Other Malignancy

The TNM classification is targeted primarily at carcinoma but also includes malignant mesothelioma, malignant melanoma, gestational tro-phoblastic tumours, germ cell tumours and retinoblastoma. This section notes the commoner non-carcinomatous cancers such as uterine and gastrointestinal smooth muscle/stromal tumours, lymphoma/leukaemia and sarcoma. Summary diagnostic and prognostic criteria are given, where relevant.

Ancillary techniques

Various ancillary techniques can be useful in the histopathology of surgical cancer and should be employed as appropriate. Some of these are commented on at various points in the protocols, e.g. under sections 2. Histological type and 8. Other pathology.

Cytology

Fine needle aspiration cytology (FNAC) using 25-22-G needles has become the first-order investigation in many cancers due to its speed, cost-effectiveness, proficiency and convenience for both clinician and patient. It can not only provide specific inflammatory (e.g. Hashimoto's thyroiditis) and malignant diagnoses (e.g. thyroid papillary carcinoma) but can sort patients into various management groups: viz, inflammatory and treat, benign and reassure, atypical and further investigation (by core/open biopsy or excision) or malignant with specific therapy (surgery, chemotherapy, radiotherapy). It can be used to refute or confirm recurrence in patients with a known previous diagnosis of malignancy and to monitor response to therapy or change in grade of disease. It provides a tissue diagnosis of cancer in patients unfit for more invasive investigations or when the lesion is relatively inaccessible, e.g. in the lung periphery, mediastinum, abdomen, pelvis and retroperitoneum. It must be integrated with the clinical features and investigations (serology, radiology) and can be complemented by other techniques, e.g. core biopsy. It potentially provides material for routine morphology, histochemical and immunohistochemical techniques, electron microscopy, cell culture and flow cytometry. The direct smear and cytospin preparations can be augmented by formalin-fixed paraffin-processed cell blocks of cell sediments and needle core fragments (mini-biopsies), which can combine good morphology (the cores providing a tissue pattern) and robust immuno-histochemistry. It can be applied to many organs: salivary gland, thyroid gland, palpable lymphadenopathy, breast, skin, prostate, subcutaneous tissues and deep connective tissues, although in some cases, e.g. breast cancer, a lack of locally available cytopathological expertise has resulted in a trend back towards core needle biopsy. Radiologically guided FNAC is useful for non-central respiratory cancers and tumours in the mediastinum, liver, pancreas, kidney, retroperitoneum, abdomen and pelvis. Endoscopic FNAC is also being used more frequently, e.g. transbronchial, transrectal, transduodenal and transgastric/transoesophageal for lymph node staging or tumours covered by intact mucosa. Body cavity fluid cytology (both aspirates of free pleural, pericardial and peritoneal fluid and peritoneal/pelvic washings) continues to play an active role in the diagnosis, staging and monitoring of cancer. Yield of information is maximized by a combination of morphology and immunohistochemistry on direct smear/cytospin preparations (using air-dried Giemsa and wet-fixed Papanicolaou/H&E stains) and cell blocks (cell sediments and fragments).

Exfoliative cytology along with cytological brushings and washings is also pivotal in the assessment of various cancers, e.g. lung cancer, where the information obtained is complementary to that derived from direct biopsy and aspiration cytology. It can provide diagnostic cells not present in the biopsy specimen (for reasons of sampling error, tumour type or accessibility), correlate with it or allow subtyping that is otherwise obscured in artefacted biopsy material. Common sites of application are bronchus, mouth, oesophagus, stomach, bile duct, large intestine, bladder, renal pelvis and ureter.

Liquid-based preparations with good morphology and preservation of immunogenicity are increasingly complementing or replacing traditional cytological methods.

Frozen sections

There has been a dramatic reduction in breast pathology due to the triple approach of clinical, radiological and cytological examination (supple mented by wide core needle biopsy) resulting in preoperative diagnosis and appropriate planning of treatment. Frozen section is contraindicated in impalpable screen-detected lesions. Other uses are:

— check excision of parathyroid glands vs. thyroid nodules or lymph nodes in hyperparathyroidism.

— operative margins in gastric carcinoma, partial hepatectomy, head and neck and urinary cancers.

— cancer vs. inflammatory lesions at laparotomy.

— lymph node metastases in head and neck, urological, and gynaecological cancers prior to radical dissection.

— Mohs' micrographical surgery in resection of basal cell carcinoma of the face.

— frozen sections should be used sparingly due to problems of interpretation and sampling in the following cancers: malignant lymphoma, ovarian carcinoma, minimally invasive thyroid carcinoma, pancreas and extrahepatic bile duct carcinoma.

Histochemical stains

Histochemical stains are appropriately mentioned and can be valuable, examples being: PAS ± diastase or mucicarmine for adenocarcinomatous differentiation, PAS-positive inclusion bodies in malignant rhabdoid tumours and alveolar soft part sarcoma, PAS-positive glycogen in renal cell carcinoma.

Immunohistochemistry

Immunohistochemistry has become the surgical pathologist's "second H&E" and is invaluable in assessing tumour type, prognosis and potential response to treatment.

Tumour type

— further detail is given in their respective chapters but typical cancer type immunoprofiles are given in Table A.

— select antibody panels are also of use in differential diagnosis in a number of circumstances (Table B).

— the cytokeratin subtypes CK7 and CK20 have an important role to play in tumour characterization (Table C).

Prognosis

— Her-2, p53 oncogene expression, Ki-67 (MIB-1) proliferation index. Potential treatment response

— oestrogen/androgen expression and hormonal response in breast (e.g. tamoxifen) and prostate cancer.

— Her-2 expression and Herceptin (trastuzumab) therapy in breast cancer.

— CD20 expression and Rituximab therapy in non-Hodgkin's malignant lymphoma.

— CD117 expression and imatinib (Glivec STI 571) therapy in GISTs.

Table A Immunoprofile of cancer types

System

Tumour/condi tion

Marker panel

Head and neck

Salivary gland tumours Thyroid gland carcinoma

Gastrointestinal Oesophageal carcinoma

Barrett's oesophagus Gastric adenocarcinoma Small bowel adenocarcinoma Colorectal adenocarcinoma Hepatocellular carcinoma Pancreaticobiliary carcinoma Gastrointestinal stromal tumours Neuroendocrine carcinoids Respiratory Small cell carcinoma

Non-small cell carcinoma

Malignant mesothelioma

Gynaecological Ovarian carcinoma

Sex cord stromal Uterus, mesenchymal

Endometrial carcinoma Cervix—CGIN Cervical adenocarcinoma Hydatidiform mole

Epithelium: AE1/AE3 and myoepithelium: S100, Sm actin, calponin, CK 5/6

Papillary and follicular: thyroglobulin, TTF-1, CK19, galectin 3. Medullary: calcitonin, CEA, chromogranin Squamous cell: AE1/AE3

Adenocarcinoma: CAM5.2, CK7, ± CK20 (>50%) Villin, cyclin Dl, p53, Ki-67 CEA, EMA, CK7, ± CK20 (> 50%), CDX-2 CEA, EMA, CK20, ± CK7 (50%)

CEA, EMA, CK20, CDX-2, ± CK7 (5-10% particularly with poor differentiation or MSI-H) AFP, Hep Pari, CEA (polyclonal/canalicular), CD 10, CAM5.2, CK8, CK18 CEA, CAI9-9, CAI25, CK7, CK19, ± CK20, CDX-2

CD 117, CD34, vimentin, Ki-67, ± Sm actin, desmin, S100, protein kinase c theta

Chromogranin, synaptophysin, CD56, ± CAM5.2, Ki-67, gastrin, insulin, glucagon CAM5.2 (paranuclear dot), CD56, synaptophysin, chromogranin, TTF-1, Ki-67 Adenocarcinoma: CAM5.2, Ber EP4, CEA, CK7, TTF-1, PE10, CD 15, MOC 31 No specific type: AE1/AE3, CK5/6, 34|3E12

Positive: CAM 5.2, AE1/AE3, CK5/6, CK7, calretinin, WT1, thrombomodulin, HBME1, p53, EMA

Negative: CEA, BerEP4, CD 15, MOC 31 Serous: CK7, CA125, WT1

Mucinous/endometrioid: CEA, CK7, ± CK20, CDX-2

Inhibin, vimentin, calretinin, CD99, ± CAM5.2, AE1/AE3, EMA, Ki-67

Leiomyomatous: desmin, h-caldesmon, oxytocin, Sm actin, ± CD 10, Ki-67

Stromal: CD 10, Ki-67, ± desmin, h-caldesmon, Sm actin

ER, vimentin, CAM5.2, AE1/AE3, CK7, ± CK20, CD 10, p53 (serous)

CEA, CK7, ± CK20 (ER/vimentin negative), pl6 p57 kip2 in partial/complete (present/absent)

Genitourinary

Breast Soft tissue

Skin

Haematopoietic

Renal clear cell carcinoma Renal papillary carcinoma Renal chromophobe carcinoma Transitional cell carcinoma Prostate carcinoma Testicular germ cell tumour

Breast carcinoma Spindle cell sarcoma Small round blue cell tumours Adrenal carcinoma Phaeochromocytoma Malignant melanoma Merkel cell carcinoma Malignant lymphomas

CAM5.2, AE1/AE3, CD 10, EMA, vimentin, CD 15, RCC ab As above but also CK7, Ber EP4

As above including CK7, Ber EP4, E-cadherin, MOC 31 (and decreased CD 10, vimentin, RCC ab)

34J3E12, AE1/AE3, CK7, CK20, p53, uroplakin

PSA (polyclonal), PSAP, AMACR (P504S) but 34|3E12, p63 basal cell negative Seminoma: PLAP, CD 117, HCG, inhibin (syncytiotrophoblast giant cells) Embryonal carcinoma: CAM5.2, CD30, ± PLAP Yolk sac tumour: CAM5.2, AFP Choriocarcinoma: HCG, CK7 (cytotrophoblast)

ER, PR, Her-2/neu, CK7. Also Sm actin, CK5/6, CK14, CK8/18, E-cadherin (see Chapter 22) Vimentin, CD34, Sm actin, desmin, h-caldesmon, CAM5.2, AE1/AE3, EMA, S100, HHV8 CD45, tdt, SI00, CD99, Flil, desmin, myogenin, WT1, NB84, CAM5.2, CD56, CK20

Inhibin, melan-A, synaptophysin (cytokeratin, EMA, CEA negative) Chromogranin, SI00 sustentacular cells (cytokeratin negative) S100, melan-A, HMB 45, Ki-67 CD56, CK20, CAM5.2

CD45, CD20, CD3 and CD4, CD5, CD8, CD 10, CD15, CD21, CD23, CD30, CD56, CD57, K and X, cyclin Dl, bcl-2, bcl-6, bcl-10, ALK, Ki-67, LMP1, EBER, granzyme B, myeloperoxidase, mum 1

Sm actin, smooth muscle actin; TTF-1, thyroid transcription factor; CK, cytokeratins: specific (e.g. CK7, 20) or cocktails: CAM5.2, CKs 8, 18, 19; 34ßE12, CKs 1, 5, 10,14; AE1/AE3, CKs 10, 15, 16, 19/1-8; AFP, alphafetoprotein; HCG, human chorionic gonadotrophin; PLAP, placental alkaline phosphatase; Hep Pari, hepatocyte antibody; RCC ab, renal cell carcinoma antibody; CD56, neural cell adhesion molecule (NCAM); Ki-67, MIB 1; ER, oestrogen receptor; PR, progesterone receptor; PSA, prostate specific antigen; PSAP, prostate specific acid phosphatase; AMACR, alpha-methy-lacyl co-enzyme A racemase; tdt, terminal deooxynucleotidyltransferase; ALK, anaplastic lymphoma kinase; LMP1, latent membrane protein (EBV); EBER, EBV encoded RNA (in-situ hybridization); MSI-H, high level of microsatellite instability.

Adapted from McManus DT. Miscellaneous specimens. In Derek C Allen, R Iain Cameron (eds). Histopathology Specimens. Clinical, Pathological and Laboratory Aspects. Springer, London 2004

Queries about immunohistochemical staining may be answered at Immuno Query (http://www.ipox.org/login.cfm).

Table B Select antibody panels in differential diagnosis

Differential diagnosis

Antibody panel

Poorly differentiated tumour

• carcinoma/melanoma/lymphoma/germ cell tumour/sarcoma (epithelioid variants)

Mesothelioma/pulmonaiy adenocarcinoma

Small round cell tumour

• small cell carcinoma/Merkel cell carcinoma/lymphoma/ leukaemia/Ewing s: PNET/rhabdomyosarcoma/ neuroblastoma/intra-abdominal desmoplastic small cell

Bladder/prostate carcinoma

Renal carcinoma/adrenal cortical neoplasm/ phaeochromocytoma

Hepatocellular carcinoma/cholangiocarcinoma/metastatic colorectal carcinoma

Paget's disease of nipple/melanoma/Bowen s disease

Metastatic adenocarcinoma of unknown primary site: site indicative antibodies

CAM5.2, AE1/AE3, S100, melan-A, HMB-45, CD45, CD30, ALK, PLAP,

CD 117, desmin, CD34 AE1/AE3, CK5/6, calretinin, thrombomodulin, EMA, CEA, Ber EP4, MOC 31, TTF-1

CAM 5.2, CD56, CK20, CD45, tdt, CD99, desmin, myogenin, synaptophysin, WT1, Ki-67

CAM5.2, AE1/AE3, CD 10, EMA, RCC ab, inhibin, melan-A, synaptophysin, S100, chromogranin CAM5.2, AE1/AE3, AFP, Her Par 1, CEA (polyclonal/canalicular), CD 10, CK7, CK20, CDX-2

CAM5.2, AE1/AE3, EMA, CK7, Her-2, S100, melan-A, (CK20 for anovulval Paget's)

Thyroglobulin, TTF-1: thyroid carcinoma and lung adenocarcinoma

CEA, calcitonin: medullary carcinoma thyroid

PSA (polyclonal), PSAP: prostate carcinoma

CDX-2: gastrointestinal carcinoma

CA125: ovary and sometimes pancreas, breast carcinoma GCDFP-15, ER, PR: breast carcinoma CA19-9: pancreas, upper gastrointestinal carcinoma PLAP, CD 117, CD30: germ cell tumour

AFP, Hep Par 1, CEA (polyclonal/canalicular): hepatocellular carcinoma RCC ab, CD 10: renal cell carcinoma CK7, CK20: see Table C

Table C CK7, CK20 tumour expression

Immunoprofile Carcinoma

Gastric adenocarcinoma Pancreatic adenocarcinoma Transitional cell carcinoma Ovarian mucinous adenocarcinoma

Lung adenocarcinoma Breast adenocarcinoma

Ovarian serous and endometrioid adenocarcinoma Endometrial/endocervical adenocarcinoma (usually CK20 negative) Mesothelioma

CK7 - CK20 + Colorectal adenocarcinoma

CK7 - CK20 - Prostate adenocarcinoma

Renal clear cell adenocarcinoma

Hepatocellular carcinoma

Lung carcinoma (non-adenocarcinoma types)

Antibodies should not be used in isolation but a panel employed with positive and negative in-built and external controls. This is due to a spectrum of co-expression seen with a number of antibodies, e.g. EMA (carcinoma, plasmacytoma, Hodgkin's disease and anaplastic large cell lymphoma) and CD 15 (Hodgkin's disease and lung adenocarcinoma). Interpretation should also be closely correlated with the morphology. The above are only part of a rapidly enlarging spectrum of new generation, robust antibodies that can be used with formalin-fixed paraffin-embedded tissues and show enhanced demonstration of expression by heat-mediated antigen retrieval techniques such as microwaving and pressure cooking, and highly sensitive polymer-based detection systems. It is important to determine that the immunopositive reaction is in an appropriate location (e.g. membrane staining for Her-2/neu, nuclear staining for ER, TTF-1), is not simply related to entrapped normal tissues (e.g. infiltration of skeletal muscle fibres), and is of appropriate staining intensity. In some circumstances the number of positive cells is important, e.g. Ki-67.

Electron microscopy

Electron microscopy has a diagnostic role to play where morphology and immunochemistry are inconclusive. Specific features can be sought in:

— carcinoma (tight junctions, short microvilli, secretory vacuoles, intermediate filaments).

— vascular tumours (intra-cytoplasmic lumina, Weibel-Palade bodies).

— neuroendocrine carcinoma (neurosecretory granules).

— mesothelioma (long microvilli).

— smooth muscle/myofibroblastic tumours (longitudinal myofilaments with focal dense bodies).

— rhabdomyosarcoma (basal lamina, sarcomere Z line formation).

— perineural/meningeal lesions (elaborate complex cytoplasmic processes).

Molecular and chromosomal studies

Evolving areas of diagnostic use of molecular and chromosomal studies are clonal immunoglobulin heavy chain and T-cell receptor gene rearrangements in the confirmation of lymphoma, and the characterization of various cancers (particularly lymphoma, sarcoma and some carcinomas, e.g. renal) by specific chromosomal changes. Gene rearrangement studies can be carried out on formalin-fixed paraffin-embedded material but fresh tissue put into suitable transport medium is required for chromosomal analysis—although RT-PCR methods are being developed for paraffin material. Genotypic subtypes of various malignancies, e.g. rhabdomyosarcoma, have been defined with differing clinical presentation, prognosis and response to therapy. Some examples are:

follicle centre lymphoma, follicular t(14;18) mantle cell lymphoma t(11;14)

synovial sarcoma t(X:18)(p11.2;q11.2)

myxoid liposarcoma t(12;16)(q13;p11)

In-situ hybridization techniques may be used to detect viral nucleic acid (e.g. EBV in post transplant lymphoproliferative disorders, HPV subtyping in cervical biopsies), lymphoid clonality (k, % light chain mRNA) and karyotypic abnormalities such as Her-2/neu amplication in breast cancer and n-myc in neuroblastoma.

Quantitative methods

There is an expanding literature regarding the use of quantitative methods as diagnostic aids. These include stereology, morphometry, automated image analysis, DNA cytophotometry and flow cytometry. In general, adverse prognosis is related to alterations in tumour cell nuclear size, shape, chromasia, texture, loss of polarity, mitotic activity index, proliferation index (Ki-67 or S-phase fraction on flow cytometry), DNA aneuploidy and spatial density. Most of these techniques show good correlation with carefully assessed basic histopathological criteria and, rather than replacing the pathologist and microscope, serve to emphasize the importance of various parameters and sound morphological technique. Areas of incorporation into pathological practice are:

— morphometric measurement of Breslow depth of melanoma invasion, osteoid seams in osteomalacia and muscle fibre type and diameter in myopathy.

— mitotic activity index in breast carcinoma.

— DNA ploidy in partial vs. complete hydatidiform mole.

With the advent of more sophisticated computers and machine-driven technology, artificial intelligence and automated tissue analysis are being explored:

— automated cervical cytology.

— inference and neural networks in prostatic cancer and colonic polyps.

— Bayesian belief networks and decision support systems in breast cytology.

— MACs (malignancy associated changes) in prostate cancer based on alterations in nuclear texture.

— bioinformatics facilitates analysis of gene and tissue microarrays used to test the level of expression for multiple genes in relatively few samples, and the staining pattern of relatively few markers on a large number of samples, respectively. This allows more standardized scoring of current prognostic markers on samples from multiple patients, and also leads to the discovery of new prognostic cancer biomarkers.

This whole area is rapidly developing and evolving, and it remains to be resolved as to which facets will eventually be incorporated into routine practice.

Error, audit, quality assurance, clinical governance and digitized microscopy

Errors in a subjective discipline such as diagnostic pathology are inevitable, but rates are surprisingly low (1-2%). Whether cognitive (oversight or interpretational) or operative (non-analytical), they may be purely academic (e.g. a difference in nomenclature) or clinically significant (e.g. a false-positive diagnosis of cancer). Any surgical pathologist hopes to avoid the latter and the potential consequences for the patient. Errors are discovered by various routes: inconsistency in clinical outcome with individual case review, review at regular multidisciplinary cancer meetings, topic related audit, systematic selective surgical case review, or prospective in-house or external case referral for opinion. Clinical governance defines standards of care with open acknowledgement, communication and correction of errors. Professionals are encouraged to quality control, sometimes double report, and check their work in a team context supporting colleagues and identify any indicators of under-performance. In the UK the Royal College of Pathologists Professional Standards Unit publishes protocols and advises on issues of professional performance with the capacity to investigate and recommend remedial action in individual cases. Consequently, most pathologists adopt several strategies to maintain standards, including participation in continuing professional development (CPD) and external quality assurance (EQA) schemes. CPD entails attendance at local, national and international conferences and seminars, journal reading and other educational activities relevant to the pathologist's practice with reinforcement of strengths and identification of knowledge gaps. This approach is inherent to annual appraisal (and potentially revalidation), which should be consolidative and developmental in nature. EQA schemes are general or specialist in type with precirculation of slides and clinical clues. The pathologist submits diagnostic answers which are marked in comparison with the participants' consensus diagnoses. Results are confidential to the patho logist but an individual with repeated outlying marks will be flagged up to the scheme co-ordinator so that appropriate advice can be given. Definition of what constitutes a specialist pathologist is complex but at least involves spending a significant amount of professional time practising in the relevant area and participation in an appropriate EQA scheme. Pragmatically, experience tells who to send the referral case to and just "who can do the business". The ever-improving technology of digitized virtual microscopy will develop a role as an alternative to slide circulation in EQA schemes and case referral to experts, as potentially will telepathol-ogy for live remote diagnostic reporting to supplement its current use as a tool to facilitate cross site multidisciplinary meetings. Automated immunocytochemistry, multiblocks and microarrays will augment morphology by assessment of multiple prognostic markers. Thus the way ahead is charted for the surgical pathologist, and in all of this there may well still be no substitute for showing a slide around to colleagues whose opinion you value.

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