NEOPLASIA I- INTRODUCTION TO NEOPLASIA

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I. INTRODUCTION TO NEOPLASIA
II. CLINICAL FEATURES OF MALIGNANCY
III. HISTOLOGIC (ARCHITECTURAL) AND GROSS FEATURES OF MALIGNANCY
IV. GRADING AND STAGING OF TUMORS
V. THE LABORATORY DIAGNOSIS OF MALIGNANCY

NEOPLASIA DIAGNOSIS OVERVIEW:

The focus of this week of lectures and Wednesday's laboratory will be the diagnosis of neoplasia. The format is to present this section to you as you will see and diagnose malignancy in your patients. Thus, after a brief introduction to the vocabulary of neoplasia, the clinical features of malignancy will be discussed. The next step in the patient's workup is often cytologic, with material obtained by fine needle aspiration (FNA) or by exfoliative cytology (urine, sputum, etc.). Tissue is often obtained in the form of biopsy or tumor resection (histologic and gross features of malignancy). Grading and staging of tumors will be important in the treatment options offered. Finally, laboratory diagnosis of malignancy will familiarize you with the role additional laboratory testing can play in the clinical work up of a patient. The neoplasia laboratory on Wednesday will expose you to the cytologic, histologic and gross features of three very common cancers (breast, lung and colon). Although our handout in the syllabus is fairly extensive, the reading in Robbins will help provide a framework which you will find most useful. Next week the molecular basis of neoplasia will be presented.

I. INTRODUCTION TO NEOPLASIA

• Vocabulary of Malignancy
  
  
• Clinical Features of Malignancy

Neoplasm: (Literally: New growth) An abnormal tissue mass whose growth exceeds and is uncoordinated with that of adjacent normal tissue and persists after cessation of the stimuli that provoked it.

A neoplasm results from the derangement of normal growth control mechanisms. In some manner, the balance between cell division and cell death is upset. Hyperplasias can result as a reaction to environmental influences such as chronic inflammation, but a neoplasm's growth is partially or totally independent of such external influences.

Benign Neoplasm:

A neoplasm that grows without invading adjacent tissue or spreading to distant sites. It is usually fairly well-circumscribed due to the lack of invasion of surrounding tissues.

Malignant Neoplasm:

A neoplasm that invades the surrounding normal tissue and usually spreads to distant sites given sufficient time.

Differentiation:

The tissue type represented by the tumor. Well-differentiated tumors resemble the tissue of origin while poorly differentiated tumors may only be identifiable by the expression of cell markers or by extremely focal and subtle histologic and/or cytologic findings.

Anaplasia:

Loss of differentiation

Dysplasia:

Atypical proliferation of cells characterized by nuclear enlargement and failure of maturation and differentiation, short of malignancy.

• Dysplasia is recognized by alterations in the appearance of cells (cytology). Dysplastic cells have some of the features of malignant cells but the changes are less pronounced (see section on cytology of malignancy below). This makes sense, because dysplasias have to undergo a series of genetic changes to become malignancies. As the dysplasia progresses, the nuclei of cells become more hyperchromatic and the nuclear membranes become more irregular; the size of the nucleus increases and the cytoplasm does not increase proportionately, so the nuclear:cytoplasmic ratio increases.
  
  
• A dysplasia may regress, persist, or progress.
  
  
  • The more advanced the dysplasia, the less likely regression will occur.
  • Currently, there are few molecular markers available to identify dysplasias. The abnormality in the microscopic appearance of the cells is the major marker we have.

Not all of the lesions we call dysplasias are neoplasias that will go on to become carcinomas. Use of the term dysplasia defines a risk of progression.

Reactive atypia:

An abnormal cellular appearance and an increased mitotic rate associated with a reparative state due to environmental influences such as inflammation. Reactive atypias are non neoplastic.

• Once the environmental influence goes away, the atypia disappears.
  
  
• The atypia general involves enlargement of both the cytoplasm and nucleus and an increased prominence of nucleoli. These features reflect the increased metabolic activity of the cells.
  
  
• Malignant criteria such as extremely irregular nuclear membranes are absent.

Carcinoma in situ (cis):

Full-thickness dysplasia extending from the basement membrane to the surface of the epithelium. Applicable only to epithelial neoplasms. If the entire lesion is no more advanced than CIS, then the risk of metastasis is zero. This is because there are no blood vessels or lymphatics within the epithelium above the basement membrane.

Invasion:

Growth into the surrounding tissue by direct expansion.

Metastasis:

Spread of tumor to distant sites by lymphatic or hematogenous routes.

Primary tumor:

Tumor at the site of origin (vs. metastasis)

Neoplasms are composed of 1) neoplastic cells and 2) stroma and vessels. Tumors induce an expansion of stroma (desmoplasia) and greater numbers of vessels (angiogenesis). In desmoplasia, there is a hyperplasia of fibroblasts that appear activated and an abundant collagenous stroma that often has a more bluish appearance than normal stroma on hematoxylin/eosin stained sections.

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Differentiation:

Differentiation can be defined by architectural or cytoplasmic features. Examples of architectural features include gland formation by adenocarcinomas and follicle (germinal center) formation by B cell lymphomas. Cytoplasmic features include mucin vacuoles in many adenocarcinomas and immunoglobulin production by B cell lymphomas.

A well-differentiated tumor is easily recognized by histology or cytology as arising from a specific tissue type. A poorly-differentiated tumor is barely recognizable as arising from a specific tissue type. Moderately differentiated tumors are somewhere in between. The term "undifferentiated" is used when no trace of differentiating features are present.

The Vocabulary of Neoplasms

ORIGIN		BENIGN	MALIGNANT
I. Epithelial
	Stratified squamous	Squamous cell papilloma	Squamous cell carcinoma
	Basal cells of skin		Basal cell carcinoma
	Epithelial lining from glands or ducts	Adenoma (e.g. of colon)	Adenocarcinoma (e.g. of colon)
	Hepatocytes	Hepatocellular adenoma	Hepatocellular carcinoma (also called "hepatoma", a confusing germ that should be avoided)
	Melanocytes	Nevus	Melanoma (or malignant melanoma)
	Renal	Renal cell adenoma	Renal cell carcinoma
	Urinary Epithelium (transitional)	Transitional cell papilloma	Transitional cell carcinoma
II. Mesenchymal Origin
A. Connective Tissue	Bone	Osteoma	Osteosarcoma
	Cartilage	Chondroma	Chondrosarcoma
	Fibroblast	Fibroma	Fibrosarcoma
B. Hematopoietic	Erythroid		Erythroid leukemia
	Myeloid		Myelogenous leukemia
	Lymphoid		Lymphocytic leukemia malignant lymphoma
C. Muscle	Smooth muscle	Leiomyoma	Leiomyosarcoma
	Striated (skeletal) muscle	Rhabdomyoma	Rhabdomyosarcoma
D. Vascular		Hemangioma	Hemangiosarcoma
III. Origin from a single germ layer with more than one type of differentiation
	Renal anlage (primitive mesodermal tissue)		Wilm's tumor
	Salivary glands	Pleomorphic adenoma (mixed tumor of salivary gland)	Malignant mixed tumor of salivary gland
IV. Origin from more than one germ layer
	Germ cells of gonads	Teratoma (dermoid cyst or teratoma)	Teratocarcinoma

The Importance of Tumor Differentiation:

• Site of origin in metastatic disease: If a lymph node is biopsied and there is squamous cell carcinoma in it, tissues with squamous differentiation are implicated. Sites to examine would include the lungs, upper respiratory tract, the gyn tract, and the skin.
  
  
• Prognosis: Prognosis varies with tumors arising at the same site, and with different levels of differentiation (well-differentiated often has better prognosis than poorly-differentiated tumors).
  
  
• Treatment: Treatment varies: adenocarcinomas may be treated with different regimens than squamous cell carcinomas.

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II. CLINICAL FEATURES OF MALIGNANCY

• Location and impingement on adjacent structures: Mediastinal masses can compress the trachea causing respiratory distress. Colon tumors can cause obstruction.
  
  
• Functional activity such as hormone synthesis: Parathyroid tumor may secrete parathyroid hormone resulting in hypercalcemia. Pheochromocytoma can elaborate catecholemines.
  
  
• Bleeding and secondary infections: Colon cancer often presents with melena (blood in the stool).
  
  
• Initiation of acute symptoms caused by rupture or infarction: Ovarian tumors can experience torsion and present with acute pain and even rupture as the venous drainage becomes blocked before the arterial supply.
  
  
• Cancer Cachexia: Cachexia refers to a wasting syndrome consisting of progressive loss of body fat accompanied by profound weakness, anorexia and anemia. The cause of cancer cachexia is not known. Probably results from actions of cytokines produced either by the tumor or by the host in response to tumor.
  
  
• Paraneoplastic Syndromes: Refers to symptom complexes in cancer patients that cannot be readily explained. Approximately 10% of patients with advanced malignancies have paraneoplastic syndromes. (Table 7-9 p.296 Robbins, 5th Ed.) It is worthwhile to recognize these syndromes as they may represent the earliest manifestation of malignancy in your patient. Some common paraneoplastic syndromes include:
  
  
  • Cushings syndrome (excessive ACTH or ACTH-like peptides) Lung cancer, Pancreatic cancer
  • Hypercalcemia (osteolysis or calcemic humoral substances) Lung cancer
  • Neuromyopathic syndromes such as myasthenia gravis (immunologic?) Lung cancer
  • Acanthosis nigricans (? immunologic) Gastric, Lung, Uterine cancer
  • Clubbing of fingers and hypertrophic osteoarthropathy (unknown) Lung cancer
  • Trousseau's syndrome (tumor products activate clotting) Pancreas, Lung cancer

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Cytologic Features of Malignancy

	Benign	Malignant
Cell arrangement in groups	Uniform; regular spacing Cohesivel rounded groups	-Disturbed polarity of cells (nuclei are oriented in different directions and are irregularly spaced)- Molding of nuclei- Loss of cohesiveness (cells falling apart from another
Cell uniformity in groups	Benign tissue: uniform Reactive states: nuclear size may vary but not as in malignancy	-Pleomorphic- Variation in size, shape, and numbers of nucleii
Nuclear: Cytoplasmic ratio (N:C ratio)	May be increased in reactive conditions	Usually increased
Nuclear Size	Size increased and variable in reactive conditions	Increased Usually more variable than in reactive conditions
Cell molding	Not common	Common (cells fit into each other; concavity into convexity, like a jigsaw puzzle. Can involve the nuclei as well as the cytoplasm
Chromatin
Hyperchromasia	Bland, not hyperchromatic	HyperchromaticL may be due to increased chromosome number
Distribution	Even	Irregular clumping and irregular areas of clearing
Nucleoli
Size	Small or not visible normally; prominant in reactive atypia	Often prominent
Shape	Round	Round or irregular
Numbers	Reactive cells may have several, but numbers do not vry widely among neighb	Sometimes numerous and variable numbers
Nuclear Membranes
Contour	Smooth	Irregular and angular bites
Thickness	Regular	Thickness varies ( a manifestation of irregular chromatin clumping on or near the nuclear membrane)
Mitoses
Number	Seen in normal cells; greater numbers in reactive cells	Mitotic rate may be low or high
Morphology	Normal	Abnormal mitoses may be seen (e.g., quadripolar or dispersed)

Nuclear features are more important in cytology than architecture in providing the diagnostic features of malignancy. Cytoplasmic and architectural features provide information on differentiation. In histology, architecture can provide more information on the questions of malignancy and differentiation.

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III. HISTOLOGIC (ARCHITECTURAL) AND GROSS FEATURES OF MALIGNANCY

• Cytology versus Histology in the Diagnosis of Malignancy
  
  
  • The cytologic features of malignancy can be applied to histologic sections. Although fine nuclear detail may be lost by formalin fixation and processing necessary for histologic sections, nuclear features of malignancy should still be discernable. Cohesiveness of cells is more easily evaluated on cytologic material.
    
    
  • Histologic sections provide added information:
    • Architecture
    • Relationship of cancer cells to normal structures (depth of invasion, etc.)
      
      
• The Architectural Features of Malignancy
  
  
  • Invasion of the underlying or surrounding tissue: In carcinomas, (epithelial origin) invasion is defined by extension of tumor beyond the basement membrane. In mesenchymal tumors (sarcomas) invasion is defined by an irregular front penetrating the surrounding tissue.
    
    
  • Stromal changes: The change that occurs in the stroma as tumor invades is called desmoplasia. Desmoplasia refers to the stroma composed of connective tissue and blood vessels that surrounds the infiltrating tumor. The spindle shaped cells that make up the desmoplasia are not themselves neoplastic. Desmoplasia is a response to invasion of tissue by malignant tumor cells.
    
    
  • Loss of normal structure: As tumors become less and less differentiated, they resemble the tissue of origin less and less. For example, well differentiated prostatic adenocarcinoma shows small round glandular structures, while poorly differentiated tumors show complicated glandular patterns and sheets of tumor cells.
    
    
  • New structures: Some tumors will create structures such as complicated "cribriform" gland structures (colon, endometrium cancers) or papillary structures (thyroid, bladder cancers) Sarcomas may form "herringbone" arrangements.
    
    
  • Necrosis: Although present in inflammatory and infectious processes, necrosis may also be present in malignancies and often indicates that the tumor has outgrown its blood supply.
    
    
  • Angiogenesis: Because tumors cannot grow without a blood supply, angiogenesis factors are involved in tumor vessel growth.
    
    
  • Inflammation: Tumors often incite inflammation.
    
    
• The Gross Features of Malignancy
  
  
  • Irregular borders: Malignant tumors often have irregular borders due to the progressive infiltration, invasion and destruction of surrounding tissue. In general, they are poorly demarcated from the surrounding tissue and a well-defined cleavage plane is lacking.
    
    
  • Necrosis: Seen on histologic sections, necrosis can also be identified on gross examination of tissues. It is not specific for malignancy, but can be associated with a malignant tumor that has outgrown it's blood supply.
    
    
  • Metastasis: Metastases are tumor implants discontinuous with the primary tumor. With few exceptions (some types of brain and skin tumors), all malignancies can metastasize. With extremely few exceptions, metastasis marks a tumor as malignant because benign tumors do not metastasize.
    
    
• Metastasis and Pathways of Spread
  
  
  • Seeding of body cavities and surfaces: This occurs when a malignant neoplasm penetrates into a natural "open field" such as peritoneal cavity, pleural space, pericardial cavity, etc. Most common examples, ovarian carcinoma and mucin secreting ovarian and appendiceal carcinomas (pseudomyxoma peritonea).
    
    
  • Lymphatic spread: This is the most common pathway for dissemination of carcinomas (although sarcomas can also use this route). The pattern of lymph node involvement follows the natural routes of drainage.
    
    
  • Hematogenous spread: This pathway is typical of sarcomas. Arteries are more difficult for tumor to penetrate than veins. With venous invasion, the blood-borne cells follow the venous flow draining the site of the tumor. Liver and lungs are frequently involved.

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IV. GRADING AND STAGING OF TUMORS

• Grade: The grade of a tumor is based on the degree of differentiation of the tumor cells, the degree of cytologic atypia and the number of mitoses within the tumor.
  
  
  • In general, low grade tumors are well differentiated, have minimal cytologic atypia and low mitotic rates. High grade tumors are poorly differentiated, have marked cytologic atypia and high mitotic rates.
    
    
• Stage: The stage of a tumor is based on the size of the primary tumor, the extent of invasion into surrounding tissue, the spread to regional lymph nodes and the presence or absence of blood-borne metastases. The staging system used here is the American Joint Committee (AJC) on Cancer Staging. The staging scheme varies by tumor site, but all tumors are assigned a "T" stage referring to tumor size, degree of penetration of surrounding tissue; a "N" indicative of presence of lymph node involvement and a "M" which indicates the existence of metastases.

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V. THE LABORATORY DIAGNOSIS OF MALIGNANCY

• Diagnostic Electron Microscopy
  
  
  • Although cytologic and histologic features of malignancy can be appreciated at the ultrastructural level, the main use of diagnostic electron microscopy is involved not with the question of whether a tumor is malignant or not, but with the issue of tumor classification. Cytoplasmic organelles such as melanosomes are indicative of melanocytic lesions such as malignant melanoma, the presence of desmosomes points to an epithelial tumor (carcinoma), and structures such as myosin and actin filaments arranged in "Z" bands are indicative of skeletal muscle differentiation and hence if found in a tumor would suggest a rhabdomyosarcoma. Neurosecretory dense core granules are found in tumors with neuroendocrine differentiation.
    
    
• Immunocytochemistry
  
  
  • Antibodies directed against antigens found in specific tumors types can be used to help diagnose poorly differentiated tumors. Tumors which look similar on hematoxylin-eosin stained sections can be differentiated from each other using these "special stains". Examples include using anti-cytokeratin antibodies to distinguish poorly differentiated carcinoma (keratin positive) from a high grade lymphoma (keratin negative). Monoclonal antibody directed against HMB45 is very helpful in the diagnosis of malignant melanoma.
    
    
  • Immunocytochemistry is also used commonly in the diagnosis of lymphomas. Specific antibodies directed against B and T cell markers as well as against kappa and lambda antigens are available.
    
    
  • Another use of immunocytochemical stains is for the detection of molecular changes. Mutations of the p53 gene leads to accumulation of the mutant protein which can be detected using immunocytochemical stains.
    
    
• Serum Tumor Markers
  
  
  • Tumor markers include cell surface antigens, cytoplasmic proteins, enzymes and hormones which indicate the presence of tumor. Many tumor markers are also used as immunohistochemical stains. A tumor marker is measured in serum or other body fluid in the clinical laboratory, often utilizing immunoassays. The following are several clinically important tumor markers.
    
    
  • Prostate specific antigen (PSA): A serine protease produced by normal as well as neoplastic prostate epithelium. In normal men, only small amounts circulate in the serum. Elevated blood levels are associated with prostatic cancer. Benign conditions such as prostatic hyperplasia can also raise the serum PSA level, but to a lesser degree.
  • Carcinoembryonic antigen (CEA): A complex glycoprotein that is normally produced in embryonic tissue of the gut, pancreas, and liver. It is elevated in some colorectal and pancreatic neoplasms. Increased serum levels can also sometimes be present in patients with gastric and breast cancers. Non neoplastic causes of increased serum CEA include smoking, alcoholic cirrhosis and ulcerative colitis.
    
    
  • Ca-125: A glycoprotein expressed by coelomic epithelium during fetal development. Increased serum levels can be present in patients with ovarian cancer. Other malignancies such as pancreatic carcinoma may also have increased levels. Non malignant causes of high serum Ca125 include pregnancy, endometriosis and liver failure.

Tumor markers may:
Suggest/support a diagnosis of malignancy (ie Prostate Specific Antigen PSA)
Predict relapse (ie Carcinoembryonic antigen CEA)
Determine the response to therapy (ie CA-125)

• Flow Cytometry
  
  
  • Flow cytometry is a technique used to measure individual cell characteristics such as membrane antigens and DNA content of tumor cells. The classification of leukemias and lymphomas is based on cell surface antigens which can be easily identified by flow cytometry. DNA ploidy appears to correlate with prognosis in a variety of tumors. In general, aneuploidy seems to be associated with a poorer prognosis in early-stage breast cancer, bladder, lung, colorectal and prostate cancer.
    
    
• Molecular Diagnosis of Malignancy
  
  
  • This topic will be covered next week. Briefly, molecular techniques such as polymerase chain reaction, the Southern blot analysis of DNA and the Northern blot analysis of RNA are used in tumor diagnosis. Southern blots are used in identifying clonal rearrangements of receptor genes of B and T cell lymphomas. Northern blots can identify amplification the N-myc oncogene in neuroblastomas by analysis of tumor RNA.

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Neoplasia II: Molecular Pathobiology of Neoplasia
Neoplasia III: Molecular Aspects of Neoplasia in Hematology

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