Articles on Medical Diseases and Conditions

Tag: DNA probes

  • Other Congenital Diseases

    There are a large number of congenital and genetic disorders, too many to include all in this book. If such a condition is suspected, in general the best procedure is to refer the patient or family to a university center that has an active genetics diagnosis program. If the state government health department has a genetic disease detection program, it can provide useful information and help in finding or making referral arrangements.

    Some Genetic Disorders Diagnosable with DNA Probes

    Huntington’s chorea
    Adult polycystic disease
    Alpha and beta thalassemia
    Congenital adrenal hyperplasia
    Duchenne’s and Becker’s muscular dystrophy
    Fragile X syndrome
    Hemophilia A and B
    Myotonic dystrophy
    Osteogenesis imperfecta
    Alpha-1 antitrypsin deficiency
    Cystic fibrosis
    Sickle cell hemoglobinopathy
    Retinoblastoma
    Familial hypertrophic cardiomyopathy

  • Diseases of Mineral Metabolism

    Wilson’s disease (hepatolenticular degeneration). Wilson’s disease is a familial disorder of copper metabolism transmitted as an autosomal recessive trait. It most often becomes manifest between ages 8 and 30 years; symptoms usually do not develop before age 6 years. About 30%-50% of patients initially develop hepatic symptoms, about 30%-40% begin with neurologic symptoms, and about 20%-30% initially are said to have psychiatric abnormalities such as schizophrenia. A few patients develop a Coombs’-negative hemolytic anemia. Children are more likely to be first seen with hepatic symptoms, although symptoms may occur at any age. In children, these most commonly take the form of chronic hepatitis, although in some patients the test results may resemble acute hepatitis virus hepatitis. A macronodular type of cirrhosis develops later and is usually present in patients with late-stage Wilson’s disease, whether or not there were symptoms of active liver disease. Some patients present with minimally active or with nonactive cirrhosis. Neurologic symptoms typically originate in the basal ganglia area (lentiform nucleus) of the brain and consist of varying degrees of incoordination, tremor, spasticity, rigidity, and dysarthria. There may also be a peculiar flapping tremor. Some young or middle-aged adults develop premature osteoarthritis, especially in the knees.

    Wilson’s disease is characterized by inability of the liver to manufacture normal quantities of ceruloplasmin, an alpha-2 globulin that transports copper. For reasons not entirely understood, excessive copper is deposited in various tissues, eventually producing damage to the basal ganglia of the brain and to the liver. The kidney is also affected, leading to aminoaciduria, and copper is deposited in the cornea, producing a zone of discoloration called the Kayser-Fleischer ring.

    Clinical diagnosis. The triad of typical basal ganglia symptoms, Kayser-Fleischer ring, and hepatic cirrhosis is virtually diagnostic. However, many patients do not have the textbook picture, especially in the early stages. The Kayser-Fleischer ring is often grossly visible but in many cases can be seen only by slit lamp examination. All patients with neurologic symptoms are said to have the Kayser-Fleischer ring as well as about 50% (range, 27%-93%) of those with hepatic symptoms. The Kayser-Fleischer ring is present in only about 20% (range, 0%-37%) of asymptomatic patients detected during family study investigation or at the beginning of symptoms from hepatic disease without neurologic findings. Overall, about 25% of patients (range, 22%-33%) do not have a demonstrable Kayser-Fleischer ring at the time of diagnosis. Patients with primary biliary cirrhosis or, occasionally, other types of chronic cholestatic liver disease may develop a corneal abnormality identical to the Kayser-Fleischer ring.

    Plasma ceruloplasmin assay. Laboratory studies may be of value in diagnosis, especially in the preclinical or early stages. Normally, about 90%-95% of serum copper is bound to ceruloplasmin, one of the alpha-2 globulins. The primary excretion pathway for serum copper is through bile. The serum ceruloplasmin level is low from birth in 95% (range, 90%-96%) of homozygous patients, and is considered the best screening test for Wilson’s disease. About 10% (range, 6%-20%) of Wilson’s disease heterozygotes have decreased serum ceruloplasmin. However, normal newborn infants usually have decreased ceruloplasmin levels, and the test is not considered reliable until 3-6 months of age. Although a normal ceruloplasmin level (over 20 mg/100 ml; 200 mg/L) is usually interpreted as excluding Wilson’s disease, about 5% (range, 4%-10%) of homozygous Wilson’s disease patients have values greater than 20 mg/100 ml. This is more likely to be found in younger children and in those with hepatic disease. Estrogen therapy, pregnancy, active liver disease of various etiologies, malignant lymphoma, and occasionally various acute inflammatory conditions (since ceruloplasmin is one of the “acute reaction” proteins) can raise ceruloplasmin levels in variable numbers of cases. Smoking is reported to raise ceruloplasmin levels about 15%-30%. Although a decreased ceruloplasmin level is usually considered suggestive of Wilson’s disease, about 5% of normal persons may have values less than 20 mg/100 ml (200 mg/L), and values may be decreased in hereditary tyrosinemia, Menke’s kinky hair syndrome, the nephrotic syndrome, malabsorption syndromes such as sprue, and in various liver diseases (about 20% of cases in one study. However, it is possible that some patients with liver disease and decreased ceruloplasmin levels actually have Wilson’s disease).

    Liver biopsy has also been used for diagnosis. The microscopic findings are not specific, and most often consist of either macronodular cirrhosis (often with some fatty change and occasionally with Mallory bodies) or chronic active hepatitis (10%-15% of patients with Wilson’s disease). The most typical finding is increased hepatic copper content by special stains (or tissue analysis, if available). For histologic staining of copper, fixation of the biopsy specimen in alcohol rather than the routine fixatives is recommended. Here again, it is advisable to wait 6-12 weeks after birth. Increased hepatic copper content is not specific for Wilson’s disease, since some degree of copper increase has been reported to occur in some patients with postnecrotic cirrhosis due to hepatitis virus hepatitis, in patients with primary biliary cirrhosis, and occasionally in patients with other chronic cholestatic syndromes. Also, increased hepatic copper content is not present in all patients with Wilson’s disease, especially in small-needle biopsy specimens.

    Serum and urine copper. Total serum copper levels are decreased in 85%-90% of Wilson’s disease patients. However, serum copper not bound to serum ceruloplasmin is usually normal or increased. Twenty-four-hour urine copper excretion in symptomatic Wilson’s disease is increased in 90% of patients. However, 24-hour copper excretion is often normal in presymptomatic patients. Increased urine copper excretion is not specific for Wilson’s disease and may be found in various types of cirrhosis, especially those with some degree of cholestasis and in 10%-30% of chronic active hepatitis patients. However, these conditions usually have normal or elevated serum ceruloplasmin levels.

    DNA probes. The gene affected in Wilson’s disease has been found on the long arm of chromosome 13, close to the gene responsible for retinoblastoma. DNA linkage probes for Wilson’s disease have been reported. In some cases, the retinoblastoma probe has been used.

    Other laboratory abnormalities. Besides abnormalities in copper metabolism, over 50% of patients (78% in one study) have a low serum uric acid level, a finding that could arouse suspicion of Wilson’s disease if supporting evidence is present. Other laboratory findings that may be encountered in some patients are low serum phosphorus levels, thrombocytopenia (about 50%; range, 22%-82%, due to cirrhosis with secondary hypersplenism), aminoaciduria, glucosuria, and uricosuria. A Coombs’-negative hemolytic anemia occurs in a few patients.

    Hemochromatosis. Hemochromatosis is an uncommon disease produced by idiopathic excess iron absorption from the GI tract, which leads to excess deposition of iron in various tissues, especially the liver. There still is dispute as to which iron storage diseases should be included within the term hemochromatosis. In this discussion, hemochromatosis refers to the hereditary iron storage disorder and hemosiderosis to nonhereditary (secondary) forms. Hemochromatosis is transmitted as an autosomal recessive trait with the gene being located on the short arm of chromosome 6 close to the class I histocompatibility antigen (HLA) locus. Males are affected more often than females (3:2 in one series), and males seem overall to have more severe disease than females. HLA-A3 antigen is present in 70%-80% of patients (vs. 20%-30% in the normal population).

    Clinical onset of the disease is usually between ages 40 and 60 years. Signs, symptoms, and laboratory abnormalities depend on the stage of disease and (probably) whether there is also a significant degree of alcohol intake. Cirrhosis, diabetes mellitus, and bronze skin pigmentation form a classic triad diagnostic of hemochromatosis. However, this triad is a late manifestation, and in one study including more early cases it was present in less than 10% of the patients. The most frequent symptom is joint pain (47%-57% of patients; 50%-75% in patients with severe disease), which can be confused with rheumatoid arthritis. Hepatomegaly is present in 54%-93% of patients, cirrhosis on liver biopsy in 57%-94%, heart failure in 0%-35%, hypogonadism (in males) in 18%-61%, skin pigmentation in 51%-85% (not really noticeable in many patients), and clinically evident diabetes in 6%-72%. Alcoholism (15%-50%) or poor nutrition was frequent in some series. Hepatoma has been reported to develop in 15%-30% of patients.

    Laboratory findings include the expected blood glucose abnormalities of diabetes (chapter 28) in those patients with overt diabetes, and decreased glucose tolerance in some of those without clinical diabetes. AST levels are elevated in 46%-54% of cases, reflecting active liver cell involvement. In one series, AST, alkaline phosphatase (ALP), and gamma-glutamyltransferase were normal or only mildly elevated unless the patient was alcoholic.

    Laboratory iron studies. The body iron abnormality is manifested by actual or relative increase in serum iron levels and decrease in total iron-binding capacity (TIBC), producing increased saturation (% saturation) of the TIBC. In addition, hemosiderin very often can be demonstrated in the urine sediment by iron stains. The most sensitive laboratory test for hemochromatosis is percent saturation of TIBC (or of transferrin), which is greater than 60% (reference range, 16%-50%) in over 90% of male homozygotes and the 60% of females who have iron loading but which misses the 40% of females who do not have iron loading. Transferrin saturation of 50% detects most males or females with or without iron loading. Therefore, it has been proposed that the screening cutoff point should be 60% for males and 50% for females. Serum iron level is increased in more than 80% of patients and serum ferritin level is increased in more than 72% of patients; both of these tests are usually abnormal in affected males but much more variable in females. However, in one report about one third of patients with chronic hepatitis B or C also had elevated serum iron, ferritin, and percent saturation, and serum ferritin is often increased by various acute inflammatory conditions. Liver biopsy demonstrates marked deposition of iron in parenchymal cells and frequently reveals cirrhosis.

    The most widely used screening test is serum iron. Elevated values raise the question of hemochromatosis. About 2.4% of normal persons are reported to have elevated serum iron values that spontaneously return to the reference range within 1-2 days. The effect of serum diurnal variation and day-to-day variation must be considered. Serum iron levels can also be increased in chronic hepatitis B or C infection (46% of cases in one study) and in hemosiderosis (nonhereditary iron overload) due to blood transfusion, chronic severe hemolytic anemias, sideroblastic anemias, alcoholic cirrhosis, parenteral iron therapy, and considerably increased iron intake. Several other conditions that may be associated with increased serum iron levels are listed in Table 37-2. Various conditions can lower the serum iron level (especially chronic iron deficiency and moderate or severe chronic disease without iron deficiency), and if one of these conditions is superimposed on hemochromatosis, the serum iron level might be decreased sufficiently to reach the reference range area.

    As noted earlier, the best screening procedure is percent saturation of transferrin. This is calculated by dividing the serum iron value by the TIBC value. However, like serum iron, increase in percent transferrin saturation is not specific for hemochromatosis, since there are other conditions that decrease percent saturation, especially alcohol-related active cirrhosis. One study found that drawing specimens after an overnight fast considerably decreased false elevation of percent saturation. In addition, there is considerable variation in the literature as to the percent saturation cutoff point that should be used (50%-80%, with the majority using either 50% or 62%). The lower levels increase sensitivity in detecting hemochromatosis; the higher levels eliminate many patients who do not have hemochromatosis.

    Definitive diagnosis is made by liver biopsy and measurement of hepatic iron content. Even liver biopsy iron may not differentiate hemochomatosis from hemosiderosis in some cases, and the liver cells of patients with cirrhosis but without demonstrable abnormality of iron metabolism may display some degree of increased iron deposition.

    Family member screening. Hemochromatosis rarely becomes clinically evident before age 30, so that screening family members of patients has been advocated to detect unrecognized homozygotes to begin therapy before clinical symptoms develop. One study found that percent transferrin saturation detected about 90% of occult homozygotes, whereas assay of serum iron levels detected about 85% and assay of serum ferritin levels detected about 50%.

  • Breast

    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.

  • Lyme Disease

    Lyme disease is caused by the spirochete Borrelia burgdorferi by means of several tick vectors, the principal one in the Northeast and North Central United States being the deer tick Ixodes dammini and in the Pacific Coast states, Ixodes pacificus, the Western black-legged tick (both morphologically “hard” ticks). The three major affected areas in the United States are the northeastern states (New Jersey to Connecticut), the far western states, and the upper midwestern states. However, cases have been reported elsewhere and also in Canada, Europe, and Australia.

    Ixodes dammini has a 2-year, three-form life cycle. The very young ticks (called larval stage, although the organism has a tick shape) feed on a vector organism, usually the white-foot mouse, and then are dormant until the following spring. The larval ticks are very small and have only three pairs of legs, like insects. The following year in the spring the larval tick changes to the nymph stage, which has four pairs of legs like the adult stage.

    In 50%-80% of patients, about 1 week (range, 3-68 days) after the tick bite, a reddish macular expanding lesion with central clearing (“erythema chronicum migrans”) develops on the skin at the inoculation site often followed by similar skin lesions in some other areas. This usually fades within 2-3 weeks (range, 1 day-4 weeks) and is usually accompanied by low-grade fever, weakness, fatigue, and regional lymphadenopathy. Although this characteristic skin lesion should strongly suggest Lyme disease, only 20%-30% of patients recall such a lesion. Migratory arthralgias and myalgia are frequently present. About 10% of patients develop anicteric hepatitis. In the second stage of illness, CNS (most often aseptic meningitis) or peripheral nervous system abnormalities (Bell’s palsy or Bannwarth’s polyneuritis syndrome) occur about 4 weeks (range, 2-8 weeks) after the tick bite in about 15%-20% of patients (range, 11%-35%). About 7% (range, 4%-10%) of patients develop transitory ECG abnormalities or myocardial inflammation, usually about 5 weeks after the tick bite (range, 4 days-7 months). In the third stage of illness, about 40% (range, 26%-60%) of patients develop recurrent arthritis. This is the most famous manifestation of Lyme disease and involves one or more joints, most commonly the knee, beginning about 6 weeks-6 months after the tick bite (range, 4 days-2 years).

    Laboratory test abnormalities include elevated erythrocyte sedimentation rate in about 50% of cases. Peripheral blood WBCs are elevated in only about 10%; fluid aspirated from arthritic joints is similar to that from patients with rheumatoid arthritis. CSF in patients with meningeal or peripheral nerve symptoms usually show increased numbers of WBCs with lymphocytes predominating, normal glucose and mildly increased protein levels, oligoclonal bands similar to those of multiple sclerosis, and CSF-IgM antibody present.

    Culture can be done from biopsy of the erythema migrans (ECM) skin lesion; best results are obtained from the advancing edge of the lesion. Transport of the specimen and specimen culture in the same special BSK culture media plus incubation for several weeks if necessary has produced best results; but even so the spirochetes were isolated in less than 45% of cases (range, 5%-71%). Warthin-Starry silver stains on ECM lesion biopsy demonstrates spirochetes in less than 40% of cases. Blood cultures may be positive in the second stage of illness but only in 2%-7% of cases and therefore is not cost-effective. Culture of CSF in second-stage symptomatic patients may be positive in about 10% of patients. DNA probes with PCR amplification have been reported to have a sensitivity of 80% when performed on a biopsy of the ECM skin lesion, the same or better than the best culture results. However, thus far, DNA probe for Borrelia antigen in blood has not substantially improved serologic test results.

    Currently, the most helpful procedures are serologic tests. IgM antibody levels rise about 2-4 weeks after onset of ECM, peak about 6-8 weeks after ECM onset, and usually become nondetectable by 4-6 months after onset. However, some patients have persistent IgM levels, presumably due to continued infection or reinfection. IgG antibody levels rise about 6-8 weeks after onset of erythema migrans and peak at about 4-6 months after onset of erythema migrans, but may not peak until later or even more than a year. The highest IgG levels tend to occur when patients develop arthritis. IgG levels typically remain elevated for life. The most commonly used tests are immunofluorescent and ELISA methods. False positive results can be obtained in patients with other spirochetal diseases, such as syphilis, relapsing fever, and leptospirosis, and according to one report also in subacute bacterial endecarditis (SBE). Some of the ELISA tests attempt to adsorb out some of these antigens if they are present. Both test methods can give about the same results, although investigators generally seem to have a more favorable opinion of ELISA. In the earliest stage of the disease (ECM present 1-7 days), serologic tests are rarely positive. Later in the first stage, 3-4 weeks after onset of ECM, the tests are positive in about 40% of patients. In the second stage of illness (coincident with systemic symptoms) about 65% are positive, and in the third (arthritic) stage, about 90%-95% (range, 80%-97%) are positive. This suggests that negative serologic tests in clinical stages one and two may have to be repeated 3-4 weeks later. ELISA tests using recombinant flagellar proteins as antigen somewhat improve IgM test specificity and may increase sensitivity a little in early disease compared to ELISA tests using whole organism alone. Sensitivity of IgG antibody is significantly greater than IgM in the second and third stages of Lyme disease because disseminated (second stage) infection raises IgG more than IgM (which has already peaked or has already started to decline).

    Evaluation of different kits has shown considerable variation in sensitivity and specificity between different kits, between laboratories, and even between evaluations in the same laboratories when the same specimen was repeated later. Western blot testing is commercially available or performed with homemade reagents. This has the advantage of visually identifying which proteins are reacting to patient antibodies. Unfortunately, there still is little agreement how to interpret the Lyme Western blot test. Some of the proteins that are rather frequently detected are shared with other organisms. Some of the more specific proteins (outer coat proteins A and B) may not appear until relatively late in some patients. Nucleic acid probe testing has recently been reported, with or without PCR amplification, mostly using homemade reagents. Although results have been more sensitive than some standard ELISA or fluorescent antibody kits, DNA probes so far have not increased usable sensitivity as much as has been achieved in some other diseases. Finally, some studies have reported that some patients with Lyme disease have a reactive antinuclear body (ANA) test, usually the speckled type. One report found that the VDRL or RPR test for syphilis is usually nonreactive.

    In one report from a Lyme disease referral center, of 788 patients with positive serologic test results for Lyme disease, 23% had active Lyme disease, 20% had previous Lyme disease, and 57% were judged not to have evidence of Lyme disease.