RECENT ADVANCES IN PANCREATIC PATHOLOGY

Presented by: Surgeon Lt Cdr N DograGUIDE: COL R B BATRA

• Classification• Precursor lesions• Pancreatic endocrine neoplasms• Familial endocrine neoplasms• Pancreatic cytopathology• CAP protocols• Advances in acute pancreatitis pathogenesis• Pancreatic transplant

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 1year 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.(Pancreatic Cancer: eMedicine Oncology Pancreatic Cancer

Author: Richard A Erickson, MD, FACP, FACG, Professor of Medicine, Division of Gastroenterology, Department of Internal Medicine, Texas A&M University Health Science Center; Director, Scott and White Clinic and Hospital Updated: Apr 7, 2009)

(eMedicine Specialties > Oncology > Carcinomas of the Gastrointestinal TractPancreatic CancerAuthor: Richard A Erickson, MD, FACP, FACG, Professor of Medicine, Division of Gastroenterology, Department of Internal Medicine, Texas A&M University Health Science Center; Director, Scott and White Clinic and Hospital) Updated: Apr 7, 2009 )

The pancreatic lesions can be divided into

Endocrine Exocrine

Diabetes Neoplasms Acute & NeoplasmsMellitus chronic

pancreatitis

CLASSIFICATION

WHO CLASSIFICATION OF EXOCRINE OF TUMOURS OF PANCREAS(Pathology and genetics, Tumours of digestive system IARC WHO CLASSIFICATION no. 2, Hamilton, S.

R , Aaltonen, L.A)

• Epithelial tumorsBenignSerous cystadenomaMucinous cystadenomaIntraductal papillary mucinous adenomaMature teratomaBorderline (uncertain malignant potential)Mucinous cystic neoplasm with moderate dysplasiaIntraductal papillary mucinous neoplasm with moderate dysplasiaSolid pseudopapillary neoplasm

• MalignantDuctal adenocarcinoma Mucinous noncystic carcinoma Signet-ring-cell carcinoma Adenosquamous carcinoma Undifferentiated (anaplastic) carcinoma Undifferentiated carcinoma with osteoclastlike giant cells Mixed ductal-endocrine carcinomaSerous cystadenocarcinomaMucinous cystadenocarcinoma Noninvasive Invasive

Malignant tumors (cont)

• Intraductal papillary mucinous carcinoma Noninvasive Invasive (papillary mucinous carcinoma)Acinar cell carcinoma Acinar cell cystadenocarcinoma Mixed acinar-endocrine carcinomaPancreatoblastomaSolid pseudopapillary carcinomaOtherNonepithelial tumorsSecondary tumors

PANCREATIC PRECURSOR LESIONS

• 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

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 with moderate dysplasia PanIN-3: Carcinoma in situ, intraductal carcinoma, severe ductal dysplasia, high-grade dysplasia, ductal hyperplasia grade 3, atypical hyperplasia

• 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.

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

• 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 – over expression is implicated in tumour cell growth, invasion, angiogenesis, and prognosis

• Cyclooxygenase 2 overexpression -potential target for chemotherapy by selective COX-2 inhibitors.

•

• Other preinvasive lesions-intraductal papillary mucinous

neoplasms-mucinous cystic neoplasms

• 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.

PANCREATIC ENDOCRINE NEOPLASMS

• 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.

• 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

Immunohistochemistry• General endocrine markers-

- labeling with atleast one of the general endocrine markers - synaptopyhsin, chromogranin.

- poorly differentiated endocrine tumors - usually negative for Cg A but retain their synaptophysin labeling

- circulating CgA can also be used as a tumor marker for PENs

• 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

• 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

• 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

Genetic studies in PENs

• MEN type 1:- pancreatic microadenomatosis -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

• Hereditory syndromes like VHL, Tuberous sclerosis - No precursor lesion identified

• 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.

• 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

Gene expression alterations• Protein coding RNAs – assessment of the

expression levels of particular genes

• Regulatory micro RNAs - 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

FAMILIAL PANCREATIC CANCER

• 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

• Surgically resected pancreas with strong family history - multifocal PanINS with lobulocentric atrophy.

• Appear as heterogenous mass on EUS and CT

• At risk individuals can be screened - early curable neoplasms detected before they progress to invasive cancer.

Screening at risk patients for early pancreatic neoplasia

• Combination of computed tomography, EUS, ERCP

• FAMILIAL CASES-harbour more multifocal precursor lesions, can be confirmed using KRAS gene mutation analysis

• Lobulocentric atrophy- common finding in familial cases.

PANCREATIC CYTOPATHOLOGY

• Pancreatic cytology has become very important due to advanced imaging technology and ESU

• The combination of cytology and radiology

• Under the multimodal approach to pancreatic tumor diagnosis-cytology is presently combined to radiological techniques

• CT,USG, MRI, Magnetic resonance cholangiopancreaticography, ERCP (esp.for obstructive jaundice and bile duct stricture) with biliary brushings.

• EUS (Endoscopic ultrasound guided)-FNA-( esp for patients with small mass lesions in the body and tail and pts with cystic lesions).

• Lymph node staging and the detection of metastatic lesions are essential aspects of pancreatic cancer staging.

.

• 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 aspirates under CT or USG-can be used if above are unavailable

• Brushings and intraductal aspirates procured by ERCP can be processed via monolayer technology

Well differentiated ductal adenocarcinoma Undifferentiated pancreatic carcinoma

• 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 -utilised for diagnostic purposes -parallels the sensitivity of cytologic

analysis

.

• Other potential markers - aberrantly expressed proteins and mucins identified through mRNA expression profiling

• Specific markers -prostrate stem cell antigen, mesothelin, and MUCI

• Barriers - reliance on presence of substantial

diagnostic material- imperfect specificity-cost

CAP PROTOCOLS

EXOCRINE NEOPLASMS

T4

Regional lymphnodes

Regional L.N (contd.)

ENDOCRINE NEOPLASMS OF PANCREAS

• 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

• Ancillary techniques

ACUTE PANCREATITIS

• Initiating cellular events - acinar cell injury• Co-localization of zymogens with lysosomal

hydrolases,

• Premature enzyme activation • Pathological exocytosis of zymogens into the

interstitial space.

• Accentuation of cell injury• Triggering of acute inflammatory mediators

• Intensification of oxidative stress• Compromise of microcirculation • Activation of a neurogenic feedback

• Localized events progress to a systemic inflammatory response

• Multiorgan dysfunction syndrome

PREMATURE ACTIVATION OF PANCREATICENZYMES WITHIN THE PANCREATIC ACINAR CELL

• Hyperstimulation model – cholecystokinin

• Missorting & co-localization of zymogens with lysosomal cathepsin B within large cytoplasmic vacuoles

• Trypsinogen cleavage and activation

BASOLATERAL EXOCYTOSIS INTO THE INTERSTITIAL SPACE

Misdirection to the basolateral plasma membrane

Shown recently by real-time imaging of single ZG exocytosis in supra-maximal CCK-stimulated rat pancreatic acinar cells

Molecules mediating basolateral exocytosis have been elucidated

(SNAP) receptor (SNARE) hypothesis

Cytosolic N-ethylmaleimide-sensitive factors & soluble SNAPs bind SNAREs on donor vesicles & target membranes

Multimolecular complexes formation

The union between v- and t-SNAREs

Fusion of the two membranes

• There is specificity of membrane fusion events

• Compartmental specificity of distinct sets of v-SNARE and t-SNARE proteins

• Accessory proteins regulate the SNARE complex assembly

ROLE OF INFLAMMATORY MEDIATORSAcini undergoing injury release zymogens, particularly trypsin

Induce macrophages to synthesize and release proinflammatory cytokines

Neutrophil recruitment and activation within the pancreatic tissue

Acinar cells themselves can synthesize cytokines, which amplify the local inflammatory response

• TNF-α has direct actions on acini - activation and disruption of the actin cytoskeleton

• TNF-α can also induce apoptosis

NF-κB is a transcription factor that promotes the expression of proinflammatory cytokines

Supramaximal CCK stimulation in vitro or in vivo, causes NF-κβ activation within acinar cells

NEUROGENIC INFLAMMATION

Stimulation of primary sensory neurons trigger autonomous arc reflexes (AARs)

• Acute, neurogenic inflammatory response initiated in the pancreatic tissue

• AARs integrate the duodenum autonomic nerve fibres with those from the celiac ganglion and bulbar-hypothalamic nuclei

• Sensory neurons in the pancreas contain unmyelinated, capsaicin-sensitive (type C) nerve fibres release sensory peptides

Substance P, neurokinin A and vasoactive intestinal peptides

Peptides act on mast cells, causing release of histamine and proinflammatory mediators

• New imaging modalities have enhanced the understanding and management of acute pancreatitis

• Radiographic assessment of the extent of inflammation and necrosis within 48 hours

• It allows for a more standardized diagnosis, severity assessment, treatment plan, and complication identification

PANCREATIC TRANSPLANTATION

• 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

• Forms of rejection, including the grade of severity (0 to V), need to be separated from nonimmunologic causes of allograft dysfunction

2007 Banff Schema for Grading of Acute Pancreas Allograft Rejection

Category Histopathology CommentsNormal 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)

1. Any venulitis or ductitis qualifies for at least mild acute rejection (or more depending on other features)2. 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

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 diagnosis1. Acinar inflammation may contain variable lymphocytes, eosinophils, and neutrophils as well as edema and/or hemorrhage2. Moderate/severe intimal arteritis consists of more frequent subendothelial lymphocytes with evidence of intimal injury, such as cell swelling, fibrin leakage, etc.3. Necrotizing arteritis may also occur in antibody-mediated rejection and C4d stain should be performed.

Chronic Active Cell-Mediated Rejection

Chronic active cell-mediated rejection

Arterial luminal narrowing due to intimal proliferation of fibroblasts, myofibroblasts, smooth muscle cells, with admixed T lymphocytes and macrophages ("active" transplant arteriopathy)

1. May represent transition between intimal arteritis and chronic transplant arteriopathy related to suboptimal immunosuppression2. Rarely seen in needle biopsies, more often seen in allograft resection related to chronic rejection

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

1. 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. 2. If C4d and only 1 of the other 2 features is found, then the diagnosis"suspicious for" antibody-mediated rejection is more appropriate3. 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 posttransplant weeks; histology varies from normal to margination of neutrophils and mononuclear cells to thrombosis and necrosis;

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Sternberg's Diagnostic Surgical Pathology, fifth edition, 2009• Caroline S Verbeke, Review-Endocrine tumours of the

pancreas; Histopathology 2010, 56, 669-682• Ralph H, Hruban, MD, Giuseppe Zamboni et al, Special issue-

Insights and Controversies in Pancreatic Pathology; Arch Pathol Lab Med, March 2009, vol 133: 147-399

• Kay Washington, Jordan Berlin et al , Protocol for the Examination of Specimens from Patients with Carcinoma of the Endocrine Pancreas and Exocrine Pancreas; AJCC/UICC TNM, 7th edition (Protocol web posting date: October 2009)

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• Drachenberg CB et al, Banff schema for grading pancreas allograft rejection:working proposal by a multi-disciplinary international consensus panel, Am J Transplant 2008; 8 :1-13