Since the pioneering demonstrations of the association of chromosomal abnormalities with human diseases like Down's syndrome, Turner's syndrome, CML, APL, and so forth as described earlier, several other congenital disorders and cancers have been now linked with specific chromosomal abnormalities. With this knowledge and combined by the effective cyto-genetic analysis tools, chromosomal banding and, among the sensitive molecular assays, FISH, genomic disease management is seeing the light of the day. The implications of cytoge-netics in congenital disorders are primarily seen in diagnosis and in determining family history or, on a larger scale, in population genetics, whereas a significant advance is seen in cancer, where cytogenetics are used for diagnosis, prognosis, in selection of therapy, as well as for monitoring residual disease through and after therapy.
7.1. CORRELATION OF CYTOGENETICS WITH PATHOBIOLOGY OF THE DISEASE The gene dosage effect causing several microdeletion syndromes has already been remarkably associated with definite phenotypic manifests. (for review, see ref. 61). Fortunately, for many of these syndromes,
Fig. 4. Tyramide amplification of FISH signals. Interleukin-8 (IL-8) mRNA was detected in bladder carcinoma cell line 5637 using a HRP-conjugated specific oligonucleotide probe and a biotin-tyramide-antibody-fluorescein system as described previously (59). On the background of the red counterstain resulting from the ISH for ribosomal RNA using Texas Red-labeled oligonucleotide probe, a clear cytoplasmic labeling (yellow) is seen localizing IL-8 mRNA. In addition, please note the green signals (arrow) for minor levels of IL-8 mRNA in the nucleus detected during synthesis in situ. Original magnification:x1000. (Courtesy of Dr. Anton K. Raap, Leiden University Medical Center, Leiden, The Netherlands). (Figure appears in color in insert following p. 172.)
commercial FISH tests are available to help in their definitive diagnosis. Similarly, certain chromosomal abnormalities have been found to be pathognomonic in certain hematological disorders. In general among all the cancers, a pathogenetic involvement of chromosomal abnormalities is well characterized in hematological malignancies compared to solid tumors. In cancer, both balanced and unbalanced aberrations have been described and it is proposed that these two types could be functionally very distinct, in that primary directly disease-related changes might be balanced and the secondary tumor progression-related changes might be unbalanced (67). It appears that, today, knowledge of more than 600 neoplasia-related balanced chromosomal alterations exists, most of which, as mentioned earlier come from hematological malignancies (63).
A direct link of a cytogenetic abnormality with specific tumor pathobiology has served to be pathognomonic in the diagnosis of some malignancies. As indicated by Mitelman (63), indeed many of these typical aberrations result in a chimeric abnormal protein. The well-known examples of such chimeric proteins are constitutively active tyrosine kinase activities resulting from t(9;22) in CML(bcr-abl), or t(5;12) with the fusion of TEL-PDGFR in chronic myelomonocytic leukemia (CMML), or a transcription factor AML1/ETO resulting from t(8;21) in acute myeloid leukemia (AML) (63,64). On the other hand, some aberrations lead to undesirable and untimely activation of a protein, such as bcl2, being turned on in follicular B-cell lymphoma as a result of t(14;18). Bcl-2, being antiapoptotic, causes a lymphoaccumulation disorder that eventually culminates into frank malignancy. Alternatively, sometimes it is the inactivation of protein that could result in increased survival and/or proliferation of cancer cells. In APL, t(15;17) inactivates the PML protein on chromosome 15, leading to a loss of its proapoptotic activity. Simultaneously, its fusion partner on chromosome 17, the retinoic acid receptor a (RARa) is rendered dysfunctional, causing a differentiation block in myeloid cells (65). A direct pathological bearing of the cytogenetic aberrations in these disorders warrants their assessment by sensitive molecular techniques like FISH as a diagnostic aid. In addition, rapidity of FISH testing also would serve greatly for diagnostic purposes. A confirmation of the adherence of the abnormality to the expected chromosome, though, needs to be supported by the use of appropriate built-in chromosome localizing controls. The cytogenetic analysis would be invaluable especially when the biopsy histology is inconclusive such as the case reported for low-grade astrocytomas, which sometimes could pose a challenge for differential diagnosis from reactive gliosis. In this study, albeit anecdotal, chromosomal aneusomy was diagnostic in 30% inconclusive cases that were reported malignant only in a follow-up biopsy (66).
7.2. RELATIONSHIP OF CHROMOSMAL ABNORMALITIES WITH DISEASE PROGNOSIS It is well established that the clinical manifests of a malignant disease vary tremendously in individual patients. Largely, such variability stems from a biological genotype of the malignant clone that determines the kinetics of disease progression and overall disease prognosis in an individual patient. Among the established biological clonal identifiers, cytogenetic abnormality is perhaps the most widely used marker besides the cell surface markers. Routine technique employed for this purpose is FISH. In myelodysplastic syndromes (MDSs), nearly 50% of the patients demonstrate single or complex cytogenetic abnormalities with frequent involvement of 5q- or -5, +8, -7, and 20q-. With the exception of 5q-, which shows low rates of leukemic transformation and better survival, all other abnormalities are known for poor survival. Also, it appears that an evolving complex kary-otype corresponds with rapid leukemic transformation and poor survival (67). Interestingly, although whole or partial chromosome losses or gains have been described for MDS, translocations have not been frequent, except for t(5;12) described recently in CMML, as mentioned earlier. However, often the metaphase analysis would note the presence of a marker chromosome. As shown in Fig. 3, the application of M-FISH certainly now helps in identifying the origin of such marker chromosome, further unfolding the biology of MDS. Because of such a cogent relationship of cytogenetic aberrations with leukemic trasformation and overall survival, cytogenetics are one of the three criteria considered in the recently proposed International Prognostic Scoring System (IPSS) for MDS risk classification (68). In a recent report, similar differences in survival rates have been shown for distinct cytogenetic alterations in multiple myeloma in a large series of patients (n = 351) studied by the Eastern Cooperative Oncology Group (ECOG) of the United States (69). In contrast to MDS, translocations involving 14q32 are very common in multiple myeloma. A remarkable drop in survival rates were noted in patients with t(4;14)(p16;q32), t(14;16)(q32;q23), and -17p13. On the other hand, barring the -13q14 cases (intermediate group), all other cytogenetic abnormalities including t(11;14)(q13;q32) demonstrated nearly two fold higher survival rates and better prognosis. (69) Among solid tumors, a direct association has been shown between HER2/neu gene amplification on chromosome 17q and high rates of disease relapse and poor survival in node-positive metastatic breast cancer (for review, see ref. 70)
7.3. CORRELATION OF KARYOTYPE WITH RESPONSE TO THERAPY HER2/neu amplification indeed seems to be a significant determinant in the response to different therapeutic regimens for breast cancer (70). Generally, in both node-negative and node-positive breast cancer, HER2/neu amplification relates to a resistance to preoperative chemotherapy with cyclo-phosphamide, methotrexate, and 5-fluorouracil (CMF). In contrast, the HER2/neu-amplified tumors could be more sensitive to the anthracyclin-based therapy regimens (doxorubicin + cyclophosphamide). Further, in a prolonged (20 yr) follow-up study on node-negative breast cancer cases treated with tamox-ifen as adjuvant therapy in a randomized trial, HER2/neu amplification correlated with low estrogen receptor status, larger tumor size, and lower overall as well as disease-free survival (71,72). The most significant implication of HER2/neu amplification has been demonstrated recently in the treatment of node-positive metastatic breast cancer with Herceptin®, a specific humanized antibody to HER2/neu protein receptor, as adjuvant chemotherapy. A remarkably higher overall response rate and disease-free survival was shown with Herceptin in combination with chemotherapy in cases of HER2/neu-amplified status (73).
Interestingly, in APL, the patients showing typical t(15;17) respond very well to all-trans retinoic acid (ATRA) differentiation therapy. However, the RARa at 17q12-21 in about 10% of APL cases shows variable partners (viz. promyelocytic leukemia zinc finger [PLZF] at 11q23, or nucleophosmin gene at 5q35, or nuclear mitotic apparatus gene at 11q13, or STAT5b on chromosome 17). It appears that the cases with t(11;17)(q23;q21) resulting in PLZF/RARa fusion do not respond to ATRA therapy as well unless combined with granulocyte-colony-stimulating factor (G-CSF) or hydroxyurea. The PLZF/RARa also do not seem to respond to arsenic trioxide (for a review of APL therapy, see ref. 65). Clearly, cytogenetic analysis could guide the therapy selection at least for some cancers at this time.
7.4. CYTOGENETIC ANALYSIS IN DISEASE MONITORING Detection of a cytogenetically marked clone in a disease is of significant value in monitoring the effects of therapy, residual disease, and relapse. The major aim in such attempts is to determine biologic remission and to detect relapse in advance, thus bringing in a precision of much sought after tailor-made therapy. Both t(9;22) resulting in bcr-abl chimera in CML as well as t(15;17) causing PML-RARa fusion in APL have been successfully used to monitor effects of recently developed imatinib mesylate (Gleevec) therapy for CML and, as mentioned earlier, ATRA therapy for APL. As reviewed elegantly by Grimwade and Coco (65), it was apparent from the UK Medical Research Council ATRA trial in APL, that persistence of PML-RARa at the end of the third course of therapy led to a poorer 5yr survival and showed almost twice as rapid rates of disease relapse in 5 yr. Furthermore, it has been shown that a pre-emptive therapy at the point of molecular relapse detected during postconsolidation therapy monitoring significantly improved a long-term survival than commencing therapy at hematological relapse, which was seen to occur within a range of 1-14 mo. The value of cytogenetic analysis in predicting the recurrence of disease has been seen in solid tumors as well. In a recent multicenter trial detecting aneusomy of chromosomes 3, 7, and 17 and loss of 9p21 in a single FISH assay in patients with prior diagnosis of transitional cell carcinoma of bladder, it was observed that finding cytogenetically abnormal transitional cells in voided urine corresponded to significantly high rates of recurrent disease. Interestingly, the predictive value of FISH in this respect was found to be superior to the most currently available other tests, including cytology (74).
The cytogenetic response criteria are most well defined for CML based on the proportion of reduction in the size of the abnormal clone with t(9;22). A complete response relates to undetectable abnormality, a partial response to 1-34% cells with t(9;22), minor response to 34-94% detection, and no response to a persistence of over 95% abnormal cells following therapy (75). A cytogenetic response to interferon-a treatment was shown to correlate with prolonged survival (76), and FISH appears to be the assay of choice strongly recommended for monitoring such responses to CML therapies (75). In the phase I trial of a new therapeutic agent for CML, a tyrosine kinase inhibitor called STI 571, imatinib mesylate (Gleevec), 33% of the responding patients showed cytogenetic response evaluated by FISH (77). However, in young CML patients with bone marrow transplant as the front-line therapy, highly sensitive RT-PCR has been recommended for posttransplant disease monitoring despite some concerns of false-positivity (75).
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