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Date posted: November 29, 2011

Dr G K Swethadri.
Prof. Deaprtemnt of Pathology
Fr. Muller Homeopathic Medical College
Mangalore.

As is your pathology so is your practice. sir william Osler
Historical aspects
Philosophy.
Renaissance
Clean water, asepsis, immunization
Rapid developments in the 20th century
Super-specializations

Fast pace of life is demanding early detection and cure. Fortunately the role of pathologist is well defined and needed and accepted by the surgeon.

Relevance of pathology to surgeons is immense.let me try to analyse these aspects.

All organs can suffer.

  • Inflammation
  • Degeneration
  • Immune insult
  • Hemodynamic derangements
  • Nutritional ,chemical,radiation injury
  • Trauma
  • Neoplasia
  • Genetic defects.

Consequence.

  • Changes in the organ and body as a whole.
  • Changes are either gross or microscopic .they are called morphological changes.The changes all begin in the cell.
  • ’it is called THE CELLULAR BASIS OF DISEASE”by Virchow.
  • Briefly Inflammation,degeneration,vascular changes and neoplasia are the features one looks for.
  • THE MICROSCOPE helps seek these changes
  • acute appendicitis-neutrophils
  • parkinsonism-lewy body
  • thrombus in a vessel in gangrene
  • cancer cells in a tumour

SURGEON WANTS THE PATHOLOGIST TO IDENTIFY THE CELLULAR/TISSUE CHANGES.that forms the clinicopathlogical correlation.

Some basic aspects

  • Hypertrophy of the left ventricle
  • Hypertrophy (micro)
  • Hyperplasia of endometrium
  • Atrophy of thymus gland
  • Morphology of cell injury
  • Protein accumulation – Hyaline
  • Hyaline degeneration – structureless, glassy, extra or intracellular material.
  • Lipids accumulation
  • Fatty change – intracellular accumulation of droplets of neutral fat (macro- and microvesicular).
  • The most common cause is poisoning and hypoxia.
  • The proteins of the cell are denatured and the cell becomes coagulated or clotted.
  • Cytoplasm becomes hyper-eosinophilic and the nuclear changes appear.
  • The cells persist as cell ghosts
  • Dead tissue appears firm and pale.

Gangrene

  • Gangrene is necrosis affecting the areas connected with the air and adde putrefaction.
  • Dry gangrene – ischemia without infection
  • If infection (clostridial) and oedema – wet gangrene or gas-gangrene.

An understanding pathologic basis must.

  • Once clinicopathlogical correlation is established then patient management becomes easy and objective.
  • That is why pathology is needed for the surgeons.
  • Let us study a few examples of CPC

It took a long time for histopathology to be accepted as a prerequisite for diagnosis

•Well it is easy.if not….

Let us study the example -breast

The expectations have changed.

  • Earlier we used to see huge cancer,bilateral,late stage.
  • Now self examination,mammography,needle aspirations have made women seek screening.even obese and those on OCP are seeking early detection.
  • Expectation of surgeon on pathologist is increasedearly diagnosis is the order of the day.

SCIENTIFIC RETHINKING  HALSTEAD RECOMMENDED RADICAL MASTECTOMY,

As histopathologists become aware that lympo-vascular emboli are seen in sections ,they wondered at the possibility of dissemination .Then why radical; save the organ as much as possible.now mastectomy,quadrantectomy are being practiced with radiotherapy

Not only diagnosis but also grading staging ,prognostification, decision making for therapy is coming in the perview of pathologist.

Breast conservation is the order of the day.a precise diagnosis and extent of disease are essential for this.

  • Steps of diagnosis
  • Clinical exam
  • Mammography
  • Ultra sound
  • MRI
  • FNAC
  • Tru-cut biopsy
  • Excision
  • Clinical+cytology+mammography=99.9 accuracy

Nottingham prognostic index

  • Index=0.2xsize+grade+node.(no=1,1-3 nodes=2,4>=3)
  • Take for example 6 cm tumour,4 positive lymph nodes and moderate grade(2)
  • (.2×6)+2+3=6.2.
  • Look at the prognosis.
  • Good=<3.4;mod=3-4.5;poor=5.4.
  • Our example is of a poor prognosis i.e.13%15 yr survival.

Organize your teaching.

  • Definition
  • Classification
  • Pathogenesis
  • Pathology-gross and microscopy
  • Diagnostic investigations
  • Differential
  • Prognosis
  • Research
  • the need for precise diagnosis;
  • verruca v/s Cervix:
  • Infiltrating Carcinoma Cx:
  • Squamous Carcinoma:
  • Melanma

Clark’s levels

  • 1.confine to epidermis and appendages-in situ
  • 2.papillary dermis-microinvasive; between papillary and reticular dermis a few melanoma cells
  • 3.pap dermis and impinge upon ret dermis
  • 4.impinge and invade ret dermis
  • 5.subcutaneus fat invasion

Some examples are illustrated

  • Mystery and happiness
  • Intrigue.
  • The mimic
  • Let us see how to teach a topic,for example’ BREAST PATHOLOGY”
  • Given as separate power point presentation.
  • Intra op diagnosis in CNS.note.described as a separate paper.
  • Frozen.
  • Sheets of round to spindle cells
  • Cartilagenous lobules & spindle cell areas & infiltration to bony trabeculae with destructon
  • Chondrosarcomatous areas
  • Pleomorphic cells in chondroid matrix
  • Distinct chondroid and spindle areas

WHAT IS IT.THE SURGEON ASKS

  • The mystery is solved to some extent by the pathologist.
  • Then the larger question;how can we together help people?
  • Registries;
  • Large medical force can contribute by educating and screening common man about cancer.
  • Aspects of curable-treatable cancer and spread of optimism.if detected early it can be cured.

Evolution of screening

  • One out of 10-13 people in India will be struck by cancer in their lifetime.
  • Tobacco,alcohol,diet,occupation,occupational disease,sexual hygiene.education and prevention.
  • NCCP was started in 1975-76. tobacco educaion,small family,18 yrs as marriageble age for female,use of condom
  • Secondary;cervical pap smear,pre invasive diseae is 100% curable.35% in advanced.
  • Detection centers in medical colleges(105 colleges),post partum centers,district hospital level is in the experimental stage.
  • In 1000 females-2.5-13.7 dysplasia was detected
  • Married ,asymptomatic,<40 yr,<20 yr married life,may be excluded from screeing.
  • Visual exam.13.7%of 67416 had unhealthy cervix.
  • FNAC for breast can cancer screen
  • Yearly occult blood for cancer.
  • 2 yearly videoscopic multiple biopsies for GIT

Chemoprevention.

  • Premalignant
  • Head and neck-isotretinoin
  • NSCLC         13-cis-retinoic acid.
  • HCC.HEP B VACCINE,INTERFERON
  • Ca colon-high fibre diet.calcium 1 g.in diet.
  • Pt with adenoma;folic acid and vit-c.
  • Anal cancer-homosexuality
  • Breast;tamoxifen in high risk by 35 yr

Investigations in cancer

  • Good clinical exam.routine blood tests
  • BRCA-1 from leucocytes.electrophoresis.MM
  • Bacterial,viral,parasitic studies
  • Tumour markers.laparotomy.B.M.biopsy
  • Tissue biopsy.frozen section.
  • U.S and CT guided needle biopsy,endoscopic,video assisted biopsy,IHC,EM,Cytogenetics.
  • Tissue diagnosis,grading,staging

Surgeon looks at pathologist for

  • Anemia-GIT,Leukemia,MPP,polycythemia,renal bleeding gum-coagulation
  • Liver-PT,PTT,Platelets,bleeding disorders
  • Microbiology.H.pylori,AIDS.kaposi,NHL,genital cancer
  • HPV cervix cancer,schistosoma-bladder tumour.

Serum markers.

  • PSA,CA-125,HCG,AFP
  • FOR DIAGNOSIS AND FOLLOW UP.
  • Hormones
  • BM aspiration and biopsy.

Different types of tissue biopsy.

  • Core needle biopsy;cylinder of tissue 3-4 m wide and 1-2 cm long.liver,lung,mediastinum,lung,pancreas,breast,prostate. tru-cut
  • Do BT,CT,PT,PTT before biopsy.trans jugular 16 gauge 85 cm needle biopsy if bleeding.
  • Excision.can be diagnostic and curative.skin,sub cutis,breastmelanoma,BCC.
  • Incision biopsy.if biopsy is difficult or dangerous a part of tissue is taken.ex.soft tissue.
  • Laparascopic biopsy.intraperitoneal organs.
  • Frozen section;brain breast to prevent second operation.
  • Endoscopic biopsy;nasal,pharyngeal,bronchial,esophageal,gastric,bile duct,colon etc.cystoscopy.
  • Spiral 3d CT can be used to guide fibroptic steriotactic biopsy forceps in glioma paracentesis
  • A tumour can present as an ascitis.repeat paracentesis is common in malignancy,study of effusion by cytology is of great help to dfferniate,transudate,exudate,tuberculosis,malignant ascitis.
  • Flow cytometry
  • Hematolymphoid neoplasms.detection and characterization,clonality,lineage,residual disease.

PCR

  • 1.CML,ALL,APL,ALCL
  • Ph CHROMOSOME-prognostic
  • Soft tissue.Ewings,PNET,alveolar rhabdo.
  • Minimal residual diseae.relapse.
  • FISH.
  • Down’s,AML,ALL.MDS,small round cell tumour.response to treatment.

PET scan

  • Using fluorodeoxyglucose.FDG.
  • Breast cancer for detection of metastasis.
  • Soft tissue.intralesional change,grade
  • Lung cancer.coin lesion.determining malignancy.
  • HD.treatment planning.

Cancer of unknown primary

  • 20%will never know about their primary.detection of primary also depends on history,clinical exam,endoscopy,FNAC,biopsy,
  • The microscopic feartures may give a clue about the primary.
  • STAGING-usefulness of biopsy
  • NSCLC.surgery for stage i,ii,iiiA.
  • Chemotherapy-III and IV stages
  • SCLC.TNM staging is not used.
  • Oesophagus.if adventitia is involved stage iii.
  • iii and iv-chemotherapy.
  • Stomach.<6mm endoscopic resection
  • i,ii,iii with minimal LN.intestinal type give 5 mm margin.if diffuse type give 8 mm margin.
  • Margin should be free
  • Role of immunocytochemistry.

HE / IHC

Basic antibody panel

Lymphoma panel

Pan-hematolymphoid marker

  • •CD45 (CD45RB):
  • –Leukocyte common antigen
  • –Positive in all normal lymphoid cells (B, T or NK lineage) except plasma cells
  • –Reed-Sternberg cells (Hodgkin lymphoma) are CD45-
  • –But usually not necessary to stain for LCA in a definite lymphoma: can proceed directly to lineage markers (e.g. CD20 + CD3)

Common B cell lymphomas

B lineage markers

  • •CD20 (e.g. L26):
  • –Very sensitive (>95% B cell lymphomas positive), although B-CLL may not stain well and B-cell lymphoblastic lymphoma may be negative
  • –Typically negative in plasma cells, plasmacytomas and plasmablastic lymphomas (thus CD20 negativity is a helpful clue to the diagnosis)
  • –Nucleolar staining alone should not be considered positive. With heat-induced epitope retrieval, any cell can show this phenomenon

B lineage markers

•CD79a (mb-1):

  • –B cell receptor complex: sensitive and specific B cell marker (normal plasma cells are positive; germinal center cells are stained weakly)
  • –~50% plasmacytomas are positive
  • –But can be positive in T-lymphoblastic lymphoma/leukemia
  • –Antibody expensive: Use as back-up
  • •Immunoglobulin (cytoplasmic and/or surface Ig)
  • •Other B-associated antibodies (LN1, LN2, MB1, MB2, 4KB5) are no longer useful

T-lineage markers

•Cytoplasmic CD3 (polyclonal against CD3e, monoclonal also available, e.g. PS1):

  • –Highly sensitive and specific for T lineage
  • –Staining stronger in perinuclear space, often accentuating the nuclear foldings
  • –Histiocytes may show faint cytoplasmic staining, but with no perinuclear accentuation
  • –Stains >90% of T and NK cell lymphomas
  • –May add other T markers (e.g. CD45RO, CD2) if CD3 is negative and yet T-cell lymphoma is suspected

T-lineage markers

  • CD45RO (e.g. UCHL1, A6):
  • –Highly sensitive for T lineage; some T cell lymphomas are CD3- CD45RO+
  • –But also stains histiocytes/myeloid cells and their tumors
  • •CD43 (e.g. MT1, DFT1, Leu22):
  • –Highly sensitive for T lineage, but not very specific
  • –Also stains histiocytes/myeloid cells and their tumors
  • –Expression in B cells can be upregulated in EBV infection
  • –More useful for diagnosis of small B-cell lymphomas (looking for aberrant expression)

T lineage markers

  • •CD4, CD8:
  • –Infrequently required for study of T cell lymphomas
  • •CD2:
  • –Sensitive and specific pan T cell marker
  • –More expensive than CD3 antibody: use as back-up
  • CD7:
  • –Pan T cell marker expressed early in T cell development
  • –Particularly helpful for demonstrating T lineage in lymphoblastic lymphoma (which can be CD3-)

Further typing of lymphoma

  • •Marker panel helpful for diagnosis of small B-cell lymphomas
  • –CD5
  • –CD23
  • –IgD
  • –Cyclin D1
  • –bcl-6 or CD10

Further typing of lymphoma

  • •Bcl-2:
  • –NOT helpful in classification of B-cell lymphomas, because many different lymphoma types are bcl-2 positive
  • –Only useful for distinguishing between follicular lymphoma (bcl-2+) and reactive follicular hyperplasia
  • •Diagnosis of plasmacytoma:
  • –LCA (commonly positive with sensitive immunostaining techniques, although classically considered negative)
  • –L26(-), CD79a(+/-), CD138(+), Oct-2(+), Bob.1(+), Ig(+)

Further typing of lymphoma

  • •CD10
  • –CD10 is the common acute lymphoblastic leukemia antigen, normally expressed by follicle center B cells and follicle center T cells
  • –Some spindly stromal cells are also CD10+ (serving as internal positive control)
  • –Value in lymphoma typing:
  • Follicular center cell differentiation (follicular lymphoma, Burkitt lymphoma, some large B-cell lymphomas)
  • Angioimmunoblastic T-cell lymphoma

Further typing of lymphoma

  • •Bcl-6
  • –Normally expressed (nuclear staining) in germinal center B cells, CD30+ perifollicular cells, and rare subpopulation of T cells
  • –Positive in the following lymphoma types:
  • •follicular lymphoma
  • some large B cell lymphomas
  • Burkitt lymphomas
  • some anaplastic large cell lymphomas

Further typing of lymphoma

MUM1

  • –Valuable marker for understanding and characterizing histogenesis of B-cell lumphoma(identification of the transition from Bcl6 positivity to CD138 expression)
  • –Excellent marker for Hodgkin’s Reed-Sternberg cells of classical Hodgkin’s disease in combination with CD30
  • –Has prognostic value since the expression correlates with clinical outcome of patients

Further typing of lymphoma

  • Follicular dendritic cell markers (such as CD21, CD35, and more recently, clusterin) can aid in the diagnosis of:
  • –Follicular lymphoma
  • –Mantle cell lymphoma (loose or dispersed meshworks)
  • –Angioimmunoblastic T cell lymphoma (extrafollicular meshworks)
  • Diagnosis of anaplastic large cell lymphoma
  • –BerH2/CD30, ALK1

CD30

  • •A marker for activated lymphoid cells; some CD30+ cells are found in perifollicular region
  • •CD30+ cells are increased in lymphoid hyperplasia
  • •Staining should be in the cell membrane +/- Golgi zone; pure diffuse cytoplasmic staining should not be considered positive
  • •CD30 is positive in classical Hodgkin lymphoma, anaplastic large cell lymphoma and some large cell lymphomas

Anaplastic Large Cell Lymphoma

Further typing of lymphoma

  • Proliferation marker Ki67: can aid in diagnosis of Burkitt lymphoma (~100% cells positive)
  • Cytotoxic markers, e.g. TIA1, granzyme B, perforin: may add in the diagnosis of NK/T cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma
  • Most useful marker for diagnosis of lymphoblastic lymphoma/leukemia: TdT

TdT (terminal deoxynucleotidyl transferase)

  • Polyclonal antibody generally gives stronger staining than monoclonal antibody
  • Outside the thymus, there are practically no TdT+ cells except:
  • –Tonsil (present in small numbers, especially at the base)
  • –Bone marrow (hematogones)
  • –Lymph node in children (scattered cells)

PROGNOSIS AND STAGING OF LYMPHOMA

  • For some lymphoma types, immunohistochemical markers may provide additional information important in prognosis
  • –Proliferation markers, e.g. high Ki67 index is associated with worse prognosis in mantle cell lymphoma
  • –Anaplastic large cell lymphoma: ALK+ subset has a much better prognosis than the ALK- subset

Summary: Most useful antibodies for identifying normal cell types and corresponding tumors

  • Mantle zone cell                                IgD
  • Immature precursor cell                     TdT
  • Plasma cell                             CD20-, CD138+
  • Histiocyte                              CD68 (PGM1 >KP1)
  • Follicular dendritic cell         CD21 + CD35
  • Langerhans cell                     S100, CD1a

Summary: Important markers for classification of lymphomas

  • Precursor lymphoblastic lymphoma: TdT
  • B-CLL: CD5, CD23
  • Mantle cell lymphoma: Cyclin D1
  • Follicular lymphoma: CD10 (or bcl-6)
  • Burkitt lymphoma: Ki67 (~100% proliferation index)
  • Angioimmunoblastic T cell lymphoma: CD10, follicular dendritic cell markers (extrafollicular meshworks)
  • Anaplastic large cell lymphoma: CD30, ALK

Carcinoma panel

“Specific” carcinoma markers

  • PSA              Prostate carcinomas
  • P504S     Stains both premalignant and malignant  prostate neoplasms,hepatocellular-, renal cell-, urothelial cell-, gastric- and                          colon carcinomas
  • TTF-1                  Thyroid and lung carcinomas
  • Thyroglobulin        Thyroid carcinomas
  • ER/PGR                 Breast, ovary, endometrial and cervical   carcinomas
  • Calcitonin               Thyroid medullary carcinomas
  • Chromogranin A   Neuroendocrine differentiation
  • Synaptophysin       Neuroendocrine differentiation

Antibody panel for carcinomas and mesotheliomas

  • BerEP4
  • Calcitonin
  • Calretinin
  • CEA
  • Chromogranin A
  • CK 5/6
  • CK 8/18
  • CK 20
  • CK 7
  • ER
  • Pan CK
  • PgR
  • PSA
  • PLAP
  • Synaptophysin
  • Thyroglobulin
  • TTF-1
  • VIM

Melanoma panel
Sarcoma panel

Antibody panel for sarcomas

  • CD117
  • CD31
  • CD34
  • CD45
  • CD57
  • CD99
  • Desmin
  • MyoD1
  • Pan CK
  • S-100
  • SMA
  • VIM

Conclusion

  • No immunostaining is 100% specific or sensitive
  • No panel is 100% specific or sensitive
  • No general rule is 100% applicable
  • Each specific diagnostic problem has to be addressed on individual basis
  • Each immunostaining has to be interpreted in its specific diagnostic context
  • Conclusion.pathologists approach to disease and how multidisciplinay approach is needed in the interest of the patient is discussed in brief

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