not present in such ductules. All cases (14 cases)with focal nodular hyperplasia (FNH) and primary biliary cirrhosis (PBC) (9 cases) belonged to this category and a few additional samples with various diseases such as primary sclerosing cholangitis (PSC), Budd-Chiari syndrome, peritumoral reaction and autoimmune cholangitis had similar staining patterns.

Type (D)ifferentiating reaction (CD56+/EMA+/CD10+)

All three antibodies decorated the majority of ductular structures in a large portion of the cases. CD10 and EMA staining were apical, whereas CD56 reactions resulted mostly in membranous positivity, similarly to Type P ductules (Figure 2 and3). HE morphology of the ductules was different in the two large groups of diseases with this type of ductular reaction.

Large intermediate hepatobiliary cells were frequently present in samples of fulminant liver failure (FHF) (12 of 15 cases had this phenotype) (Figure 3D). In cirrhotic livers (11 of 14 cases), ductules were often narrow bands virtually without a lumen, similarly to "atypical" ductular reactions (Figure 2D). Intermediate hepatobiliary cells were scarce, yet they were positive with these three antibodies.

Type (O)bstuctive reaction (CD56-/EMA+/CD10-)

In the case of Type O reactions, beside the consistent strong EMA positivity, CD10 and CD56 staining remained negative (Figure 4.). This phenotype was identical with to that of normal interlobular bile ducts. Morphologically, ductules with Type O immunophenotype look like differentiated biliary structures with an obvious lumen, but their number is increased. Biliary obstruction was common in all the diseases (acut biliary obstruction, biliary atresia, secundaer biliary cirrhosis) which lead to this type of biliary proliferation.

Ten of the 69 ductular samples (14%) did not be fit in the above classification.

Statistical analysis

The diagnoses were adjusted with Fisher's exact test with the types of the ductular reaction (P,D,O) and the expression of the investigated markers (CD56, EMA, CD10) (Table 1 and2).

The correlation was highly significant (p<0,001). Our results on normal liver samples are in line with previous publications. CD56 is generally considered as a marker for neuroendocrine differentiation. The canals of Hering, the potential niche for hepatic stem cells, have been reported to express this antigen. [START_REF] Van Den Heuvel | Expression of anti-OV6 antibody and anti-N-CAM antibody along the biliary line of normal and diseased human livers[END_REF][START_REF] Strain | Human liver-derived stem cells[END_REF] We were able to detect CD56 positive ductules on normal, frozen liver sections by immunofluorescence, but the presently applied method on paraffin embedded tissues did not seem to be sensitive enough. CD10 decorates several normal tissues and tumors. This antigen is an established marker for hepatocytes [START_REF] Xiao | cDNA arrays and immunohistochemistry identification of CD10/CALLA expression on hepatocellular carcinoma[END_REF][START_REF] Shousha | CD-10 immunostaining of bile canaliculi in liver biopsies: change of staining pattern with the development of cirrhosis[END_REF] in the liver, as well as is EMA for interlobular bile ducts. [START_REF] Sonzogni | Isolated idiopathic bile ductular hyperplasia in patients with persistently abnormal liver function tests[END_REF] CD56 staining, in the case of Type P reactions, may indicate that these ductules derive from the canals of Hering. There are no morphological signs of differentiation in this type of ductules, and the lack of EMA and CD10 staining does not support large bile duct or hepatocyte directed commitment either. This is surprising in the case of FNH, because occasionally there seems to be continuity between hepatic ductules and the tubularly arranged documented, only with different morphology, namely by the formation of hepatocyte "buds". [START_REF] Falkowski | Regeneration of hepatocyte "buds" in cirrhosis from intrabiliary stem cells[END_REF] This situation is similar to the one observed in rat liver, where identical oval cells differentiated into hepatocytes in distinct spatial distributions depending on the applied experimental conditions. [START_REF] Paku | 2-acetylaminofluorene dose-dependent differentiation of rat oval cells into hepatocytes: confocal and electron microscopic studies[END_REF] That is, this immunophenotype may be characteristic for hepatic ductules with the capacity/commitment to differentiate into hepatocytes, featuring even those ductules which do not show any morphological sign (e.g. enlarged polygonal cells with abundant cytoplasm, pale CK7 staining) of such differentiation. Interestingly, hepatocytic differentiation of stem/progenitor cells in the rat liver is preceded by the appearance of the biliary marker CK7. [START_REF] Paku | Immunohistochemical analysis of cytokeratin 7 expression in resting and proliferating biliary structures of rat liver[END_REF] The EMA staining in human ductules may indicate a similar process.

The immunophenotype of Type O reactions corresponds to normal interlobular bile ducts and morphologically these ducts/ductules are also very similar. The etiology of this reaction type further confirms that it is equal with to the bile duct proliferation induced by the ligation of the common bile duct in rats. [START_REF] Cameron | Ligation of the common bile duct[END_REF] The previously proposed classifications, based on clearly morphological features e.g. "typical vs. atypical" gained no general acceptance, probably due to poor reproducibility. Therefore we tried a more "objective" approach, based on the immunophenotype of the ductules.

The 3 types of ductular reactions, distinguished by their immunophenotype show a great deal of similarities to the classification used by Desmet et al. [START_REF] Desmet | Ductular reaction in the liver[END_REF] more than 10 years ago. The most important difference is that immunohistochemistry clearly distinguishes type P from type D reactions, while ductules corresponding to this latter category "ductular reaction in (sub)massive hepatic necrosis" is only a subtype of the atypical ductular reaction in Desmet et al's review. [START_REF] Desmet | Ductular reaction in the liver[END_REF] We do not know if the different immunophenotypes of type P and D reactions indicates a distinct stage/orientation of differentiation or dissimilar histogenesis but it very likely marks some basic differences. It is well known that ductular reactions are heterogeneous. Careful analysis of Zhou et al. [START_REF] Zhou | Identification of hepatocytic and bile ductular cell lineages and candidate stem cells in bipolar ductular reactions in cirrhotic human liver[END_REF] revealed tremendous immunophenotypic heterogeneity even within individual ductules.

It would be almost impossible to perform such detailed analysis on numerous specimens. In order to evaluate our immunoreactions, we assumed that there was a dominant type of ductular reaction in each liver sample, and we strove after its recognition, not to pay attention to the "background noise". Such a reductionist approach may help to recognize a general overview, although important details might be missed occasionally. A little more than 10% of the investigated ductular reactions do not fit in any of our proposed categories. It certainly can not be ruled out that there are more than three types of ductular reactions and the applied markers are not able to identify them. Another or an additional explanation is that our assumption, namely that there is one dominant type of ductular reaction in each sample, may not be true in all cases. This latter suspicion is supported by our observation that it was easy to classify "simply" hepatic diseases (e.g. PBC, FNH), since staining of ductules was relatively homogeneous. Whereas in "complex" diseases (e.g. PSC, cirrhosis), where biliary obstruction can be combined with other biological reactions like hepatocyte directed differentiation, different types of ductular reactions can be present together. Indeed, the immunophenotipic pattern of the ductules was not so clear in these cases. This may be explaining why ductular reactions in PSC and cirrhotic samples do not have identical immunophenotypes in each case.

The key issue is: is there any point classifying ductular reactions? The extreme diversity in the terminology of ductular reactions reflects the variable morphological appearance, origin and potential function of these histological alterations. However, the nomenclature was not consistent and the applied names were not defined. Therefore, we think it was make a sense to collect them under the broad name "ductular reaction". [START_REF] Roskams | Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductulr reactions in human livers[END_REF] At the same time, it is also obvious that very different biological reactions are put together in this category. Nobody debates that the oval cells in rat liver [START_REF] Santoni-Rugiu | Progenitor cells in liver regeneration: molecular responses controlling their activation and expansion[END_REF] , induced by chemical carcinogens, are different from the proliferating bile ducts following the ligation of the common bile duct. [START_REF] Cameron | Ligation of the common bile duct[END_REF] Oval cells are able to differentiate into hepatocytes while cholangiocytes of the elongated biliary ducts are not. Of course, nobody would investigate the origin, function or growth regulation of these reactions together, although both of them are "ductular reactions". The situation is easy in the case of rat liver, since a simple AFP immunostaining can clearly distinguish between the two reactions. [START_REF] Santoni-Rugiu | Progenitor cells in liver regeneration: molecular responses controlling their activation and expansion[END_REF] Unfortunately, so far we have no such "magic bullet" for the human liver, in addition, the morphological differences are not so distinct as in the rat either. However, this does not mean that we can spare a rational classification.

Dumping together data coming from all sorts of "ductular reactions" would result only in confusion. Detailed analysis, like Zhou et al's [START_REF] Zhou | Identification of hepatocytic and bile ductular cell lineages and candidate stem cells in bipolar ductular reactions in cirrhotic human liver[END_REF] , performed afterward, on well defined types of ductular reactions would be more informative. Similarly to lymphomas or soft tissue tumors, where the reproducible classification elicited a huge step in our understanding of these diseases.

In conclusion, we immunostained 69 human liver specimens containing ductular reactions and the majority of the ductular reactions could be classified into three categories based on the distribution of three antibodies. This classification is similar to previously described types of ductular reactions, but probably more reproducible. We propose these categories as an initiation, and our intention is to stimulate further similar studies in the hepatopathologists' community using different approaches and methodology (e.g. gene expression profiling by microarrays), which will hopefully lead to the rational classification of ductular reactions in human liver. We do not think that various liver diseases can be diagnosed purely on the immunostaining of the ductules but rational sorting is inevitable if we want to understand the biology behind the various reactions and to apply our knowledge to understand the role of the different ductules in hepatic lesions.

  of antibodies, chosen mostly from the above mentioned studies, to determine whether immunohistochemical characterization can be used for their rational, reproducible classification. The ductules in certain specimens did show neither hepatocytic nor biliary commitment, we called them P(rimitive) type. The cells constructing other ductules were characterized by specific hepatocytic features, these ductules were named as D(ifferentiating).While the ductules mostly of obstructive hepatic lesion, which were similar to normal bile ducts were mentioned as O(bstructive).

Immunohistochemistry

  Traditional immunohistochemical reaction was performed on paraffin embedded histological sections. Primary antibodies were: CK 7 (Biogenex MU 255-UC, 1:50); Epithelial Membrane Antigene (EMA): (Novocastra EMA-C-CE, l:1:50); CD 10 (Novocastra NCL-CD10-270, 1:10); CD56 (BD Pharmingen 559 043, 1:50). Reactions were visualized by Vectastain Elite Kit using DAB as chromogen. Each section was evaluated blindly, without knowing the diagnosis and the results of the other immunohistochemical reactions. Ductular reactions are inherently heterogeneous. To simplify the assessment, results for each reaction were given on a bipolar scale, positive or negative, depending on the dominant staining pattern of the ductules.

  in strong apical reaction on interlobular bile ducts and no positivity was observed in other locations. (Data not shown) Type (P)rimitive reaction (CD56+/EMA-/CD10-)

DISCUSSION

  We stained 69 liver specimens containing ductular reactions with several antibodies. The combination of three immunostainings seems feasible to classify the majority of ductular reactions into three groups, based on their immunophenotype.

  small hepatocytes of the hyperplastic nodules on HE stained sections. Although, intermediate hepatobiliary cells are never present in FNH ductules. The Type D reaction is more complex. Besides the CD56 staining, which may indicate the origin of canals of Hering, both the hepatocytic CD10 and the cholangiocytic EMA markers are turned on. Two major groups of diseases belong to this category: liver regeneration following fulminant liver necrosis and cirrhosis. The frequent occurrence of intermediate hepatobiliary cells clearly indicates hepatocyte directed differentiation in FHF. Although these cells are rare in cirrhosis, the hepatocytic differentiation of reactive ductules is also well

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Immunohistochemical characterization of Type P ductular reaction.

  of the British Division of the International Academy of Pathology

Table 1 . Contingency table to compare the diagnosis with the immunophenotype

 1

	Diagnosis	P	D	Ductular reaction type O	Other
	FHF	0	12	0	3
	Cirrhosis	0	11	0	3
	FNH	14	0	0	0
	PBC	9	0	0	0
	Obstructive	0	0	8	0
	PSC	2	0	0	3

Table 2 . Contingency table to compare the diagnosis with the Immunohistochemical markers

 2

	Diagnosis	CD56+	Immunohistochemical positivity EMA+ CD10+
	FHF	13	15	13
	Cirrhosis	12	13	14
	FNH	14	0	0
	PBC	9	0	0
	Obstructive	0	8	0
	PSC	5	3	0

Published on behalf of the British Division of the International Academy of Pathology Histopathology


Professor Kikkeri N Naresh

Philippa C May

Alistair G Reid

Alexandra J Marks

Donald Macdonald

Edward Kanfer

Philippa Reid

Alistair Marks

Alexandra Macdonald

Alexandra J Marks

T-cell lymphoblastic leukaemia / lymphoma associated with a microenvironment of thymic asteroid-B-cells in the bone marrow

Keywords: Thymus, T-ALL, T-cell lymphoblastic lymphoma, Bone marrow trephine, Asteroid B-cells bone marrow trephine biopsy

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The tumour microenvironment has been well studied in many haematological malignancies such as follicular lymphoma and diffuse large B-cell lymphoma. [START_REF] Dave | Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells[END_REF][START_REF] Lenz | Stromal gene signatures in large-B-cell lymphomas[END_REF] With respect to T-ALL/LBL, in 2001 Winter et al showed the importance of interactions between lymphocyte function-associated antigen-1 (LFA-1) on T-ALL/LBL cell lines and adhesion molecules present on bone marrow stromal cells such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. [START_REF] Winter | Enhanced Tlineage acute lymphoblastic leukaemia cell survival on bone marrow stroma requires involvement of LFA-1 and ICAM-1[END_REF] The bone marrow mesenchymal cell / microenvironment niche may play an important role in growth and survival of leukemic cells, and can contribute to tumour cell proliferation, dormancy, selfrenewal or resistance to treatment. Several cytokines secreted by the tumour cells or the microenvironment contribute towards this interdependency. [START_REF] Iwamoto | Mesenchymal cells regulate the response of acute lymphoblastic leukemia cells to asparaginase[END_REF][START_REF] Williams | Cytokine-dependent imatinib resistance in mouse BCR-ABL+, Arf-null lymphoblastic leukemia[END_REF][START_REF] Campana | Molecular determinants of treatment response in acute lymphoblastic leukemia[END_REF] Among such cytokines, interleukin-8 (IL-8) is highly expressed in T-ALL/LBL cells refractory to chemotherapy. The mechanisms of IL-8 overexpression by T-ALL/LBL cells have been recently explored. It appears that this is dependent on the expression and secretion of the CXCL12 chemokine by bone marrow stromal cells, which then bind to CXCR4 on the leukaemic cells, which in turn upregulates IL-8 production through the nuclear factor-B and Jun-N-terminal kinase (JNK)/activating protein (AP)-1 signalling pathways. [START_REF] Scupoli | Bone marrow stromal cells and the upregulation of interleukin-8 production in human T-cell acute lymphoblastic leukemia through the CXCL12/CXCR4 axis and the NF-kappaB and JNK/AP-1 pathways[END_REF] The understanding of the molecular mechanisms underlying the dependence of T-ALL/LBL cells on the microenvironment will help in unravelling the natural history of T-ALL/LBL and in designing alternative therapeutic strategies. Among Philadelphia chromosome-positive (Ph(+))B-LL a subpopulation of leukaemic cells express endothelial markers like VE-cadherin, PECAM-1, and Flk-1and make them responsive to microenvironment-derived self-renewing and proliferative signals. The presence of dendritic B-cells with the morphology and immunophenotype of thymic asteroid-B-cells was serendipitously observed in the bone marrow stroma / microenvironment of a patient with relapsed T-ALL/LBL. Similar cells were then identified in the lymph node (LN) sample at the time of relapse. Thymic asteroid B-cells are large B-cells with short dendritic cytoplasmic processes and are normally present in the thymic medulla. [START_REF] Hofmann | Thymic medullary cells expressing B lymphocyte antigens[END_REF] Following this the bone marrow trephine biopsy (BMTB) and LN samples from seven other patients with T-ALL/LBL were re-evaluated, and asteroid-B-cells were found in the marrow stroma of one other patient. In this communication, we describe the findings and discuss the implications.

Case report: A 47 year old man presented with a four week history of worsening shortness of breath, dry cough, sweats and fevers. Blood tests showed a normal full blood count. Chest

x-ray revealed a large mediastinal mass with impending SVC obstruction. He also had a 1cm supraclavicular lymph node on the left side. CT guided Tru-cut biopsy showed T-ALL/LBL. His bone marrow examination showed no evidence of lymphoma or leukaemia. He was HIV negative, and his LDH was 1377u/L. The patient was treated with UKALL XII protocol.

Fourteen months after the initial presentation he relapsed with a mediastinal mass infiltrating the sternum and with bone marrow involvement.

Bone marrow trephine biopsy (BMTB) at relapse:

There was near-complete replacement of the marrow by sheets of blastic cells. The blasts expressed CD3, TdT, CD99, CD43, and CD10. They are negative for CD20, CD79a, CD34, CD117, CD68, CD68R, myeloperoxidase and HLADR. In addition, there were clusters and singly scattered B-cells, which were identified on the immunostains. These cells were unusual and showed dendritic processes.

They were positive for CD20, CD79a, CD23, PAX5, IgD, CD44, OCT2 and BOB.1. The cells were negative for CD21, CD38, GCET, CD138, CD76, IgM, IgG, IgA and IgE. Immunostains CD20, CD79a, CD23 and IgD highlighted the dendritic processes. The cells had features of asteroid-B-cells normally seen only in the thymus (Figure 1).

Cytogenetic analysis on bone marrow at relapse: G-banded chromosome analysis of bone

marrow showed complex structural and numerical abnormalities. The karyotype was defined as 47~48,XY, del(2)(p16), der(3)add(3)(p24)add(3)(q26), -5,del(6)(q22), der(7)t(7;?)(q34;?), t(8;11)(p1;q2), add(9)(q34), +16, +19, +20[cp5]/46, XY [7]. Notably, the abnormalities observed included rearrangement of the long arm of one homologue of chromosome 7, which prompted further investigation by interphase fluorescence in situ hybridization (FISH). FISH analysis using a dual colour "break-apart" probe system specific for the TCRB locus at 7q35 (Dako, Denmark) revealed a split hybridisation signal, consistent with rearrangement of TCRB, in 94% of interphase cells examined.

Metaphase analysis using the same probe revealed the presence of the 3'TCRB sequences on the long arm of a chromosome identified by reversed DAPI banding as chromosome 9 (Figure 2), while the 5'TCRB probe was observed on a small F group-sized marker chromosome. Unexpectedly, neither component of the rearranged TCRB was present on the derivative chromosome 7, indicating further complex rearrangement of 7q than was detectable by G-band analysis.

Mediastinal mass biopsy at relapse: Biopsy of the mass infiltrating the sternum at relapse also showed features of T-ALL/LBL. The lymphoid cells were positive for CD2, CD3, CD5, CD7, CD4, CD8 and TdT. The cells were negative for CD20, CD79a, PAX5, CD34 and CD117. Similar to the bone marrow asteroid-B-cells were identified on CD20 and CD79a; but these were fewer in number as compared to the bone marrow sample.

Biopsy of the mediastinal mass at presentation:

The biopsy showed essentially similar features and asteroid-B-cells were identified. Revisit of additional cases: Samples of LN and BMTB from one and BMTB only from 6 other cases of T-ALL/LBL were revisited. All the BMTBs showed extensive involvement by tumour. One of the cases with bone marrow disease showed presence of asteroid B-cells in the bone marrow microenvironment and this case is discussed in more detail.

Case report of the additional case identified on review: A 38-year old man presented with B symptoms and cough, which had not resolved on antibiotics. A LN biopsy performed elsewhere was diagnosed as non Hodgkin lymphoma and staging CT was carried out showed mediastinal and cervical lymphadenopathy and he was therefore classified as Stage 2B. He was initially treated elsewhere with six cycles of CHOP chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisone). With a partial remission to chemotherapy, he developed new left-sided cervical lymphadenopathy and the CT scan and the PET scan confirmed presence of residual active disease in the mediastinum and neck, with new disease in the left cervical region. A bone marrow aspirate and trephine was performed, and it confirmed involvement by T-ALL/LBL. He was then commenced UKALL-12 protocol achieving complete response after the 2nd cycle, then followed by 3 courses of high-dose methotrexate. This was followed by LACE (lomustine (CCNU), cytarabine (Ara-C), cyclophosphamide, etoposide) conditioned autologous stem cell transplant. However, he relapsed within three months in the mediastinum and two months later in the bone marrow.

Bone marrow investigations on the additional case at relapse:

The BMTB was hypercellular with a cellularity of nearly 100%, and the marrow showed sheets of blastic cells amounting to over 90% of the marrow cells. They expressed CD3, CD10, TdT and CD99 and were of the cells are negative for CD20, CD43, CD34, myeloperoxidase and CD68R. In addition, there were B-cells with short dendritic processes, and these were positive for CD20 and CD79a.

Flow cytometry performed on the bone marrow aspirate showed blasts representing 75% of the bone marrow cells and they expressed cytoplasmic CD3, CD10, TdT, CD5, CD7 and

Rest of the cases reviewed:

The other six cases included four male and two female adult patients in the age range of 22-32 years. The bone marrow in all the cases and LN in one case showed typical features and immunophenotype of T-ALL/LBL, and the patients were appropriately managed.

Discussion: We report the first case of T-ALL/LBL wherein certain aspects of thymic microenvironment are simulated at the bone marrow and LN sites of disease involvement.

The case also showed a similar microenvironment in the mediastinal site at the time of relapse.

Although the thymus is predominantly a T-cell organ, B-lymphocytes are present in the thymic medulla. A proportion of thymic B-cells are asteroid-shaped larger cells with short dendritic cytoplasmic processes. These asteroid-B-cells express CD20 and CD23, and inconsistently express CD19, CD22, CD37 and IgM. The asteroid-B-cells are located in the cortico-medullary region of the foetal thymus. However, in the adolescent and adult thymus they are randomly distributed in the medulla and are seen in greater numbers close to Hassall's corpuscles. They also form rosettes with T-cells and possibly represent the earliest interaction of maturing T-cells with B-cells. [START_REF] Hofmann | Thymic medullary cells expressing B lymphocyte antigens[END_REF] Their close association with T-cells and medullary epithelial cells suggests a functional role for the asteroid-B-cells in the thymus. [START_REF] Fend | Phenotype and topography of human thymic B cells. An immunohistologic study[END_REF] However, the exact nature of interaction between the asteroid-B-cells and other cells in the thymus is poorly understood. More recently, the tumour cells of primary mediastinal large Bcell lymphoma of thymic origin were hypothesised to be related to asteroid-B-cells. Asteroid-B-cells are not seen outside of the thymus, though similar large B-cells with a dendritic morphology have been described in the interfollicular region of the LNs. [START_REF] Marafioti | Phenotype and genotype of interfollicular large B cells, a subpopulation of lymphocytes often with dendritic morphology[END_REF] In the two cases where the asteroid-B-cells were identified in the bone marrow, it is possible that the T-ALL/LBL cells are dependent on the asteroid-B-cells for their survival. This feature of presence of asteroid-B-cells as an important component of the T-ALL/LBL was seen only in two of the 8 cases evaluated, and both had presented with mediastinal involvement. Generally, about one-half of T-ALL/LBL patients present with mediastinal disease. [START_REF] Wright | Childhood non-Hodgkin lymphomas in the United Kingdom: findings from the UK Children's Cancer Study Group[END_REF][START_REF] Boucheix | Immunophenotype of adult acute lymphoblastic leukemia, clinical parameters, and outcome: an analysis of a prospective trial including 562 tested patients (LALA87). French Group on Therapy for Adult Acute Lymphoblastic Leukemia[END_REF] It may be that in a subset of cases that have prominent mediastinal involvement the neoplastic cells may receive survival signals from the asteroid-B-cells.

We could not identify a unique immunophenotype of the neoplastic cells among the two cases of T-ALL/LBL with asteroid B-cells. Though, both the cases of T-ALL/LBL with asteroid B-cells showed co-expression of CD10 in the blasts, this feature was also noted in one of the cases without asteroid B-cells. Furthermore, CD10 expression in is reported in close to 20% of T-ALL/LBL and in some studies in over 60%. [START_REF] Consolini | Clinical relevance of CD10 expression in childhood ALL. The Italian Association for Pediatric Hematology and Oncology (AIEOP)[END_REF]16 Our index case had an aggressive disease course with disease relapsing within four months after completion of treatment. The second case that was identified on review also had an aggressive course with a short remission after high-dose chemotherapy. The question whether cases of T-ALL/LBL with asteroid-B-cells represent a distinct subset with aggressive behaviour, needs to be addressed. Our index case was associated with rearrangement of the TCRB locus (7q35). This in itself is not unique. A majority of T-ALL/LBL have cytogenetic abnormalities and among them, those involving the four TCR loci are more common. The partner genes involved in the translocations are variable and include HOX11 (10q24), HOX11L2 (5q35), MYC (8q24), TAL1 (1p32), NOTCH1 (9q34), RBTN1 (11p15), RBTN2 (11p13), LYL1 (19p13) and others. These translocations often result in aberrant expression of Figure 2: Fluorescence in situ hybridisation of a representative metaphase cell performed using Dako TCRB split signal probe (Dako, Denmark). This probe system is composed of two contigs, specific for sequences 5' and 3' of the TCRB locus, labelled in green and red, respectively. In normal cells two red-green fusion signals are observed. However, in the index case, an aberrant signal pattern was observed. One intact red-green fusion signal was present on the normal chromosome 7 homologue, marking the un-rearranged TCRB locus. In contrast, the second TCRB hybridisation signal reveals relocation and rearrangement of this region, with part of the green 5' signal hybridised at the distal end of an F group-sized marker, while the remains of the green 5' signal together with the red 3' signal are present at the distal end of the putative chromosome 9 (identified by reversed DAPI analysis). This finding is consistent with rearrangement of TCRB followed by further rearrangement of chromosome 7.

  much progress has been made in understanding the immunophenotypic subsets and molecular genetics of T-cell lymphoblastic leukaemia / lymphoma (T-ALL/LBL), relatively little is known about its microenvironment and the role it plays.

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  were negative for CD8, CD2, TCR-alpha/beta, TCR-gamma/delta, CD19, CD79a, CD22, cytoplasmic IgM, CD13, myeloperoxidase, CD34, CD15, CD117, CD33 and HLA-DR.

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Figure 1 :

 1 Figure 1: Bone marrow trephine biopsy of the index case shows diffuse infiltration by blasts (a) positive for CD3 (b) and TdT (c). CD20 immunostain identifies loosely organised asteroid / dendritic B-cells (d & e) that are also positive for CD23 (f).

  of the British Division of the International Academy of Pathology Histopathology

Published on behalf of the British Division of the International Academy of Pathology Histopathology