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.

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

9. CLASSIFICATION

10. 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

11. • 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

12. 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

15. PANCREATIC PRECURSOR LESIONS

16. ● 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.

17. • 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

19. 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.

20. • 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.

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

22. 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.

25. 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.

29. • 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.

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

31. • 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.

33. PANCREATIC
ENDOCRINE NEOPLASMS

34. • 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.

37. • 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

40. Immunohistochemistr
y
• General endocrine markers-
- Labeling with at least one of the general
endocrine markers - synaptophysin,
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

41. 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)

42. • 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) shouldbe
screened or 2000 tumor cells counted.
- Use of grid or printed microscopic picture of
selected field

43. • 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.

44. • 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

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

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

47. • 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.

48. • 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

49. 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.

50. FAMILIAL PANCREATIC CANCER

51. • 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

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

54. PANCREATIC CYTOPATHOLOGY

55. • 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.

56. • 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.

57. • 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

61. Well differentiated
ductal adenocarcinoma
Undifferentiated
pancreatic carcinoma

63. (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.

64. • 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.

65. CAP PROTOCOLS

66. EXOCRINE
NEOPLASMS

76. T4

77. Regional lymphnodes

78. Regional L.N
(contd.)

83. CAP Protocols for
ENDOCRINE NEOPLASMS
OF PANCREAS

88. • 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

89. PANCREATIC
TRANSPLANTATION

90. ● 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%

91. • 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

92. ● 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.

93. ● 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.

94. 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.

95. 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.

96. 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)
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

97. 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
1.Acinar inflammation may
contain variable
lymphocytes, eosinophils,
and neutrophils as well as
edema and/or hemorrhage
2.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.

98. 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

99. 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
appropriate
3.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;

100. 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.

101. Thank You