Recent advances in pancreatic pathology

Recent advances in pancreatic pathology

1. RECENT ADVANCES IN PANCREATIC PATHOLOGY Part - I Dr. Indranil Bhattacharya Consultant Pathologist Jagjivan Ram Hospital Mumbai

2. Classification Precursor lesions Pancreatic endocrine neoplasms Familial endocrine neoplasms Pancreatic cytopathology CAP protocols Pancreatic transplant

3. INTRODUCTION Pancreatic cancer - fourth leading cause of death among both men and women, comprising 6% of all cancer-related deaths. At the time of diagnosis - 52% of all patients have distant disease & 26% have regional spread. The relative 1 year survival - only 24%, and the overall 5-year survival rate for this disease is less than 5%. Incidence in India - less than 2 cases per 100,000 persons per year.

4. The pancreatic lesions can be divided into Endocrine Exocrine Neoplasm Diabetes Neoplasm Mellitus Acute & chronic pancreatitis

5. CLASSIFICATION

6. WHO CLASSIFICATION OF EXOCRINE TUMOURS OF PANCREAS Epithelial tumors Benign Serous cystadenoma Mucinous cystadenoma Intraductal papillary mucinous adenoma Mature teratoma Borderline (uncertain malignant potential) Mucinous cystic neoplasm with moderate dysplasia Intraductal papillary mucinous neoplasm with moderate dysplasia Solid pseudopapillary neoplasm

7. Malignant Ductal adenocarcinoma Mucinous noncystic carcinoma Signet-ring-cell carcinoma Adenosquamous carcinoma Undifferentiated (anaplastic) carcinoma Undifferentiated carcinoma with osteoclastlike giant cells Mixed ductal-endocrine carcinoma Serous cystadenocarcinoma Mucinous cystadenocarcinoma Noninvasive Invasive

8. Malignant tumors (cont) Intraductal papillary mucinous carcinoma Noninvasive Invasive (papillary mucinous carcinoma) Acinar cell carcinoma Acinar cell cystadenocarcinoma Mixed acinar-endocrine carcinoma Pancreatoblastoma Solid pseudopapillary carcinoma Non-epithelial tumors Secondary tumors

9. PANCREATIC PRECURSOR LESIONS

10. Pancreatic intraepithelial neoplasia (PanIN) is a microscopic neoplastic lesion of the pancreas that can progress to invasive ductal adenocarcinoma. Regarded as the main precursor lesion to invasive pancreatic carcinoma . Small, papillary or flat, noninvasive epithelial neoplasm characterized by variable mucin production and a spectrum / variable degrees of cytologic and architectural atypia. By definition, it is a non-tumoral form of dysplasia (intraepithelial neoplasia). Almost by default, it doesn't have any clinical manifestation.

11. Invasive pancreatic cancer develops through stepwise tumor progression. Based on pathologic, clinical, and genetic observations. The progression of normal pancreatic epithelium to infiltrating cancer - through a series of histologically defined lesions called as PanINs

12. Pancreatic Intraepithelial Neoplasias (PanIN) and Corresponding Older Synonyms Squamous metaplasia: Epidermoid metaplasia, multilayered metaplasia. PanIN-1A: Pyloric gland metaplasia, goblet cell metaplasia, mucinous hypertrophy, mucinous ductal hyperplasia, mucinous cell hyperplasia, mucoid transformation, simple hyperplasia, flat duct lesion without atypia, flat ductal hyperplasia, ductal hyperplasia grade 1, nonpapillary epithelial hypertrophy, nonpapillary ductal hyperplasia. PanIN-1B: Papillary hyperplasia, papillary ductal hyperplasia, papillary ductal lesion without atypia, ductal hyperplasia grade 2, adenomatous ductal hyperplasia, adenomatoid hyperplasia. PanIN-2: Atypical hyperplasia, papillary duct lesion with atypia, low-grade dysplasia, any PanIN lesions wit h moderate dysplasia. PanIN-3: Carcinoma in situ, intraductal carcinoma, severe ductal dysplasia, high-grade dysplasia, ductal hyperplasia grade 3, atypical hyperplasia.

13. Molecular genetic alterations are well known now Cumulative genetic disarrays - potential markers for early diagnosis & target for intervention Genetic alterations in (PanIN-1 and 2) - Telomere shortening, - Activating mutation in codon 12 of KRAS - Inactivation of CDKN2A/p16 tumour suppressor gene.

14. PanIN-3 - additional molecular alterations - inactivation of tumor suppressor gene: SMAD4/ DPC4, TP53, and BRCA2.

15. Tumor progression model of pancreatic carcinogenesis: bottom, schematic drawing; middle, in men; and top, in mice. The consecutive stages are formed by the various stages of pancreatic intraepithelial neoplasia lesions with their specific histopathology. During tumor progression there is an increasing generalized genetic disarray in which alterations in specific oncogenes and tumor suppressor genes are the major players.

16. PanIN progression model of pancreatic cancer. Each step in the progression from normal epithelium to low-grade PanIN, and on to high-grade PanIN is accompanied by accumulating genetic alterations. A normal pancreatic duct is lined by cuboidal to low-columnar epithelium with amphophilic cytoplasm. PanIN-1A shows flat epithelial lining with tall columnar cells with basally located nuclei and abundant supranuclear mucin. PanIN-1B identical to PanIN-1A except for a papillary, micropapillary, or basally pseudostratified architecture in PanIN-1B. PanIN-2 demonstrates full-thickness pseudostratification of nuclei with mild-to-moderate cytologic abnormalities. PanIN-3 is characterized by complete loss of polarity, budding of cellular tufts into the duct lumen, and significant nuclear pleomorphism.

17. Increased expression of proteins in ductal pancreatic acini in PanIN lesions. Cyclin D1 overexpression is associated with poor prognosis. Not seen in normal pancreatic ducts or PanIN 1. Cyclooxygenase 2 – a rate limiting enzyme in PG pathway – overexpression is implicated in tumour cell growth, invasion, angiogenesis and prognosis. Cyclooxygenase 2 over expression - potential target for chemotherapy by selective COX-2 inhibitors.

18. Other Pre-invasive Lesions - Intraductal papillary mucinous neoplasms - Mucinous cystic neoplasms

19. IHC labeling of genes - CDKNIA, MMP7, CLDN18, ANXA2, S100P - Progressive increase in their expression from low to high grade PanINs. Gene products - are proposed to be good candidates validated in serum and urine as early markers of pancreatic cancer.

20. PANCREATIC ENDOCRINE NEOPLASMS

21. Morphology allows distinction between poorly differentiated and well differentiated neoplasms. Well differentiated constitute >90 % of PEN - poorly differentiated are invariably high grade malignancies Well differentiated endocrine carcinomas - morphologically similar to well differentiated endocrine tumors.

22. The WHO has differentiated endocrine tumors from endocrine carcinomas Prognostic stratification of well differentiated endocrine carcinomas - not done in WHO classification European Neuroendocrine Tumor Society - proposed a TNM based staging system for gastroenteropancreatic endocrine tumors

23. Immunohistochemistry General endocrine markers- L abeling with at least one of the general endocrine markers - synaptophysin , chromogranin. P oorly differentiated endocrine tumors - usually negative for Cg A but retain their synaptophysin labeling C irculating CgA can also be used as a tumor marker for PENs

24. Grade 1 pancreatic neuroendocrine tumor (A) with Ki67 with a proliferation index of <2% (B) and phosphohistone-H3 (PHH3) showing no mitotic activity (C). The H&E stain of grade 2 pancreatic neuroendocrine tumor with two mitotic figures (arrows; D) showing increased Ki67 with a proliferation index of 3–20% (E) and increased mitotic activity by PHH3 (three mitoses per high power field; F). High-grade (grade 3) tumor (G) with >20% proliferation rate via Ki67 (H) and increased mitotic figures highlighted by the PHH3 stain (I)

25. Ki67 (MIB-1 antibody ) Proliferative activity is an integral part of the WHO classification. Assessment should be made in the ‘hot spot’. At least 40 HPF (1 HPF = 0.2 mm2) should be screened or 2000 tumor cells counted. Use of grid or printed microscopic picture of selected field

26. Hormones Confirmation of resected PET as a source of the clinically observed hormone hypersecretion. Identification of functioning PETs. Confirmation of neoplastic nature of small PETs.

27. Additional markers – Cytokeratin 8 and 18 are constantly positive, 7 & 20 are usually negative. - Cytokeratin 19 is regarded as a marker for aggressiveness – Trypsin - marker for acinar differentiation – if >25 % of neoplastic cells are positive (mixed acinar-endocrine carcinoma) – COX2, P27, CD99

28. Genetic studies in PENs MEN type 1: - pancreatic micro adenomatosis - seen in more than 80 % of MEN-1, are now considered to be precursor lesion of PENs. Loss of heterozygosity - mono hormonal islet like endocrine cell clusters found in MEN 1 pancreas - identified as fore runners of microadenomas

29. Hereditary syndromes like VHL, Tuberous sclerosis - No precursor lesion identified

30. Chromosomal anomalies – associated with tumor burden and stage of the disease DNA copy number status - proposed as the most sensitive and efficient marker of clinical outcome of insulinomas. Chromosomal instability - associated with tumor progression.

31. Chromosomal losses - more frequent than gains & amplifications Most common gains – chromosomes 5q, 7pq, 9q, 14q, 20q Most common losses - 1p, 3p, 11q, 6q Epigenetic anomalies - occur but there role in tumorigenesis not yet proved

32. Gene expression alterations Protein coding RNAs – assessment of the expression levels of particular genes. Regulatory microRNAs - particular pattern of micro RNA expression distinguishes PEN from normal pancreas and acinar carcinoma. miRNA-204 is primarily expressed in Insulinomas. miRNA-21 is high proliferation index and metastasis to liver.

33. FAMILIAL PANCREATIC CANCER

34. 5 % to 10 % of individuals with pancreatic cancer - history in close family member Several known genetic syndromes - increased risk of pancreatic cancer Known genes explain - only a portion of the clustering of pancreatic cancers Research has been on to identify additional susceptibility genes

35. RECENT ADVANCES IN PANCREATIC PATHOLOGY Part - II Dr. Indranil Bhattacharya Consultant Pathologist Jagjivan Ram Hospital Mumbai

36. PANCREATIC CYTOPATHOLOGY

37. Pancreatic cytology has become very important due to advanced imaging technology and EUS The combination of Cytology and Radiology Under the multimodal approach to pancreatic tumor diagnosis - cytology is presently combined to radiological techniques.

38. CT, USG, MRI , Magnetic Resonance cholangiopancreaticography, ERCP (esp. for obstructive jaundice and bile duct stricture) with biliary brushings. EUS (Endoscopic Ultrasound guided) FNA : For patients with small mass lesions in the body and tail of Pancreas and pts with cystic lesions). Lymph node staging and the detection of metastatic lesions are essential aspects of pancreatic cancer staging.

39. EUS-FNA allows for sampling of suspicious- appearing peripancreatic lymph nodes and liver lesions. Increases the accuracy of lymph node staging and can preoperatively stage pancreatic cancer. Percutaneous aspiration under CT or USG-can be used if above are unavailable. Brushings and intraductal aspirates procured by ERCP can be processed via monolayer technology

40. Well differentiated ductal adenocarcinoma Undifferentiated pancreatic carcinoma

41. ( A) a hypercellular smear is composed of 3 distinct cell types, including a centrally located, multinucleated osteoclastic giant cell surrounded by pleomorphic tumor giant cells and spindled and histiocytoid tumor cells (Diff‐Quik stain). (B) Tumor cells in a syncytial cluster include pleomorphic tumor giant cells and spindled cells with a multinucleated osteoclastic giant cell on the right (Papanicolaou stain). (C) Tumor giant cells are markedly pleomorphic, with nuclear irregularity and hypochromasia (Papanicolaou stain). (D) A corresponding resection sample from the same patient exhibits a mixed conventional pancreatic ductal adenocarcinoma (upper one‐half) and a circumscribed, undifferentiated carcinoma with osteoclastic giant cells.

42. Development of newer molecular markers – identification of cancer at an early stage. Common genetic alterations - activating point mutations in codon 12 of KRAS, silencing of p16, TP53 and DPC4. Similar changes in KRAS2 utilized for diagnostic purposes parallels the sensitivity of cytologic analysis.

43. CAP PROTOCOLS

44. EXOCRINE NEOPLASMS

45. T4

46. Regional lymphnodes

47. Regional L.N (contd.)

48. CAP Protocols for ENDOCRINE NEOPLASMS OF PANCREAS

49. Tumor dimensions – endocrine microadenomas (< 5 mm) Tumor multifocality – seen in majority of MEN 1 cases - careful gross examination of resected specimen with systemic sectioning at 3-5 mm

50. PANCREATIC TRANSPLANTATION

51. Pancreas transplantation is an effective treatment option for patients with either brittle or complicated diabetes mellitus (DM). A successful pancreas transplant results in disappearance of the acute complications of DM (i.e. hypoglycemia, severe hyperglycemia, and ketoacidosis). The first pancreas transplant was performed in 1966, but routine application of this procedure did not occur until the 1980s. The slower progress for pancreas transplantation in comparison to other organ transplants was related both to technical and immunological challenges inherent to the graft itself. Results of pancreas transplantation have continued to improve, with current 1-year graft survival (complete insulin independence) rates of 85% for SPK, 78% for PAK and 77% for PTA. One-year patient survival rates are excellent in all three categories, ranging from 95% to 97%

52. Increasingly accepted as a treatment for young to middle - aged adults afflicted with insulin - dependent Diabetes Mellitus (Type 1). Allograft rejection can be assessed by needle biopsies of the graft and/or cytologic studies of pancreatic juice drained from the graft. Types of rejection, including the grade of severity (0 to V), need to be separated from nonimmunologic causes of allograft dysfunction

53. Accurate determination of the cause of pancreas allograft dysfunction requires histological evaluation. Histopathological types of acute rejection, T-cell-mediated rejection (TCMR) and Antibody mediated allograft rejection (AMR), Histopathology has allowed for a better differentiation from each other and from other non-rejection-related processes. Acute TCMR is characterized by active parenchymal cellular infiltrates composed predominantly of T cells and typically involving veins, ducts, acini, and occasionally arterial branches.

54. The main differential diagnosis of TCMR includes infectious processes such as cytomegalovirus infection and EBV-related post-transplant lymphoproliferative disorder. Significant parenchymal involvement in acute AMR, is characterized by predominantly macrophagic (± neutrophilic) inflammation and typically Complement (C4d) - positive microvascular injury. Accurate diagnosis of TCMR and AMR, as well as mixed forms of rejection, requires Systematic analysis of the histological features, Evaluation of C4d staining, and Determination of circulating Donor Specific Antibody (DSA) status.

55. C4d staining in pancreas allografts. (A and B) Immunohistochemical and immunofluorescence C4d staining demonstrates comparable interacinar capillary staining. (C) Atrophic lobule in chronic active AMR shows strong C4d positivity in residual interacinar capillaries. (D) C4d staining in severe acute AMR. Due to extensive parenchymal necrosis there is nonspecific background staining with very rare recognizable positive interacinar capillaries. A thrombosed necrotic artery shows positive staining in its wall and contents.

56. Banff Schema for Grading of Acute Pancreas Allograft Rejection The Banff Foundation for Allograft Pathology also known as the Banff Foundation for Transplant Pathology is a nonprofit Swiss foundation. The goals of the Banff foundation are to facilitate knowledge generation and translation in transplantation pathology with the ultimate aim of improving patient outcomes, maintaining the Banff meeting spirit of a multinational, multidisciplinary consensus group.

57. Category Histopathology Comments Normal No inflammation OR inactive septal mononuclear inflammation not involving veins, arteries, ducts, or acini Fibrous tissue limited to septa in appropriate amounts; no injury or atrophy of acinar regions Indeterminate for Acute Rejection Active septal inflammation without other criteria for rejection (see below) Any venulitis or ductitis qualifies for at least mild acute rejection (or more depending on other features) Active inflammation refers to blastic lymphoctyes with variable numbers of eosinophils Grade I (Mild acute cell- mediated rejection Active septal inflammation with involvement of septal veins (venulitis) and/or ducts (ductitis) AND/OR focal (1-2 foci/lobule) acinar "active" inflammation with minimal/no acinar cell injury 1. Any venulitis or ductitis is sufficient for diagnosis; nerve branches usually involved but rarely sampled; focal acinar "active" inflammation alone also adequate for diagnosis

58. Grade II (Moderate acute cell- mediated rejection) Minimal intimal arteritis AND/OR multiple (3 or more foci/lobule) foci of acinar "active" inflammation with single cell injury/dropout 1. Any venulitis or ductitis is sufficient for diagnosis; nerve branches usually involved but rarely sampled; focal acinar "active" inflammation alone also adequate for diagnosis Grade III (Severe acute cell- mediated rejection) Widespread acinar inflammation with confluent areas of acinar cell injury/necrosis AND/OR moderate to severe intimal arteritis AND/OR necrotizing arteritis Any of these three findings is sufficient for the diagnosis Acinar inflammation may contain variable lymphocytes, eosinophils, and neutrophils as well as edema and/or hemorrhage Moderate/severe intimal arteritis consists of more frequent subendothelial lymphocytes with evidence of intimal injury, such as cell swelling, fibrin leakage, etc. Necrotizing arteritis may also occur in antibody- mediated rejection and C4d stain should be performed.

59. Chronic Active Cell-Mediated Rejection Chronic active cell- Arterial luminal 1. May represent mediated rejection narrowing due to intimal transition between proliferation of intimal arteritis and fibroblasts, chronic transplant myofibroblasts, smooth arteriopathy related to muscle cells, with suboptimal admixed T lymphocytes immunosuppression and macrophages 2. Rarely seen in needle ("active" transplant biopsies, more often arteriopathy) seen in allograft resection related to chronic rejection

60. Antibody-Mediated Rejection Category Histopathology Comments Hyperacute antibody-mediated rejection Widespread deposition of immunoglobulin (usu. IgG) and complement (e.g., C4d) with resultant arteritis and venous thrombosis, hemorrhagic necrosis and allograft failure usually within 1 hour after revascularization In all cases, diagnosis is dependent upon demonstration of a) graft dysfunction, b) capillary complement deposition (i.e., C4d positivity), AND c) donor specific antibodies in serum. If C4d and only 1 of the other 2 features is found, then the diagnosis"suspicious for" antibody- mediated rejection is more appropriate In cases in which vascular thrombosis is the predominant finding, the differential diagnosis lies between antibody-mediated rejection and "technical failure“. Accelerated antibody-mediated rejection Similar to hyperacute, but changes evolve over hours to days after revascularization. Acute antibody-mediated rejection Allograft dysfunction in first post-transplant weeks; histology varies from normal to margination of neutrophils and mononuclear cells to thrombosis and necrosis;

61. Take Home Message Pancreatic ductal adenocarcinoma (PDA) accounts for more than 85% of all pancreatic neoplasms. These tumors are derived from pancreatic ductal cells. PDA is one of the most genetically unstable organ cancers. Any given case often shows multiple chromosomal losses and gains. Somatic mutations of four key driver genes have been implicated in PDA: KRAS, P16/CDKN2A, TP53 , and SMAD4/DPC4 . KRAS is the most frequently identified oncogene in ductal adenocarcinoma and is seen in more than 90% of these tumors PanIN is the microscopic noninvasive precursor of PDA and shows similar genetic mutations to those seen in its invasive counterpart.

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