Articles on Medical Diseases and Conditions

Chest x-ray films. Chest x-ray films have been the usual means of detecting lung cancer. Unfortunately, best results are obtained from the less common peripheral lesions rather than the more usual bronchogenic carcinomas arising in major bronchi. In general, chest x-ray films are not an efficient means of early diagnosis, and this is especially true for the miniature films used in mass survey work.

Sputum cytology. Sputum cytology is generally considered more sensitive than x-ray films, although some studies detected about equal numbers of asymptomatic tumor with either technique. Sputum cytology yield increases if the patient is a smoker and has symptoms such as chronic cough, hemoptysis, or recurrent pneumonia. Sputum samples for cytology should be obtained once daily (before breakfast and after rinsing the mouth with water) for at least 3 days. The material should be from a “deep cough”; saliva is not adequate. Many cytopathologists recommend expectoration directly into a bottle containing a special fixative (e.g., 50% ethanol, with or without additives). This type of specimen cannot be used for bacterial culture. In patients who do not have a productive (sputum-producing) cough, aerosol induction of sputum has been recommended. Some investigators achieved better results with a 3-day collection period than with aerosol inducement when several deep-cough specimens per day were expectorated directly into sputum cytology fixative. Twenty-four-hour collections without fixative are not recommended due to cell disintegration. A good specimen is the key to success in pulmonary cytology, because tumor cells may not be present continuously, because upper respiratory tract material usually does not reflect lower respiratory tract disease, and because pulmonary cytologic interpretation is more difficult than with uterine material.

Sensitivity of various diagnostic methods for lung cancer is not always easy to determine from the literature. The detection rate of carcinoma in asymptomatic persons (occult carcinoma) is naturally lower than in patients who have symptoms. For sputum cytology, better results are found if detection rates are used (“definitely positive” plus “suspicious” diagnoses) rather than only positive diagnoses. Unfortunately, it is often not clear which reporting method is being used. There is no question that more than one sputum sample, each sample being obtained on different days, significantly increases diagnostic yield (Table 33-12). Aerosol inducement of sputum also increases diagnostic yield (by about 20%–30%). There is a difference in detectability of central lesions (higher sputum cytology sensitivity) versus peripheral lung lesions (lower sputum sensitivity). For that reason, squamous cell carcinoma is more readily detectable by sputum cytology (literature range, 58%–85%), due to its tendency for proximal bronchus origin, than is adenocarcinoma (10%–57%), which tends to be peripherally located. Small cell undifferentiated carcinoma has intermediate detectability (30%–70%).

Table 33-12 Sensitivity of sputum cytology in primary lung carcinoma

Bronchoscopy. Bronchoscopy is reported to detect about 70%–80% of cases (45%–90%), with better detection of central versus peripheral lesions and with better results from direct biopsy of visible lesions versus bronchial washings, brushings, or blind biopsy. Percutaneous needle biopsy of lung tumors visible on x-ray film is reported to verify about 65%–70% of cases (48%–90%). Scalene node biopsy detects about 10% of cases (5%–21%). Mediastinoscopy with mediastinal node biopsy is reported to provide the only preoperative tissue evidence of carcinoma in 7%–20% of cases.

When cytologic material is obtained by bronchoscopy, saline is often used for bronchial washings. It is essential to use some type of “physiologic” saline rather than “normal” saline; contrary to common belief, the two are not identical. Normal saline (0.85% sodium chloride [NaCl]) can produce cellular artifact if the slides are not prepared within 5 minutes after collection. Physiologic saline is a balanced salt preparation with other minerals besides NaCl.

Radionuclide scans. When the diagnosis of lung cancer is first made, the question frequently arises as to which tests might help delineate extent of disease and thus establish operability. Bone scan is reported to detect lesions in approximately 35%–45% of patients, the frequency correlating roughly with clinical stage of the disease. However, a smaller number of those who are asymptomatic have an abnormal bone scan (14%–36%) and some of these abnormalities may not be due to metastasis. Brain scans have produced as many as 14%–20% positive results, but most studies found less than 6% were positive if there were no neurologic signs or symptoms. Initial workup liver scans disclose 13%–19% of abnormal results but less than 6% when there is no laboratory or physical examination evidence of liver metastases.

Computerized tomography. CT has been very useful to determine operability by visualizing the size and location of the lesion, the presence of thoracic metastases, and the size of the mediastinal lymph nodes.

Cancer antigen 125. The cancer antigen 125 (CA 125) test uses an antibody against antigen from tissue culture of an ovarian tumor cell line. Various published evaluations report sensitivity of about 75%–80% in patients with ovarian carcinoma. There is also an appreciable incidence of elevated values in nonovarian malignancies and in certain benign conditions (see the box on this page). Test values may transiently increase during chemotherapy.
CA 125 has been advocated to monitor patients for recurrence of ovarian carcinoma after initial surgery, similar to the use of CEA after surgery for colon carcinoma. With both CA 125 and CEA there are sufficient normal results (at least 20%) in patients with cancer and sufficient abnormal results in other tumors and in benign conditions to preclude use of the test to screen for tumor under most circumstances. Studies in patients after therapy showed that up to 90% of patients with persistent CA 125 level elevation after surgery had residual tumor, and that nearly all patients with rising titers had recurrent disease; but 50%–61% of patients with normal levels also had recurrent or persistent tumor. Therefore, only a change from normal to abnormal or a rising titer is significant. In one study an increase in titer preceded clinical evidence of metastasis or recurrence by an average of 3 months (range, 1-11 months). CA 125 has also been useful to detect ovarian cancer cells in effusions. However, a study based on decision analysis methodology concluded that CA 125 (and ultrasound) were not cost effective as early detection screening tests for ovarian cancer.

Elevated CA 125 Levels in Various Conditions
Malignant
Epithelial ovarian carcinoma, 75%–80% (range, 25%–92%, better in serous than mucinous cystadenocarcinoma)
Endometrial carcinoma, 25%–48% (2%–90%)
Pancreatic carcinoma, %
Colorectal carcinoma, 20% (15%–56%)
Endocervical adenocarcinoma, %
Squamous cervical or vaginal carcinoma, 7%–14%
Lung carcinoma, 32%
Breast carcinoma, 12%–40%
Lymphoma, 35%
Benign
Cirrhosis, 40%–80%
Acute pancreatitis, 38%
Acute peritonitis, 75%
Endometriosis, 88%
Acute pelvic inflammation disease, 33%
Pregnancy 1st trimester, 2%–24%
During menstruation (occasionally)
Renal failure (? frequency)
Normal persons, 0.6%–1.4%

Cervix. The mainstay of screening for uterine carcinoma is the Papanicolaou (Pap) smear. For Pap examination of the cervix, material is best obtained directly from the cervix by some type of scraping technique. Vaginal irrigation or vaginal pool smears are reported to be only 50%–75% as accurate as cervical scrape for detection of cervical carcinoma. A single scrape smear has a false negative rate of about 30% (literature range, 5%–50%). Some reports indicate that the false negative rate can be reduced by one half or more by obtaining two successive cervical scrape specimens at the same examination or by obtaining both a cervical scrape specimen and an endocervical aspiration or endocervical swab specimen (making another smear with the endocervical specimen, either on the same slide as the scrape material or on another slide). Care should be taken not to contaminate the cervix area with water or lubricating jelly, both of which distort the tissue cells.

There have been several significant changes since 1980 in diagnosis of uterine cervix abnormalities. First, there has been reclassification of how degrees of cervix histologic abnormality are reported. This was previously graded in terms of degrees of dysplasia (or atypia, often used as a synonym for dysplasia, although some use atypia to describe nuclear abnormality and dysplasia to describe both nuclear and architectural abnormality). Grading of histologic abnormality included degree of dysplasia from I to III, based on abnormal changes in the lower third of the squamous cervical epithelium (dysplasia degree I), the lower third plus involvement of the middle third (dysplasia degree II), and both the lower and middle thirds plus incomplete involvement of the upper third (dysplasia degree III). Involvement of the entire thickness of the epithelium was named carcinoma in situ (CIS). The newer system was based on the concept of cervical intraepithelial neoplasia (CIN) from a philosophy that any cytologic abnormality of the cervix was potentially cancer and should not be ignored, even though the response of the physician in follow-up or treatment modality would vary. This in turn was based on studies using culposcopy that biopsied lesions from which Pap smears interpreted as Class II “nonspecific inflammation” or minimal atypia were obtained. These biopsies disclosed a substantial number of patients (12%–25%) who had what is now classified as varying categories of CIN, even including CIN III. In the past, if a patient had a class II Pap smear, the physician would follow the patient with periodic cytology specimens. However, an increasing number of investigators point toward substantial false negative rates on repeat cytology (similar to false negative initial cytology) and recommend culposcopy or cervicography after the first abnormal Pap result regardless of the degree of abnormality. In the CIN system, the previous dysplasia degree III and CIS were combined into the single category of CIN III. This eliminated the “gray zone” between total replacement of the epithelium by abnormal nuclei and replacement of nearly full thickness except for only one or two layers or rows of cells at the surface.

Another significant change was a new system of reporting cytologic changes. The long-established Pap report was based entirely on 5 categories (see the box on this page) that were primarily morphologic (with some interpretive implications). Additional interpretative comments or information that could affect interpretation were optional.

The new system was developed at a 1988 conference in Bethesda and revised in 1991. The 1991 revision of the Bethesda reporting system is shown in the box. This system mandates reporting of various technical factors or other findings that could affect interpretation (predominantly those that could produce false negative results, the presence or absence of various types of infection, and specific identification of various types of “reactive” changes. It also reduced interpretation of definite cytologic abnormality to two categories: low grade (formerly Pap Class III, mild dysplasia, CIN I) and high grade (formerly Pap Class IV and V, moderate or severe dysplasia, or CIN II and III). This implies more importance to a somewhat lesser degree of cytologic abnormality. Unfortunately, there still exist borderline or gray areas in the classification that depend on subjective decisions by the cytologist. It cannot prevent false negative results because material was not obtained from the lesion (sampling error) or because abnormal cells were missed when the slide was examined (interpretive or laboratory error).

There have also been some advances in detection of cervical abnormality. Methods have been devised to make monolayer specimen cell preparations for Pap examination. These preparations permit better staining and prevent overlooking abnormal cells in overlapping cell clumps or masses or in blood. Most of the reported methods improved overall Pap sensitivity to greater or lesser degree. Cervix specimen collection using various brushes rather than swabs or wooden spatula-type instruments have generally been reported to increase overall Pap sensitivity. Culposcopy (direct visual examination of the cervix through a viewing device called a culposcope) and cerviography (photography of the entire visually available cervix with an instrument called a cerviscope) have both been reported to increase detection of cervical lesions compared to “blind” cytology. Some investigators report better results than others for the monolayer cytologic methods, sampling devices, and viewing techniques; a few investigators found relatively little overall differences between the old and new methods.

Standard Papanicolaou Classification
Class I
Absence of atypical or abnormal cells
Class II
Atypical cytologic changes but no evidence of malignancy
Class III
Cytologic changes suggestive of, but not conclusive for, malignancy
Class IV
Cytologic changes strongly suggestive of malignancy
Class V
Cytologic changes conclusive for malignancy

Revised Bethesda System (1991)
Adequacy of the specimen
Satisfactory for evaluation
Satisfactory for evaluation but limited by . . .(specify reason)
Unsatisfactory for evaluation . . . (specify reason)
General Categorization (optional)
Within normal limits
Benign cellular changes: See descriptive diagnosis
Epithelial cell abnormality: See descriptive diagnosis
Descriptive Diagnoses
Benign cellular changes
Infection
Trichomonas vaginalis
Fungal organisms morphologically consistent with Candida spp.
Predominance of coccobacilli consistent with shift in vaginal flora
Bacteria morphologically consistent with Actinomyces spp.
Cellular changes associated with herpes simplex virus
Other
Reactive changes
Reactive cellular changes associated with:
Inflammation (includes typical repair)
Atrophy with inflammation (“atrophic vaginitis”)
Radiation
Intrauterine contraceptive device
Other
Epithelial Cell Abnormalities
Squamous cell
Atypical squamous cells of undetermined significance: Qualify*
Low-grade squamous intraepithelial lesion encompassing: HPV† mild dysplasia/CIN I
High-grade squamous intraepithelial lesion encompassing: moderate and severe dysplasia, CIS/CIN 2 and CIN 3
Squamous cell carcinoma
Glandular cell
Endrometrial cells, cytologically benign, in a postmenopausal woman
Atypical glandular cells of undetermined significance: Qualify*
Endocervical adenocarcinoma
Endometrial adenocarcinoma
Extrauterine adenocarcinoma
Adenocarcinoma, NOS
Other Malignant Neoplasms: Specify
Hormonal Evaluation (applies to vaginal smears only)
Hormonal pattern compatible with age and history
Hormonal pattern incompatible with age and history: Specify
Hormonal evaluation not possible due to: Specify
_______________________________________________________
*Atypical squamous or glandular cells of undetermined significance should be further qualified as to whether a reactive or a premalignant/malignant process is favored.
†Cellular changes of human papillomavirus (HPV)—previously termed koilocytosis, koilocytotic atypia, or condylomatous atypia—are included in the category of low-grade squamous intraepithelial lesion.

Considerable evidence has accumulated linking cervical infection by human papillomavirus (HPV) types 16 and 18 with cervical epithelial cell atypia, premalignant changes, and progression to carcinoma (Chapter 17). HPV changes can be seen in squamous epithelial cells on biopsy and on Pap smears, but the virus type cannot be specified. Nucleic acid probe is more sensitive than either visual microscopy or cytology and in addition is specific for the viral type for which the probe is constructed.

Endometrium. Screening for endometrial carcinoma can be done using endometrial suction biopsy, endometrial aspiration for cytology, endometrial washing methods, endometrial brushing, and cytology specimens taken from the endocervix or vaginal pool. Endometrial sampling methods are somewhat difficult to evaluate due to a considerable number of methods that can vary in detection rate but that are frequently lumped together in the literature. Endometrial biopsy using the Vabra aspirator generally is reported to detect about 96% (literature range, 80%–100%) of endometrial carcinomas. Endometrial biopsy with the Novak suction curette has been reported to detect about 91% of cases (range, 77%–94%). Endometrial cavity aspiration for cytology with a device known as the Isaacs cell sampler detects about 92%–94% of cases (range, 78%–100%). Endometrial lavage methods (e.g., Gravlee cell wash) detect about 80%–85% of cases (range, 66%–100%). Endometrial mechanical cell dislodgement methods, such as the MiMark and Endopap devices, are reported to detect more than 90% of carcinomas. Endometrial brush methods (another way to dislodge cells mechanically) are reported to detect 57%–92% of cases. In methods depending predominantly on cytology, hyperplasias and abnormalities other than tumor are not as easily identified as they can be with tissue specimens provided by biopsy. Endocervical aspiration detects endometrial carcinoma in about 70% of cases (range, 14%–90%). Smears from the vaginal pool in the posterior fornix of the vagina detect 30%–50% of cases (range, 18%–90%). Specimens from the cervix detect only about 35% (range, 25%–55%) of advanced endometrial carcinoma.

Follow-up of abnormal Papanicolaou smear. Abnormal or definitely positive Pap smears should be followed up with a biopsy of the site indicated to confirm the diagnosis and determine the extent and histologic characteristics of the neoplasm. For the cervix, colposcopic examination with biopsy is becoming the most widely used technique. For the endometrium, dilatation and curettage should be done.

Mammography. Until 1960 diagnosis of mammary carcinoma depended on discovery of a breast mass by physical examination, followed by a biopsy of the lesion. It has been said that, with experience, carcinoma as small as 1 cm may be regularly detected by palpation. After 1960, x-ray study of the breast (mammography) began to receive considerable attention. Several favorable reports have been published, and several mass screening surveys have been attempted. To date, available information on the status of mammography includes the following:

1. Breast carcinoma can be visualized on mammography in some cases (23%–42%) in which it is not palpable. Reports indicate that 9%–42% of visualized nonpalpable lesions are malignant, with an incidence of lymph node metastasis of 0%–38%.
2. Screening surveys utilizing mammography are reporting detection of 2-3 times the expected rate of breast carcinoma.
3. Proper technique is of the utmost importance; this calls for special training and conscientious technicians.
4. Mammography is definitely not infallible. The average good radiologist will probably miss a malignant diagnosis in about 15% of cases (range, 4%–44%) and call a benign lesion malignant in about 5%–10% (range, 4%–15%). Reported incidence of palpable lesions not visualized on mammography ranges from 5%–20%. Biopsy or some other type of tissue diagnosis is still essential for all breast lesions.
5. Mammography is best in the postmenopausal or large breast where fatty tissue predominates. In these circumstances, probably 80%–90% of malignant tumors can be diagnosed correctly, whereas the figure decreases to 55% for women under age 45 years.
6. Mammography is useful for indicating the site for biopsy when several breast masses are present, for demonstrating unexpected additional foci of tumor elsewhere in the breast, and for detecting unsuspected tumor in the opposite (contralateral) breast. Mammography has demonstrated simultaneous tumor in the contralateral breast in 2%–3% of patients and eventual development of contralateral breast carcinoma in 6%–8% of patients. However, not all such tumors are detected by mammography. Pathology studies on mastectomy specimens have disclosed multiple foci of carcinoma in the same breast in 20%–30% of cases (literature range, 13%–75%), and biopsies of the contralateral breast have detected invasive carcinoma in about 1%–2% (0.5%–16%) and lobular carcinoma in situ in about 20%–30% (10%–53%).
7. At present, mammography is not an ideal screening procedure, because only a limited number of satisfactory studies can be performed daily under present conditions in the average radiology office.

Radionuclide bone scan. When the diagnosis of breast cancer is first made or suspected, the question may arise as to which tests provide useful information that might influence type or extent of treatment. Bone scan detects lesions on initial workup in about 5% of clinical stage I lesions (literature range, 0%–30%), about 10% of clinical stage II lesions (0%–43%), about 8% of combined stage I and II lesions (1%–40%), about 25% of clinical stage III lesions (0%–62%), and about 15% of all lesions (4%–48%). Also, about one half of patients with solitary bone scan abnormalities have no demonstrable tumor at the abnormal site, the scan changes being due to benign abnormalities of bone. The incidence of overall false positive scan findings in patients with breast carcinoma is about 10% (3%–57%). About 10% (4%–33%) of breast carcinoma metastases to bone seen on x-ray film will be missed by bone scan, because breast carcinoma produces a relatively high number of osteolytic lesions, which are not as frequently seen by bone scan as are osteoblastic lesions. Bone scan detects about 20% (10%–40%) of metastases not seen on x-ray film. The small amount of information available on results of initial-visit brain scanning suggests that fewer than 5% will be abnormal if there are no neurologic signs or symptoms. Surprisingly few data are available on the contribution of liver scan to initial (pretherapy) workup, but one study found that liver scan yielded only 1% true positive results.

Fine-needle aspiration. Another diagnostic modality is fine-needle (22-gauge) aspiration of breast masses with cytologic examination of the aspirate. Studies have shown about 1% (range, 0%–3%) false positive results and about 5%–10% (range, 0%–24%) false negative results. The procedure gives excellent results when the person doing the aspiration and the cytologist are experienced. Reliable interpretation requires special training. A definite cytologic diagnosis of malignancy is much more helpful than a diagnosis of benignity due to the significant rate of false negative results. Whether this procedure should replace surgical biopsy with frozen section is controversial. In cases where the patient refuses biopsy, when surgery cannot be performed, or when the lesion is cystic, there is not the same controversy.

Prognostic tests in breast carcinoma

The earliest prognostic factors were established from gross and microscopic examination. Presence or absence of palpable axillary nodes (and to a lesser extent, the number of nodes containing metastases), tumor size (cutoff point, 2.0 cm diameter), and tumor nuclear grade (size, shape, and degree of chromatin abnormality determining low or high grade) were (and still are) important independent prognostic indicators. Later, various laboratory tests were tried in hopes of further improving accuracy either alone or in combination.

Estrogen receptor assay. Estrogen receptor assay (ERA) is widely used as an aid in selection of breast cancer therapy. It has been shown that approximately 30%–40% of postmenopausal breast cancer patients respond to oophorectomy, adrenalectomy, hypophysectomy, or estrogen therapy. Premenopausal patients respond less frequently. Certain tissues, such as endometrium, vagina, and breast, have been shown to respond to estrogen stimulation of estrogen receptor sites within their epithelial cells. ERA techniques most frequently used involve tissue slices or cytoplasm (cytosol) extracts from the tumor to be evaluated. Radioactive estradiol and another estrogen-binding substance are added, and the tumor estrogen receptors compete with the estrogen binder for the labeled estradiol. The amount of labeled estradiol bound by the tumor estrogen receptors can then be determined. This type of assay is an estimate of the number of unoccupied (“active”) estrogen-binding sites. Immunoassays of several types are also becoming available. This technique estimates total quantity of estrogen receptors, including both active and inactive receptors. According to current information, about 60%–65% (literature range, 30%–80%) of primary breast carcinomas and about 45%–50% of breast carcinoma metastases have tumor cells with estrogen receptors that bind sufficient estrogen per unit of cancer tissue to be considered estrogen receptive, or “positive.” ERA positivity does not show satisfactory correlation with presence or absence of lymph node involvement or the degree of differentiation of the tumor. Breast carcinomas in postmenopausal women are more likely to have estrogen receptors than those in premenopausal women. About 60%–70% of women whose tumors are estrogen-receptor positive respond to hormonal manipulation (estrogens, antiestrogens, endocrine ablation, or androgens). About 5%–10% of those considered ERA negative will respond (some laboratories report < 5%). Thus a negative ERA result is interpreted by many as an indication that chemotherapy is more likely to be effective than endocrine therapy. In general, metastases tend to share the same type of receptor status as the primary tumor. However, some investigators report that metastases from estrogen-treated primary tumors are frequently receptor negative.

Certain laboratory aspects of the test are important. There are several techniques for preparing tissue for the cytosol method for receptor assay, and some of the techniques differ significantly in the number of patient tumors that demonstrate apparently increased receptors. At present, it is necessary to secure at least 1 gm of tumor after eliminating all fat and normal breast tissue. The tumor must be fresh and must be frozen by dry ice within a short time (preferably within 15 minutes) after excision. The specimen must be kept on dry ice and sent to the reference laboratory on dry ice. The estrogen receptors are very labile, and failure to quick-freeze and provide adequate low temperatures produces false negative results. In addition, quality control surveys have shown considerable variation among laboratories in ability to detect low concentrations of estrogen receptors.

As the description of this test indicates, with current techniques the procedure is partially a bioassay and therefore is available only at larger institutions or reference laboratories.

Immunocytochemical estrogen receptor methods. Several investigators have developed immunologic methods to visually demonstrate presence or absence of estrogen receptors in cells within fixed tissue microscopic sections or cytology smears. Although true receptor quantitation cannot be done, this technique permits visualization of receptor distribution, that is, how many tumor cells are visually positive or negative. Many carcinomas do not have a cell population that is uniformly estrogen receptor positive or negative. Studies to date have reported about 80%–85% (range, 60%–90%) correlation with standard ERA results.

Progesterone receptor assay. Progesterone receptors can be assayed (PRA) on the same tumor tissue in a manner similar to estrogen receptors. In general, demonstration that a breast carcinoma is PRA positive adds about 10%–15% more likelihood to ERA positivity that the tumor will respond to hormonal manipulation. Thus, tumors that are both estrogen- and progesterone-receptor positive have about a 70%–80% chance of responding to hormonal therapy. Those negative for both receptors have less than a 10% chance of responding to hormones or antihormones. Eighteen percent to 49% of patients are ERA positive but PRA negative and have about a 30% chance of responding. Three percent to 13% of patients are ERA negative and PRA positive. PRA can be performed on formalin-fixed paraffin-embedded microscopic slides using immunohistochemical stains similar to those used for ERA. As in ERA, this technique does not provide a quantitative answer.

DNA ploidy. DNA (deoxyribonucleic acid) ploidy is a measure of total nuclear DNA content, usually performed by flow cytometry. About 60% of breast cancer overall are aneuploid and 40% are diploid. The amount of nuclear DNA in the active cell stage of DNA synthesis (S-phase) can be calculated and is reported as the S-phase fraction. This is a parameter of cell proliferative activity. There is now some controversy whether DNA ploidy (aneuploid vs. diploid status) provides significant prognostic information. Whereas many of the earlier studies reported that diploid breast carcinomas had significantly or considerably better prognosis than aneuploid ones, some more recent studies do not confirm this or do not find that ploidy is a significant independent risk factor. Increased S-phase activity is somewhat better accepted as an unfavorable prognostic sign. Unfortunately, SPF is technically more difficult to measure accurately and is less standardized between laboratories.

Cathepsin D. Cathepsin D is a protease enzyme found in lysosomes of cells in many tissues. In breast cancer, it appears to be regulated by estrogen. Studies using cytosol (tissue extracts) from breast cancer found that increased quantity (tumor “overexpression”) of Cathepsin D predicted shorter disease-free survival and worse overall prognosis. However, more recent studies using monoclonal antibody immunohistologic stains on routine formalin-fixed and processed tissue microscopic slides found that presence of Cathepsin D staining of tumor cells, especially with strong staining, predicted longer disease-free survival and better overall prognosis (the opposite from cytosol-based studies).

C-erbB2 oncogene amplification. C-erbB2 (HER-2/neu or HER-2) protooncogene is a gene involved with cell growth. When the gene increases (or causes to increase) production or associated C-erbB-2 protein, the gene is said to be amplified (or overproducing). This amplification has been reported in about 10%–30% of most human adenocarcinomas. The reported frequencies from individual site adenocarcinomas can be influenced by several factors: whether the gene itself is identified (usually by nucleic acid probe techniques) or the gene protein is detected (usually by immunohistochemistry), whether frozen sections (fresh tissue) or formalin-fixed paraffin-embedded sections from routine tissue slide examination are used, whether the antibody recognizes the external or internal part of the molecule, and what criteria are used to define a positive result. In breast cancer, formalin-fixed paraffin-embedded slides generally show fewer positive cells (20%–30%; range, 9%–80%) than fresh-frozen tissue (22%–49%). C-erbB2 amplification, in general, correlates with negative estrogen receptor status, higher tumor grade, and higher probability of aneuploidy, therefore suggesting poorer prognosis. However, the majority of studies found status of axillary lymph nodes to be a more powerful independent risk factor than c-erbB2 amplification.

Cell proliferation markers. In general, the more rapidly growing a tumor is, the more aggressive it is, and the prognosis becomes worse. Therefore, various indices of cell proliferation have been proposed to help estimate prognosis. One of the first was the nuclear mitotic count (or index), based on the number of mitoses per (microscope) high-power field (generally at 400x magnification). In breast cancer, this was found to have some correlation with tumor differentiation and therefore with prognosis, but prognostic usefulness was not as good as could be obtained using other tests. The SPF from cell cycle DNA flow cytometry has been discussed. This has proved (under the right conditions) to be a useful and helpful prognostic indicator. Ki-67 is a monoclonal antibody that detects a protein in cell nuclei that appears only in the growth phase of the cell cycle. In certain tumors, including breast carcinoma, abnormal quantity of Ki-67 (Ki index, by immunostaining of microscopic slides from tumor areas) correlates with less differentiated tumors, larger tumor size, increased p53, and less favorable prognosis, and to a lesser degree with negative estrogen and progesterone receptor status. There is disagreement concerning correlation with axillary node metastases.

The p53 assay. The p53 gene is a tumor suppressor gene similar to the retinoblastoma suppressor gene. Mutation of the p53 gene results in production of an altered protein that cannot function normally and may actually promote cell growth. Normally, p53 cannot be detected in breast tissue using immunohistologic stains. In breast carcinoma, about 25% of patients have detectable p53 nuclear protein. This correlates with increased cell proliferative activity and to some extent with lack of estrogen receptors.

Summary of breast carcinoma prognostic tests

At present, axillary lymph node status, tumor size, and estrogen and progesterone receptor assay are still by far the most widely used and accepted prognostic indicators. Nuclear grade could be more widely used if uniform criteria for interpretation were established. S-phase fraction would probably become more important if uniform methodology and interpretation were agreed upon. Of the other current contenders, Cathepsin-D and c-erbB2 oncogene “expression” seem to be the most likely possibilities for at least limited use. However, the prognostic test area can change rapidly.

Predisposition to colon cancer. Certain conditions either predispose to colon cancer or are frequently associated with it. These include age over 40 years; family history of cancer; the syndromes of multiple polyposis, Gardner’s syndrome, and Peutz-Jegher’s syndrome; and ulcerative colitis present for more than 8 years. The chance of having a second colon cancer simultaneously (synchronous) with one newly discovered appears to be about 4% (range, 1.5%–18%), and the chance of developing a second carcinoma at some time after resection of the first is about 5%–10%.

As a general rule, GI carcinoma is not common in persons under age 40 years (although it can occur) and increases steadily in probability after that age, as do many other types of cancer. The major symptom of colon carcinoma is change in bowel habits, either toward chronic diarrhea or constipation. However, either upper or lower GI tract carcinoma may be relatively asymptomatic until very late.

Detection methods for these GI lesions are of three kinds: tests for occult blood in the stool, x-ray examination, and direct visualization.

X-ray. Barium enema is the standard x-ray procedure for the colon. In a barium enema study the barium is washed into the colon through a tube after all feces have been eliminated by laxatives and regular enemas. The major cause of poor barium enema studies is inadequate colon preparation. If feces remain in the colon after cleansing, obviously the barium cannot fill these areas, and small lesions may be missed. Barium enema may be performed by two techniques: regular and air contrast. Air contrast is more sensitive for colon lesions than standard barium enema, especially for polyps, but is somewhat more difficult to perform well and is even more dependent on good preparatory cleansing of the colon.

Direct visualization techniques. Direct visualization techniques include gastroscopy for stomach lesions and proctoscopy, sigmoidoscopy, and fiberoptic colonoscopy for rectal and colon lesions. Proctoscopy examines the anus and rectum. Sigmoidoscopes have two variants: the rigid type permits vision only to 25 cm, whereas the flexible type can reach as high as 40-50 cm. The fiberoptic colonoscope in experienced hands can be used to view almost the entire colon. Biopsy specimens can be obtained at the same time. Simple digital rectal examination detects many rectal and prostate cancers. For this reason, rectal examination is always included as a part of any good physical examination.

Stool occult blood. The most useful laboratory screening test at present is examination of the feces for blood. Usually this blood is occult (not grossly visible), although sometimes it is grossly visible. If it is from the upper GI tract the stool is often black (“tarry”), whereas lower GI bleeding may still show unchanged blood and color the stool red. Anemia of the chronic iron deficiency type is often present, although not always, and sometimes may be severe. Occult blood in the feces can be demonstrated by simple chemical tests for hemoglobin. These are based on peroxidase activity of hemoglobin, which is detected when it catalyzes the oxidation of a color reagent by a peroxide reagent. The most popular test agents are benzidine, guaiac (as powder, tablet, or impregnated filter paper), and orthotolidine. Many studies have been done evaluating one or more of these methods. Results have often been conflicting and, at times, completely contradictory. Nevertheless, some consensus emerges. Benzidine is the most sensitive of the reagents but yields a great number of false positive results. It is currently not available in the United States. Orthotolidine (most commonly in the form of a tablet called Hematest) has intermediate sensitivity, consistently detecting 10-15 ml of blood placed in the stomach experimentally. False positive results (in patients on an unrestricted diet) are most often reported as 20%–30%. Guaiac in powder form provides surprisingly divergent results for different investigators, but the majority report a lesser degree of sensitivity than with Hematest. Guaiac-impregnated filter paper slides have been available since 1966 under various trade names, of which the earliest and best known is Hemoccult. The guaiac-impregnated filter papers are said to be approximately 25% as sensitive as guaiac powder or orthotolidine. Limits of consistent detection (>90% sensitivity) are variously reported as 6-20 mg of hemoglobin/gm of stool. Some of the discrepancies in the literature may reflect an increase in sensitivity of the newer guaiac-impregnated filter paper tests compared to the older versions. In patients on an unrestricted diet, false positive results are reported in 1%–12% of cases, most being trace or weak reactions. In vitro tests on specimens with added blood in amounts considered to be normal (usually 2 mg of hemoglobin/gm of feces) similarly yields about 10% false positive results.

Interfering substances. False positive results may be caused by ingestion of meat that has not been cooked sufficiently to inactivate its peroxidase. Plant material also may contain peroxidase. False negative results may be caused by large doses of ascorbic acid (vitamin C). This is more likely to occur with oral doses more than 500 mg/day. False negative results are frequently caused by bleeding that is intermittent rather than continuous and by blood that is not uniformly distributed within a stool specimen.

Useful precautions. Certain precautions must be taken to minimize false positive or false negative reactions and increase detection of true lesions:

1. The patient should be on a meat-free high-residue diet beginning at least 24 hours before collections of the stool. Eliminating meat decreases weak false positive reactions. In addition, some investigators advocate preparation and boiling of a fecal suspension to destroy plant peroxidases, although few laboratories do this routinely. Some protocols eliminate any vitamin C intake for at least 48 hours before the test. The diet high-residue component increases detection of significant lesions. If patients must be screened on an unrestricted diet, someone who manifests a weakly positive result could be restudied on a restricted diet.
2. At least three stool specimens should be collected, each specimen obtained at least 1 day apart but with the collection days as close together as possible. Testing should be performed on two well-separated areas from each specimen, since the blood may not be evenly distributed. A single stool specimen yields about 40%–50% positive results in colon carcinoma, whereas increasing the number of stool specimens to three or more increases sensitivity to approximately 60%–70%.
3. Stool specimens should be tested within 48 hours after collection. Conversion of positive to negative results or vice versa has been reported after storage, although data are conflicting.

Other guaiac tests. Collecting or working with stool specimens has never appealed to most persons. Compliance rates have been low in various stool guaiac programs. It is not unusual for less than 50% of specimen kits distributed to be returned to the laboratory. Several variations of the guaiac method have been devised to improve rates of patient specimen collection. One variant is a guaiac-impregnated paper that is placed in the toilet bowl water with the stool specimen. Water leaching hemoglobin from the outside portions of the stool react with the guaiac reagent in the paper to produce a blue color. Another variant obtains the specimen by wiping the anal area with a special guaiac-impregnated paper system, from which the grossly contaminated portion is discarded and the remaining area tested. Whether these techniques will substantially improve cancer detection awaits adequately controlled clinical trials.

Nonguaiac stool tests. Immunologic tests specific for hemoglobin have been reported but to date have not been widely used due to their relatively high cost and relatively long time needed for assay. An assay method specific for heme called HemoQuant has been developed based on extraction and measurement of porphyrins from hemoglobin in the patient sample. This method also has not yet been widely used, since a measured amount of specimen must first be heated in an acid reagent to extract the porphyrins, purified by extraction with another acid reagent, and then quantitated with a fluorometer.

Other considerations. Besides ulcer, polyp, or malignancy, many other conditions affecting the nasopharynx (nasal bleeding) to the anus (hemorrhoids) may produce a guaiac-positive stool. Some of the more common serious conditions include ulcerative colitis, regional enteritis, and diverticulitis.

Carcinoembryonic antigen (CEA)

CEA is a glycoprotein antigen migrating in the beta-globulin area on electrophoresis, found in gastrointestinal tract epithelium in early fetal life but not detectable in most normal persons after birth. Immunologic serum tests have been based on antibodies against CEA. The procedure was originally thought to be specific for colon adenocarcinoma, but as more experience was obtained and modifications of the original technique were developed, it became evident that although abnormal results were most frequently found in colon carcinoma, elevated serum CEA levels could be obtained in persons with malignancies in various organs, with certain benign diseases (usually involving tissue inflammation or destruction), with occasional benign tumors, and in those persons who smoke cigarettes.

Several basic immunoassay techniques have been used. The most frequent RIA method for many years was the Hansen procedure, in which Z-gel is used as a radioactivity separation agent. More recently, many laboratories have changed to shorter and more simple nonradioactive immunoassay methods, frequently using a sandwich antigen-antibody technique. Reference values for both of these techniques are approximately 0-2.5 ng/ml using some manufacturer’s kits but not others. Even when values for two manufacturer’s kits give similar results when performed on the same specimens, there are usually a moderate number of discrepancies, sufficient that serial tests on the same patient should be performed with the same manufacturer’s kit.

Test results in colon cancer and other conditions

In colon carcinoma, different investigators have published widely divergent results; with about 75% average detection of carcinoma, but with reported detection rates ranging from 59%–97% (Table 33-11). The smaller and earlier-stage tumors are less likely to give positive results. Among noncolonic tumors, using the Hansen method, more than one investigator has reported that 70%–90% of lung, 85%–100% of pancreatic, and 45%–60% of breast and gastric carcinomas produce abnormal results. Normal persons who smoke were CEA reactive in nearly 20% of cases, and conditions associated with elevation in more than 10% of patients include pulmonary emphysema, benign rectal polyps, benign breast diseases, cholecystitis or extrahepatic biliary duct obstruction, alcoholic cirrhosis, and ulcerative colitis. At CEA levels more than 5 ng/ml (twice upper limits), abnormal results in colorectal, lung, and pancreatic carcinoma decreased to 50%–60% and decreased to 30% in breast and gastric carcinoma. Most other conditions were reduced to 5% abnormality or less, except for alcoholic cirrhosis (about 25%), common bile duct obstruction (17%), active ulcerative colitis (13%), and emphysema (20%). At CEA levels greater than 10 ng/ml 4 times the upper normal limit), abnormal results were found in 35% of colorectal, 25% of lung, 35% of pancreatic, and 15%–30% of gastric and breast carcinoma. All the benign conditions were less than 1% reactive except for emphysema (4%), active ulcerative colitis (5%), and alcoholic cirrhosis (2%).

Table 33-11 Sensitivity of tests for colon carcinoma*

These data indicate that CEA results greater than 10 ng/ml are strongly suggestive of tumor. Results in the 5-10 ng/ml range are suggestive of tumor, and results less than 5 ng/ml are either equivocal or not helpful. However, since a substantial minority of colon cancers are not detected, since a varying number of patients with other tumors are detected, and since certain benign conditions may produce elevated values, most investigators do not recommend CEA as a screening test either for colon cancer or in most circumstances for cancer in general.

Prognostic value of carcinoembryonic antigen level in colon cancer. In general, CEA titers greater than 10 ng/ml (4 times the upper reference limit) in colorectal carcinoma most often occur in more advanced tumor stages and imply a worse prognosis. However, about 5%–10% (literature range, 0%–18%) of Dukes’ A lesions (tumor confined to the colon wall) and about 50% of Dukes’ B and C (local tumor extension with or without local node involvement) have CEA titers of 10 ng/ml or more, whereas about 10% (0%–16%) of Dukes’ D (distant metastases) have normal CEA levels. Therefore, a normal CEA level does not exclude far-advanced tumor, and high CEA titers do not mean that a colorectal carcinoma is unresectable, although high CEA titers are reasonable (but not conclusive) evidence against an early Dukes’ A) tumor stage (see also Table 33-11).

Use of carcinoembryonic antigen to detect recurrent tumor. The major currently recommended use for CEA is to follow the results of tumor therapy. For this, a pretherapy baseline assay is needed to determine if the CEA level is elevated. After surgical treatment, at least 4 weeks should elapse before follow-up CEA assay is performed (some cases have been reported in which CEA levels did not return to normal for 2-6 months). Thereafter, assay every 2 months for 2 years appears to be the most widely used protocol. Another problem associated with interpretation of CEA response to therapy is a transient elevation above baseline after the start of chemotherapy or radiation therapy in some patients, probably related to the destructive effect of therapy on the tumor. Return of CEA titer to the reference range or nondetectable level is fairly reliable evidence that most of the tumor has been removed. This does not guarantee that all tumor has been eliminated. Further elevation of CEA levels suggests recurrence (either local disease or metastasis). This may develop as much as 6 months before clinical evidence of recurrence. Some investigators report that CEA is more sensitive in detecting metastatic colon carcinoma to the liver than is alkaline phosphatase. However, numerous reports indicate that sporadic nonsustained CEA elevations that are not due to neoplasia may occur; sometimes they are associated with nonmalignant illnesses, but often there is no apparent cause. These elevations are usually less than 5 ng/ml. The most reliable indicator of recurrence is a sustained (on at least two occasions) elevation of at least 5 ng/ml. Even this conservative criterion is associated with about a 10% false positive rate, based on “second-look” operations. However, in some cases where tumor was not found, recurrences developed later. Another published criterion is persistent elevation in three consecutive specimens obtained during 6 weeks.

There are several problems that may confuse CEA interpretation.

1. CEA in its current format uses two antibodies in a so-called sandwich technique, one of which is a monoclonal antibody derived from mouse (murine) spleen cells. Occasionally persons have antibodies that react (or cross-react) with mouse-derived antibody (HAMA) that can falsely elevate the CEA level and various other immunoassays using similar antibody methods. However, the elevated antibody level tends to remain the same over time.
2. Twenty percent or more patients with colon carcinoma will not have a rising CEA level.
3. Colon carcinoma located in the pelvis often fails to cause CEA level elevation.
4. Smoking can elevate the CEA level (generally only mildly).
5. Small but significant fluctuations may occur (mentioned previously) because of technical reasons, acute infection or inflammation, or unknown cause.

One report indicates that the CEA level is more likely to be elevated in recurrent colorectal carcinoma with liver or retroperitoneal metastasis (about 75%) and substantially less often elevated with lung, peritoneal, or nonhepatic local or single metastases (about 45%). These investigators had very few cures on reoperation prompted by reelevation of CEA levels, therefore raising the question of the cost-effectiveness of posttherapy monitoring.

In summary, a positive CEA result does not differentiate colon tumors from those of other primary sites. There is considerable overlap between malignancy and various benign conditions in the area up to 4 times the upper reference limit (2.5-10 mg/ml, normal being 0-2.5 ng/ml), especially in the area up to twice the upper reference limit (2.5-5 ng/ml). At present, CEA use in colon cancer is limited mostly to follow-up of patients after therapy. A significant and sustained increase in titer suggests recurrence or metastasis. Since the test requires technical expertise and considerable attention to technical details, repeat assay is suggested if one individual value deviates significantly from previous values and major decisions would be based on that value.

Sensitivity of tests for colon cancer

It is important to have some idea of the sensitivity of the various tests available to detect colon cancer, since one must make decisions on the basis of these test results. This information was presented in Table 33-11. In some cases it is difficult to compile accurate data, since a technique (e.g., barium enema) may be carried out by different methods that are not specified in the report. In the case of stool guaiac (as noted previously) there is significant improvement in detection rate when more than one specimen is obtained; this is not adequately reflected in the overall statistics provided by the studies summarized in Table 33-11. Nevertheless, most reports indicate that 20%–30% of colon cancers will be missed with Hemoccult using multiple stool specimens. In fact, at least one investigator concluded that a careful history, emphasizing symptoms commonly associated with colon carcinoma (change in bowel habits toward diarrhea, constipation, or narrowing of the stool; vague abdominal pain; increased flatus or mucous discharge) was at least as sensitive as, if not more than, stool testing for occult blood in raising suspicion of colon cancer.

In summary, rectal examination and stool tests for occult blood are the best simple screening procedures for GI tumor. If these are positive or arouse strong clinical suspicion, one can proceed to x-ray studies of the area indicated. When possible, direct visualization techniques are extremely helpful. Fiberoptic colonoscopy can detect lesions throughout the colon.

The three most important pancreatic tumors are carcinoma of the exocrine pancreas (pancreatic adenocarcinoma of duct origin), islet cell tumors producing insulin (insulinoma), and islet cell tumors producing gastrin (gastrinoma) associated with the Zollinger-Ellison (Z-E) syndrome.

Exocrine adenocarcinoma. Carcinoma of the pancreas as a descriptive term usually refers to an adenocarcinoma of the exocrine pancreatic ducts, which comprises 90%–95% of pancreatic carcinomas. At the time of diagnosis about 65% are located in the head of the pancreas, about 20% in the body, about 5% in the tail, and about 10% are relatively diffuse. At the time of initial diagnosis, pain (usually abdominal) is present in about 75% of patients, weight loss in 50%–60%, bowel symptoms in 20%–30%, an abdominal mass in 5%–50%, and thrombophlebitis or thromboembolism (traditionally highly associated with pancreatic carcinoma) in 5%–10% (more common with body or tail lesions). At the time of diagnosis there is said to be local invasion or lymph node spread in about 25% of cases and distant metastases in about 60% (although literature ranges for distant metastases vary widely, depending on whether the metastases are overt or occult).

Laboratory findings include anemia in 25%–50% of cases, stool occult blood in as many as 50% of tumors in the pancreatic head, fasting hyperglycemia in about 20%, and oral glucose tolerance test abnormality in about 50% (literature range, 20%–81%, depending on criteria used). Jaundice is present in about 65% (seen in 45%–95% of pancreatic head tumors but much less common in those from the body and tail). Alkaline phosphatase level is elevated in most patients with jaundice and about one third of those without jaundice. Serum amylase level is elevated in only about 10% of patients.

Cancer antigen 19-9 (CA 19-9) and carcinoembryonic antigen (CEA) levels are elevated in a substantial percentage of patients with pancreatic carcinoma. However, at least 10%–20% of patients have normal levels of these tumor markers, and levels of both markers are elevated in a significant percentage of other tumors and nonmalignant conditions. Therefore, neither test is currently being widely used for screening, diagnosis, or therapy of pancreatic carcinoma.

Upper GI series is reported to be about 50% sensitive alone and about 80% sensitive with hypotonic duodenography for detecting carcinoma of the pancreas head. Duodenal tube drainage with secretin stimulation has an overall sensitivity of 30% (literature range, 10%–90%), which is increased to 50% (20%–84%) when cytologic study is performed on the pancreatic duodenal secretions. Ultrasound has overall sensitivity of 80% (68%–94%), with about 10%–15% of the studies attempted being technically unsatisfactory. CT scan averages about 80% overall sensitivity (60%–92%), with average sensitivity probably even better with the newest-generation scanners. Endoscopic retrograde choledochopancreatography (ERCP) is 80% sensitive (54%–96%), whereas ERCP combined with duct aspiration cytology is reported to be 85% sensitive. ERCP technical failures occur in about 15%–20% of cases. Selective angiography (celiac and superior mesenteric artery) detects 60% of carcinomas, whereas catheterization of pancreatic vessels (“superselective angiography”) may detect about 90%. Percutaneous transhepatic cholangiography is occasionally needed when jaundice is present and the other methods fail to yield a diagnosis or cannot be used.

Zollinger-Ellison syndrome. The Z-E syndrome is caused by a gastrin-producing nonbeta islet cell (G-cell) tumor of the pancreas (gastrinoma). There are multiple tumors in 70% of cases (literature range, 55%–80%). Two thirds (range, 60%–100%) of the tumors are malignant. Most gastrinomas originate in the pancreas, but occasionally they are found in an “ectopic” location (alone or in addition to the pancreas). Thus, about 10%–13% occur in the duodenum, and, rarely, they may arise in the stomach. Within the pancreas, the majority are in the head or the tail. Their microscopic appearance is similar to that of an insulinoma or carcinoid. About 10%–40% of patients with gastrinomas also have other endocrine tumors (most commonly a parathyroid adenoma); association with the MEN I syndrome is fairly common.

The major components of the Z-E syndrome are listed in the box on this page. Approximately 50%–60% of Z-E syndrome ulcers (31%–75%) are located in the proximal duodenum, which is the usual site for peptic ulcers; 25% (range, 20%–42%) are found in the distal duodenum or the jejunum; and 10% (range, 8%–15%) are found in the stomach. Multiple ulcers occur in 10%–20% of patients. About 10% (range, 7%–15%) have no ulcer. Diarrhea is found in 30%–35% of patients (range, 16%–75%). The degree of diarrhea is variable, but severe chronic diarrhea with hypokalemia is typical. Diarrhea is the only symptom of the Z-E syndrome in 7%–10% of cases. Steatorrhea occurs in 40% of cases (range, 38%–66%). However, many patients initially have symptoms very similar to ordinary peptic ulcer without the classic features of Z-E syndrome.

Signs and Symptoms Suggestive of the Zollinger-Ellison Syndrome
Intractable or recurrent peptic ulcer(s)
Multiple peptic ulcers or ulcers in unusual locations
Recurrent or marginal ulcer after complete vagotomy or partial gastric resection
Chronic diarrhea
Gastric acid hypersecretion

The Z-E syndrome has been divided into two types. Type I is caused by so-called G-cell hyperplasia, an increase in number or activity of gastric antrum G cells without gastrinoma tumor. Type II is due to gastrinoma of the pancreas or duodenum.

Gastric Analysis. The Z-E syndrome is usually accompanied by gastric hypersecretion and hyperacidity, which some consider an integral part of the syndrome. According to the old gastric analysis method using Topfer’s reagent as a pH indicator, now considered outmoded, basal gastric secretion quantity more than 200 ml/hour and basal gastric acid secretion more than 100 mEq/L/12 hours were considered suggestive of Z-E syndrome. According to the currently recommended gastric analysis methods using a pH meter or methyl red pH indicator, basal (1-hour) acid secretion greater than 10 mEq of hydrochloric acid (HCl)/hour and a ratio of basal acid output (BAO) to maximal acid output (MAO) of 0.4 or greater raise the question of Z-E syndrome; a BAO of 15 mEq/hour and a BAO/MAO ratio of 0.6 or greater are very suggestive of Z-E syndrome (although not pathognomonic) (Table 33-10).

Table 33-10 Gastric analysis in Zollinger-Ellison syndrome*

Serum gastrin. Serum gastrin assay is the method of choice for diagnosis. Fasting gastrin levels are elevated in more than 95% of gastrinomas. Serum gastrin levels more than 5 times the upper limit of the reference range (1,000 pg/ml or 1,000 ng/L) are virtually diagnostic of Z-E syndrome. Some believe that gastric analysis can therefore be omitted in these patients. However, a few patients with gastrinoma have basal gastrin levels that are within reference range, and 50% have levels that are only mildly or moderately elevated and overlap with values found in certain other conditions associated with elevated serum gastrin levels. These other conditions include diseases associated with hypochlorhydria or achlorhydria, such as atrophic gastritis and pernicious anemia (if the antrum is not severely affected), after vagotomy, in patients with retained antrum following gastrojejunostomy, in uremia, and possibly in chronic hypercalcemia. In one series, about 60% of patients with elevated serum gastrin level had hypochlorhydria or achlorhydria as the cause of the elevated gastrin level. Because of this, some investigators recommend gastric analysis in patients with mild or moderate gastrin elevations. Food ingestion has also been reported to produce a significant temporary increase in serum gastrin level. A high-protein meal is said to increase serum gastrin levels 2-5 times baseline values. In addition, some patients with peptic ulcer have mild or moderate serum gastrin elevation that overlaps with those occasional gastrinoma patients who have values that are not markedly elevated.

Gastrin stimulation tests. Since overlap in gastrin values may occur between gastrinoma and other conditions when fasting gastrin is less than 1,000 pg/ml (1,000 ng/L), stimulation tests have been devised to assist differentiation (see the box on this page). The original standard procedure was calcium infusion. Patients with gastrinomas more than double the baseline values, whereas patients with ulcers fail to do so. Patients with pernicious anemia, however, frequently respond to calcium infusion. Also, calcium infusion can produce cardiac problems, especially in patients with renal or cardiac disease. Secretin stimulation appears to be replacing calcium infusion as the confirmatory procedure of choice. Secretin seems to be a little more sensitive than calcium and appears to differentiate better between gastrinomas and other causes of elevated serum gastrin. In one study, 6% of patients reached the peak at 2 minutes, 69% at 5 minutes, 20% at 10 minutes, and 5% at 15 minutes. However, about 5%–10% of Z-E patients with fasting gastrin elevated but less than 1,000 pg/ml had negative secretin tests. In these patients, calcium infusion may be helpful, since about one third have diagnostic results with calcium. It has been reported that pernicious anemia patients do not respond to secretin stimulation.

Gastrin Stimulation Tests in Zollinger-Ellison Syndrome
Secretin (2 units/kg IV bolus): Baseline; 2,5,10,15 minutes postsecretin. Peak response of over 200 pg/ml (200 ng/L) over baseline occurs in 87%–100% of Z-E syndrome patients.
Calcium Infusion (10% calcium gluconate infusion; 5 mg calcium/kg/hr for 3 hrs): Baseline; post dose 120, 150, and 180 minute specimens. Increase over 395 pg/ml (395 ng/L) occurs in over 95% of Z-E syndrome patients; increase over 3 times baseline occurs in over 85%. Response to calcium is less specific than response to secretin.

Primary gastrinoma localization. Ultrasound is reported to demonstrate 21%–28% of gastrinomas, CT is said to detect 35%–60% (range, 18%–80%), and selective angiography can locate 35%–68%.

These are found mainly in the GI tract, although a minority are located in the lungs and a few arise in other locations. The appendix is the most frequent site of origin; these are almost always benign. Carcinoid are next most frequent in the terminal ileum and colon; these are frequently malignant. Carcinoid are considered part of the amine precursor uptake and decarboxylation (APUD) system, composed of cells derived from embryonic neuroectoderm that migrate from the primitive neural crest. These cells are located in embryonic GI tract derivatives, which include the GI tract, GI tract accessory glands (pancreas, biliary system), and organs with a very early embryonic GI source (lungs, thymus, genitourinary tract). They potentially can synthesize and secrete most body hormones (amines or peptides) except for steroids. Carcinoid cells contain fluorogenic amine substances and in certain areas characteristically contain secretory granules or material that stains with silver (argentaffin or argyrophilic). GI carcinoid cells are considered to be derived from Kulchitsky’s cells of GI epithelium.

Intestinal carcinoid may be multiple and frequently are associated with noncarcinoid malignancies (about 30% of cases, literature range, 7%–38%).

Carcinoid Syndrome. Carcinoids typically produce the vasoconstrictor substance serotonin, which induces several of the symptoms that are part of the carcinoid syndrome (Table 33-9). Carcinoid arising in foregut derivatives (bronchus, stomach, pancreas, duodenum, biliary tract) may produce the carcinoid syndrome and may be associated with the multiple endocrine neoplasia type I (MEN I) syndrome (with parathyroid, pituitary, and pancreatic islet cell tumors). Bronchial carcinoid may also secrete adrenocorticotropic hormone (ACTH) and may even produce the ectopic ACTH syndrome. These carcinoid usually do not stain with silver methods. Carcinoid of midgut origin (jejunum, ileum, appendix, and right side of the colon) typically are silver positive, and the jejunum and ileum are the most frequent source of the carcinoid syndrome. Carcinoid of hindgut origin (left side of the colon, rectum, and anus) usually do not have stainable argentaffin granules and usually do not produce the carcinoid syndrome.

Table 33-9 Carcinoid syndrome

When carcinoid cells produce serotonin, in most cases the venous drainage of the tumor is routed through the liver, which metabolizes or alters the hormone and prevents the carcinoid syndrome. If liver metastases develop in sufficient quantity or location, serotonin from carcinoid tumor in the liver bypasses hepatic portal vein drainage into the liver and exerts its effect unaltered. The same thing occurs with bronchial and ovarian carcinoid, because their venous drainage does not enter the hepatic portal vein system. Most carcinoid that produce the carcinoid syndrome originate in the intestine, and the syndrome usually does not appear until there is extensive metastasis by the carcinoid to the liver. However, the syndrome may occur without liver metastasis, especially when the primary site is the ovary. Conversely, in one third to two thirds of patients, liver metastases develop without the carcinoid syndrome.

Urine 5-hydroxyindoleacetic acid assay. Diagnosis of carcinoid syndrome can usually be made by testing for abnormal urine levels of 5-hydroxyindoleacetic acid (5-HIAA), the chief metabolic breakdown product of serotonin. Interestingly, there is very little specific information in the literature regarding the incidence of 5-HIAA elevation in either the carcinoid syndrome or in carcinoid patients without the carcinoid syndrome. In three studies of carcinoid patients (some with and some without the carcinoid syndrome), incidence of elevated urine 5-HIAA levels in those patients assayed was about 65% (range, 60%–87%). References that mention incidence of elevated 5-HIAA levels usually state that most patients with the malignant carcinoid syndrome have elevated 5-HIAA levels. However, some patients do not have continually elevated values; and in some cases repeated specimens may be necessary. Some patients with carcinoid may have elevated urine 5-HIAA levels without manifestations of the carcinoid syndrome; how often this happens is not known. Certain foods may elevate urine 5-HIAA levels. A few conditions, such as nontropical sprue and Whipple’s disease may produce mildly elevated urinary 5-HIAA in some patients. One study found that a few carcinoid patients with normal or only slightly elevated urine 5-HIAA had elevated urine serotonin (5-hydroxytryptamine). However, serotonin assay is difficult and expensive.

Tumors of the upper gastrointestinal tract The major benign disease of the upper GI tract area is peptic ulcer; those of the lower GI tract are diverticulosis and mucosal polyp. The malignant disease usually affecting either area is the same—adenocarcinoma. The major clinical symptom of peptic ulcer is epigastric pain that occurs between meals and is relieved by food or antacids. Patients with gastric carcinoma may have similar pain, nonspecific pain, or simple gastric discomfort. The most frequent malignancies arise in the stomach and the head of the pancreas. X-rays. Radiologic procedures include an upper GI series for stomach and duodenum and a barium enema for the colon. In the upper GI series, the patient swallows a barium mixture, and a series of x-ray films shows this radiopaque material filling the stomach and duodenum. Carcinoma of the stomach. Under the best conditions, x-ray examination (upper GI series) is said to be about 90% accurate in detection of gastric carcinoma. However, x-ray examination does not reveal the nature of the lesion. Gastroscopy is currently the best procedure for diagnosis, since instruments that allow visualization of most areas in the stomach and also permit biopsy are available. If gastroscopy is not available, gastric analysis for acid (after stimulation) may be helpful; achlorhydria considerably increases suspicion of carcinoma. Cytology of gastric washings is useful. However, gastric cytology is not as successful as cytology of specimens from uterine or even from pulmonary neoplasia, since small gastric tumors may not shed many neoplastic cells, and interpretation of gastric Papanicolaou smears in general is more difficult. Gastric aspiration specimens for cytology should be placed in ice immediately to preserve the cells.

Acid phosphatase-biochemical. Prostatic carcinoma often may be detected chemically because normal prostatic tissue is rich in the enzyme acid phosphatase, and adenocarcinomas arising from the prostate often retain the ability to produce this enzyme. Acid phosphatase is actually a group of closely related enzymes that share certain biochemical characteristics. Members of this group are found in several tissues (e.g., kidney, bone, prostate, platelets, and spleen). For almost 30 years, standard biochemical methods were the only assay systems available for acid phosphatase. Since enzyme quantity cannot be measured directly by these systems, it is estimated indirectly by the amount of change the enzyme produces in a measured quantity of substrate (a substance that can be changed by action of the enzyme). Several different assay systems have evolved, each of which differs somewhat in sensitivity and specificity for acid phosphatase of prostate origin. This accounts for some (although not all) of the conflicting reports in the literature regarding ability of biochemical enzyme system assays for acid phosphatase to detect prostate carcinoma.

Studies have shown that about 5%–10% (literature range, 5%–15%) of patients with prostate adenocarcinoma confined to the prostate have elevated serum acid phosphatase levels. Elevation occurs in about 20%–25% (10%–56%) of patients with extension of prostate tumor outside the prostate capsule without distant metastases, and in about 75%–80% (47%–92%) of those with bone metastases.

False positive results. Since acid phosphatase measurement is usually undertaken to detect prostate carcinoma, elevation in other conditions is usually considered to be a false positive result. Prostatic infarcts can temporarily elevate serum acid phosphatase levels. Acid phosphatase levels are reported to be elevated in about 5%–10% (0%–19%) of patients with benign prostatic hypertrophy without any evidence of carcinoma. Elevations have been reported in patients with several other diseases; patients with the most substantial percentage of abnormality include those with nonprostatic metastatic carcinomas to bone, certain metabolic bone diseases (Paget’s disease, primary hyperparathyroidism), Gaucher’s disease, and certain platelet disorders (thrombocytosis, disorders of platelet destruction). Alkaline and acid phosphatase are similar enzymes that differ predominantly in the pH at which they work best. Therefore, any condition that produces greatly increased alkaline phosphatase values may induce some elevation of acid phosphatase values because the ordinarily negligible action of the alkaline phosphatase at lower pH is magnified by a greatly increased quantity of enzyme.

There is controversy in the literature whether a rectal examination will temporarily (up to 24 hours) increase serum acid phosphatase levels. More investigators have reported no change than those who did find significant elevation, but the size of the two groups is not far apart. Therefore, the test probably should be repeated if elevated levels occur after a rectal examination. There is some evidence that elevated serum acid phosphatase values can fluctuate 25%–50% or even more during a 48-hour period.

Various methods have been used to decrease false positive results, some of which are more specific than others for prostatic acid phosphatase. However, none is completely specific. For example, it was found that various substances could inhibit either prostate or nonprostate acid phosphatase; the most widely used of these substances is L-tartrate, which inhibits prostatic acid phosphatase. Thus, acid phosphatase elevation that persists after patient serum is incubated with L-tartrate (“tartrate resistant”) is likely to be of nonprostatic origin. Unfortunately, although tartrate inhibition does increase specificity, it does not produce complete specificity. Fortunately, in most situations where prostatic carcinoma is suspected, the majority of the nonprostatic conditions that produce elevated acid phosphatase levels would be unlikely (except for occult nonprostatic tumor involving bone).

False negative results. Biochemical enzyme assays for acid phosphatase are heat and pH sensitive. Serum left at room temperature after exposure to air may exhibit significantly decreased activity after as little as 1 hour. The use of a preservative with prompt refrigeration is strongly recommended. Immunoassays can tolerate exposure of the sample to room temperature for 4-5 days.

Acid phosphatase immunoassay. In the last half of the 1970s, methods were found to obtain antibodies against prostatic acid phosphatase. This enabled the development of immunoassays (radioimmunoassay [RIA], enzyme-linked immunosorbent assay [ELISA], and counterimmunoelectrophoresis) for determining prostatic acid phosphatase levels. In general, the immunoassays have greater specificity for prostatic acid phosphatase than biochemical enzyme procedures have. The immunoassays also detect more patients with prostate carcinoma. Unfortunately, the tests uncover only about 10%–20% more patients with prostate carcinoma than the biochemical tests at comparable stages of tumor spread (Table 33-7). In addition, the immunoassays are not completely specific; for example, they become elevated in some cases of nonprostate carcinoma that is metastatic to bone. Many of the elevations of test kits found that a certain number of patients with benign prostatic hypertrophy, without evidence of tumor, had elevated results. Moreover, there has been a very disturbing variation in sensitivity and specificity of the immunoassay kits. Another drawback is that immunoassays are 3 to 5 times more costly to perform than the biochemical enzyme methods.

Table 33-7 Comparison of biochemical and immunologic assays for acid phosphatase*

Prostate-specific antigen (PSA). Prostate-specific antigen (PSA) is a glycoprotein enzyme that was isolated from prostate gland epithelial cells in 1979. There are claims that this enzyme is specific for prostate origin, although a few isolated instances of elevation in nonprostate tumors have been reported. Three manufacturers now have kits commercially available. Reports thus far agree that PSA detects more patients with prostate carcinoma than acid phosphatase by immunoassay or biochemical methods (Table 33-7). Prostate carcinoma in stage A is detected in about 55% of cases. Overall prostate carcinoma detection is about 80%–90% (range, 75%–96%). Unfortunately, detection rate for patients with benign prostatic hypertrophy is about 50%–60% (range, 10%–83%). Since prostatic glandular hypertrophy is common in the same age group as prostate carcinoma, there has been much dispute whether PSA should be used as a screening test for prostate carcinoma. Although screening with PSA detects a significant number of patients with early stage cancer, there has not yet been proof that a significantly greater number of patients will be cured. Also, about 20% (range, 16%–33%) of patients with cancer have PSA values within the usual PSA population reference range of 0.1-4.0 ng/ml (µg/L). Several ways have been suggested to improve PSA usefulness. It has been shown that PSA levels correlate reasonably well on the average with prostate weight due to benign prostate glandular hyperplasia (BPH) and prostate cancer produces about 10 times the amount of PSA on a tissue volume basis than does BPH. Studies have resulted in several formulas that at least partially correct PSA levels for effect of BPH. This is the most well established of these formulas at present: Predicted PSA level (PSA “serum density”, in ng/ml or µg/L = 0.12 Ч gland volume (in cubic centimeters [cc]) by transrectal ultrasound [TRUS]; when TRUS gland volume = prostate height Ч width Ч length Ч 0.523). A PSA serum density greater than the predicted value suggests increased possibility of carcinoma. Another parameter is the height of the PSA level. Values within reference range suggest relatively low probability of cancer (although, as noted previously, some 20% of cancers have normal PSA); values over 10 ng/ml suggest a relatively high probability of carcinoma; and values between 4-10 ng/ml suggest an intermediate possibility (this being the zone in which there is greatest overlap with elevation from BPH). Another parameter, especially for values below 10 ng/ml, is the trend of PSA values repeated in 3- or 6-month intervals. A definite upward trend increases the likelihood of cancer. Since this may also occur due to BPH, there have been efforts to find a trend formula that differentiates the two entities. However, none of the articles proposing a trend formula had sufficient number of patients to make the formula statistically valid and none have had adequate independent evaluation.

Several investigators have proposed age-adjusted PSA reference ranges, although their values differ somewhat. A Mayo Clinic study proposes a PSA upper limit of 2.5 ng/ml (PSA density [PSAD] upper limit 0.08) for ages 40–49 years, PSA 3.5 (PSAD 0.10) for ages 50–59, PSA 4.5 (PSAD 0.11) for ages 60–69, and PSA 6.5 (PSAD 0.13) for ages 70–79.

These diagnostic problems have occurred because prostate carcinoma in general is a slow-growing tumor that most often clinically becomes evident in older persons; and although there are tumor subsets that are aggressive, the majority of patients die from other causes than prostate cancer. In addition, the three current modes of therapy (radical prostatectomy, radiation therapy, and hormonal therapy or androgen deprivation) all have a significant amount of both somewhat unpleasant morbidity and also of treatment failure. Nevertheless, since it has been calculated that about 10% of U.S. males will develop clinically evident prostate carcinoma and about 3% of U.S. males will die from it, screening will continue in the hope of detecting occult disease in a curable stage.

The most common prostate screening recommendation is to obtain both PSA and direct rectal examination (DRE). Although PSA misses about 20%–30% and DRE misses about 50% (range, 14%–64%) of prostate cancer, the two together detect an additional 15%–20% or more over results from either one alone. If DRE is positive, or if DRE is negative but clinical suspicion is high, transrectal ultrasound is advised to help find a target for biopsy and to estimate prostate volume for the PSA serum density value if no TRUS area suggestive of cancer is seen. Abnormal PSA serum density could be a reason for biopsies without a clearly defined TRUS target. Incidence of prostate carcinoma is increased in African Americans (about twice the risk of European descendants). Previous vasectomy may create some increased risk.

There are some points regarding PSA that are worth mentioning. There is no diurnal variation. No preservatives are needed for the specimens. There has been some controversy whether DRE will temporarily elevate PSA; most investigators found 2% or fewer patients above 4.0 after DRE. A post-DRE specimen could be drawn less than 1 hour after the examination to minimize any examination effect. Prostate biopsy or TUR elevate PSA to varying degree in most persons. This effect varies in duration between persons, the amount of prostate injury, or the height of the PSA response, but is usually back to baseline or below by 6 weeks (in one study, 63% of persons took less than 2 weeks and 15% took over 4 weeks). The serum half-life of PSA is about 2 days. Finasteride therapy of BPH in dosage of 5 mg/day decreases the PSA to 50% of pretherapy baseline value after 6-12 months of therapy. Patient compliance can be monitored by serum dihydrotestosterone assay, which should be suppressed by more than 60% from pretherapy baseline values. One report recommends that serum PSA should be checked 6 months after beginning therapy, and if the value (in a compliant patient) is not 50% of baseline, the patient should have a workup for possible prostate carcinoma.

Serum alkaline phosphatase. Stage D prostate carcinoma metastasizes to bone in about 70% of patients (range, 33%–92%). More than 90% of these metastases have an osteoblastic component. Serum alkaline phosphatase levels are elevated in 70%–90% of patients with bone metastoses.

Renal cell adenocarcinoma (hypernephroma) is about twice as frequent in males as in females. It occurs with about equal frequency in both kidneys. About 90% of cases occur after age 40, although more than 30 cases have been reported in children. About 80% of renal cell carcinomas are located in either the upper or the lower poles of the kidney.

Clinical findings. In renal cell adenocarcinoma, symptoms and urinary findings vary according to the location, size, and aggressiveness of the tumor. Renal carcinoma often causes hematuria (as does bladder carcinoma). Painless gross hematuria is the initial symptom in about 40% of patients and occurs with other symptoms in additional cases. These episodes are often intermittent and may be misdiagnosed as urinary tract calculus or infection. Flank pain is present in about 40% of patients, and about one third have a significant degree of weight loss. A mass is noticeable to the patient in about 10%–15% of cases and is palpable by the physician in about 50% of cases. In addition, hypernephroma, on occasion, is a well-recognized cause of fever of unknown origin. It has rarely but repeatedly been associated with secondary polycythemia (about 3% of patients), although a large minority have anemia and the majority do not show hemoglobin abnormality. About 20% of patients have hypertension (range, 4%–38%). About 10% (range, 4%–28%) have no clinical symptoms suggestive of hypernephroma, and the tumor is discovered on intravenous pyelogram (IVP) performed for some other reason.

Laboratory findings. Hematuria, either gross or microscopic, is the most frequent abnormality, being detected at some time in about 60% of patients (range, 28%–80%). Unfortunately, most publications either do not differentiate between gross and microscopic hematuria or record gross hematuria only. In 38 patients with renal cell adenocarcinoma seen in my area, 39% had gross hematuria and an additional 27% had microscopic hematuria. Proteinuria may be present but is less frequent. About 30% of patients have a completely normal urinalysis on admission. The IVP is the most useful screening test for renal cell carcinoma and, if carefully done, will also detect many cases of carcinoma in the renal pelvis and ureters. Other procedures, such as kidney scanning or computerized tomography (CT), are also useful.

Other procedures. Once a space-occupying lesion is identified in the kidney, the question arises as to its nature. B-mode ultrasound, CT, and drip infusion tomography seem to be excellent methods of distinguishing a solid renal tumor from a renal cyst. However, there is coincidence of renal carcinoma and simple renal cyst in about 2%–3% (literature range, 2%–7%) of cysts explored. Selective renal angiography is also very effective and can be performed if tomography is inconclusive. No technique is infallible, however, since a few tumors may become exceptionally cystic due to internal necrosis. Urine cytology has relatively little value at present in the diagnosis of renal cell adenocarcinoma. Metastatic carcinoma or malignant lymphoma in the kidney usually does not produce significant clinical or urinary findings.