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The past 20 years have witnessed extraordinary advances in the field of cytogenetics, with the discovery that a multitude of neoplasms is characterized by identifiable chromosomal changes. The ability of Cytogenetics to aid in the identification and precise classification of a variety of neoplasms has not gone unnoticed by Cytology. In particular, Cytology has recognized Cytogenetics as a welcome companion in the evaluation of soft tissue tumors, lymphomas, renal and urothelial tumors, and mesothelioma. This relationship requires a good understanding of the proper handling of specimens for optimal evaluation by Cytogenetics. The marriage of Cytology and Cytogenetics will likely grow stronger as more solid tumors (eg, salivary gland neoplasms) are discovered that harbor characteristic chromosomal abnormalities. Cancer (Cancer Cytopathol) 2013;121:279–90. © 2013 American Cancer Society.
“Dearly Beloved, we are gathered here to join in happy matrimony…” Cytology and Cytogenetics?! Could anyone imagine a more unlikely pair? Cytology: mature, established, and unpretentious; and Cytogenetics: young, hip, flirtatious. Yet friends have been noticing the growing attraction between the two in recent years; therefore, few were truly surprised when the engagement was announced. To understand the growing connection, it helps to understand the individuals a bit better.
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In the past 2 decades, Cytogenetics, devoted to the study of chromosomes, has witnessed remarkable development with significant relevance to the science and clinical practice of oncology. For the hematologist, recurring chromosomal abnormalities are important diagnostic and prognostic indicators. The identification of specific chromosomal changes in solid tumors lagged behind hematologic malignancies for technical reasons, mostly related to obtaining sufficient representative tumor cells for study, but recent years have witnessed burgeoning discovery. Beyond the clinical relevance of cytogenetic studies for diagnosis, prognosis, and treatment, Cytogenetics is used in gene mapping and gene identification and has led to the cloning of genes responsible for specific diseases.
Currently, various approaches are used to provide evidence of chromosomal and molecular aberrations in malignancies: cytogenetic analysis, fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), and DNA sequencing. Immunohistochemistry also can indirectly detect molecular changes, for example immunostain TFE3 detects any Xp11.2 rearrangement (as in alveolar soft part sarcoma and Xp11.2 renal cell carcinoma). Currently, there is no single diagnostic approach capable of demonstrating all types of genetic events.
Conventional cytogenetic analysis depends on the success of tumor cell growth in culture and the quality of metaphase cell preparations. It cannot be performed on archival/fixed specimens. It requires skilled personnel and remains time-consuming, even with automated karyotyping systems.
Molecular cytogenetic techniques like FISH play a critical role in the diagnostic (eg, detection of submicroscopic chromosomal aberrations) and research arenas (eg, localization and mapping of chromosomal breakpoints and candidate disease genes). A major advantage of FISH is that specific DNA target sequences can be detected in the nuclei of nondividing (interphase) cells from fresh or fixed samples. Thus, this procedure can provide results when the tissue is insufficient for cytogenetic analysis (eg, some fine-needle aspiration specimens), when conventional cytogenetic analysis has failed to yield metaphase cells, when so-called cryptic rearrangements (those that are too small to be observed with conventional cytogenetics) are present, or when only paraffin-embedded tissue is available. Chromosome-specific probes most commonly are labeled with fluorescent dyes and are used to observe the chromosome or chromosomal sequence of interest. FISH is typically an overnight procedure, depending on the probes and the microscope used to observe the signal(s). It is important to note that FISH is a targeted approach that requires a priori knowledge of a suspected aberration. Probes that are frequently used in oncology include 1) sequence-specific or loci-specific probes, such as those flanking or spanning a breakpoint to detect chromosome rearrangements (eg, translocations, deletions); and 2) chromosome-specific centromeric probes (alpha-beta satellite sequences), which detect aneuploidy. The major limitation is the paucity of commercially available probes. (Clinical laboratories can and do develop “home-brew” reagents for special studies.) There are other types of probes, like whole chromosome painted probes and 24-color FISH (special karyotyping [SKY], multiplex FISH [M-FISH], and cross-species color banding [RX-FISH]), all of which are used only on metaphase cells, but the information obtained is of no significant diagnostic use. These probes are not helpful in the analysis of interphase cells, because the signal domains are so large and diffuse.
Comparative genomic hybridization (CGH) was developed originally to define regions of gains or losses across all chromosomal regions and does not require viable or dividing tumor cells. More recently, conventional CGH has been replaced by array CGH, in which tumor DNA is hybridized instead to high-density arrays of probes that cover the entire genome. The application of conventional and array-based CGH to cancer diagnosis has been limited by the inability of CGH to detect balanced translocations and its tendency to underestimate low-level copy numbers.
Over more than 5 decades, Cytology has become a simple yet powerful technique that allows a safe, accurate, rapid, and economic morphologic assessment of cells from virtually any body site. Before Cytogenetics arrived on the scene, other ancillary tools like microbiology, flow cytometry, and immunocytochemistry had already established a supplementary role to cytomorphologic evaluation. In recent years, genetic techniques like karyotype analysis, FISH, and PCR gained the attention of Cytology.[2, 3] Cytologic specimens, it turns out, are ideal for cytogenetic studies, because a large proportion of the cytologic specimen consists of disaggregated cells. No mechanical mincing is necessary to obtain free cells. Cytologic specimens can be processed for karyotypic analysis and/or FISH at multiple steps in their preparation. Depending on the technique used, the substrate for Cytogenetics can be the fresh cell suspension, which is obtained by rinsing the biopsy needle with sterile saline or a culture medium, cytocentrifuge preparations, thin-layer slides, and cell blocks (Fig. 1).
Figure 1. Triaging cytology for cytogenetics is illustrated. The cytologic sample can be apportioned for cytogenetic analysis at several different points, depending on the cytogenetic technique used. A fresh, unfixed sample is needed for a karyotype; this decision is best made at the time of on-site adequacy evaluation. Either fresh or fixed cells can be used for fluorescence in situ hybridization (FISH).
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What Makes the Marriage Work?
- Karyotype analysis needs fresh cells, eg obtained by centrifuging the needle-rinse fluid, placing the sediment in tissue culture medium for short-term culture.[4, 5]
- FISH can be performed on a formalin-fixed cell pellet, direct smear, cytocentrifuge preparation, and thin-layer preparation. Routine cytology preparations are ideal for FISH, because the preparations leave single cells intact. FISH can be performed on sections from a paraffin-embedded “cell block” that is created from the congealed sediment after formalin fixation.
A conventional karyotype is useful because it provides an overview of the entire genome.
- A technically unsuccessful karyotype is nearly always because of an inadequate (sparsely cellular and/or not viable) sample. It is important to make sure there is enough material in the needle rinse.
- A normal karyotype does not exclude malignancy. It may result from the growth of normal cells (eg, fibroblasts), because the tumor cells were not sampled or the cells were not viable.
FISH analysis detects specific chromosomal abnormalities, including subtle or cryptic rearrangements, and small deletions and amplifications in interphase nuclei.
- FISH requires a priori knowledge of a suspected aberration.
- Applicability of FISH depends of the availability of probes.
- A normal FISH result is uninformative, because: 1) there may have been too few neoplastic cells in the sample; and 2) the tumor cells do not have the rearrangement you are probing for (they may have another rearrangement which cannot be detected by the probe used).
Specific genetic abnormalities have been described in a large number of tumors.[7, 8] Cytogenetic analysis is particularly useful in certain tumors for which a large number of specific probes are available. These include soft tissue tumors, lymphomas, renal tumors, mesothelioma, and urothelial cancers. A shared interest in these entities, friends say, is what brought Cytology and Cytogenetics together.
Soft Tissue Tumors
The family of soft tissue tumors is very heterogeneous, and diagnosis by fine-needle aspiration cytology alone is especially challenging. Cytologic evaluation begins by identifying the principal cytomorphologic pattern: adipocytic, myxoid, spindle cell, epithelioid, small round cell, and pleomorphic. Cytogenetics is a useful adjunct to cytology if applied properly, because large numbers of soft tissue tumors have specific genetic aberrations.[7, 10] Specific genes have been implicated in the most frequently encountered translocations, facilitating their detection by FISH analysis using commercially available probes (Table 1).
Table 1. Soft Tissue Tumors, Their Associated Chromosomal Changes, and FISH Probes
|Tumor||Cytogenetic Aberration(s)||FISH Probe(s)|
|Angiofibroma of the soft tissue||t(5;8)(p15;q13)||NA|
|Angiomatoid fibrous histiocytoma||t(12;16)(q13;p11)||FUS|
|Alveolar soft part sarcoma||der(X)t(X;17)(p11;q25)||TFE3|
|Clear cell sarcoma||t(12;22)(q13;q12)||EWSR1|
|Dermatofibrosarcoma protuberans/giant cell fibroblastoma||r(17;22)(q21;q13)/t(17;22)(q21;q13)||PDGFB|
|Desmoid fibromatosis||Trisomy 8 and 20||CEP8/CEP20|
|Desmoplastic small round cell tumor||t(11;22)(p13;q12)||EWSR1|
|Ewing sarcoma/primitive neuroectodermal tumor||t(11;22)(q24;q12)||EWSR1|
|Extraskeletal myxoid chondrosarcoma||t(9;22)(q22;q12)||EWSR1|
|Inflammatory myofibroblastic tumor||2p23 rearrangement||ALK|
|Lipoblastoma||8q12 rearrangement or polysomy 8||NA|
|Lipoma, ordinary||12q14.3 rearrangement||HMGA2|
|Lipoma, spindle cell and pleomorphic||Deletions of 13q and 16q||NA|
|Liposarcoma, well differentiated/dedifferentiated||Ring/giant marker chromosomes from chromosome 12q||HMGA2/MDM2|
|Liposarcoma, myxoid/round cell||t(12;16)(q13;p11)||DDIT3/FUS|
|Low-grade fibromyxoid sarcoma||t(7;16)(q34;p11)||FUS|
|Myoeptheilioma, soft tissue||t(19;22)(q13;q12)||EWSR1|
|Myxoinflammatory fibroblastic sarcoma||der(10)t((1;10)(p22;q24)||NA|
| ||t(1;13)(p36;q14), double minutes||FOXO1|
| ||2q35 rearrangement||NA|
|Rhabdomyosarcoma, embryonal||Trisomies 2q, 8, and 20||NA|
| ||Loss of heterozygosity at 11p15|| |
|Schwannoma||Deletion of 22q||NA|
|Tenosynovial giant cell tumor||t(1;2)(p13;q37)||NA|
Most adipocytic tumors, both benign and malignant, are characterized by specific chromosomal abnormalities (Table 1). Most benign adipocytic tumors, like spindle cell/pleomorphic lipoma and hibernoma, can be diagnosed based on clinical and cytomorphologic findings, but many liposarcomas need ancillary studies to confirm the diagnosis. Conventional cytogenetic analysis is often noncontributory, however, because a normal karyotype is usually obtained due to the outgrowth of normal fibroblasts over tumor cells, particularly for the well differentiated lipogenic neoplasms. FISH, conversely, can provide conclusive evidence for well differentiated liposarcoma/atypical lipomatous tumor (WDLPS/ALT), dedifferentiated liposarcoma (DDLPS), and myxoid/round cell liposarcoma (see also Myxoid tumors, below). WDLPS/ALT and DDLPS are characterized by the presence of supernumerary ring and/or giant chromosomes, with amplifications of 12q13-15 involving genes including MDM2 and cyclin-dependent kinase 4 (CDK4). Although demonstration of MDM2 and CDK4 protein overexpression by immunohistochemistry is helpful to distinguish WDLPS/ALT from its benign mimics and DDLPS from other high-grade sarcomas, FISH that reveals MDM2 amplification is more sensitive and specific in establishing the diagnosis of WDLPS/ALT and DDLPS.[12, 13]
Precisely classifying myxoid soft tissue tumors is often difficult, because many yield hypocellular smears with overlapping, nonspecific cytomorphology, and they usually have no distinctive immunoprofile. Specific chromosomal translocations, which can be detected by FISH, however, can help to identify a subset of myxoid tumors, including myxoid liposarcoma, low-grade fibromyxoid sarcoma, and extraskeletal myxoid chondrosarcoma.[14, 15] In contrast to myxofibrosarcoma, a superficially located sarcoma with pleomorphic neoplastic cells and myxoid stroma, most myxoid tumors with specific translocations are deep-seated and monotonous/uniform in appearance, useful clues to the differential diagnosis that should serve as an indication for cytogenetic studies.
Spindle cell tumors
“Spindle cell” soft tissue tumors are a large group of benign and malignant mesenchymal tumors characterized by fascicular growth pattern. The diagnosis of these tumors is usually straightforward based on the integration of clinical data, cytomorphology, and a distinctive immunoprofile. Examples include desmoid fibromatosis, schwannoma, leiomyoma/leiomyosarcoma, and gastrointestinal stromal tumor. Specific chromosomal translocations/FISH tests can help to confirm the diagnosis of synovial sarcoma, dermatofibrosarcoma protuberans (Fig. 2), inflammatory myofibroblastic tumor, and dedifferentiated liposarcoma, particularly when these tumors occur at an unusual location and/or have unusual morphology and an inconclusive immunoprofile.
Figure 2. (A) In dermatofibrosarcoma protuberans, a fine-needle aspiration smear reveals scattered, relatively monomorphic spindle cells with myxoid stroma (Romanowsky stain; original magnification, ×400). The immunoprofile was relatively nonspecific. (B) The karyotype obtained from the needle-rinse sample reveals numerous aberrations (open arrows). The hallmark of this tumor is represented by the derivative chromosome 22 (solid arrows), which contains additional genetic material from the long arm of chromosome 17.
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Epithelioid tumors include the epithelioid variants of many benign and malignant soft tissue neoplasms (eg, epithelioid schwannoma, epithelioid angiosarcoma) as well as specific, well defined entities like epithelioid sarcoma, clear cell sarcoma, and alveolar soft part sarcoma. Immunohistochemical studies play a decisive role in establishing the diagnosis of most epithelioid soft tissue tumors as well as excluding metastatic carcinoma, melanoma, and large cell lymphomas. Specific chromosomal translocations/FISH tests can help to establish the diagnosis of some clinically, morphologically, and genetically well defined tumors. For example, the demonstration of an EWSR1 rearrangement by FISH is essential to separate clear cell sarcoma from metastatic melanoma, because these are indistinguishable by cytomorphology and immunoprofile.
Small round blue cell tumors
Small round blue cell tumors comprise a group of malignant neoplasms that occur mainly in children and young adults. Most of them are clinically aggressive and responsive to chemoradiation therapy; therefore, a definitive, specific diagnosis made in a timely fashion is crucial for proper clinical management. The marriage between Cytology and Cytogenetics makes perfect sense in this regard, because fine-needle aspiration biopsy of this group of tumors usually yields cellular smears and sufficient material for both immunohistochemistry and cytogenetic studies. FISH on unstained Cytospin slides can be performed and reported within 24 hours (faster than on nuclei extracted from paraffin-embedded tissue). In addition, conventional karyotyping is often successful and diagnostically meaningful. The detection of cytogenetic alterations by karyotype/FISH analysis can aid in providing not only a specific diagnosis for tumors like Ewing sarcoma and desmoplastic small round cell tumor, but also prognostic information for tumors like alveolar rhabdomyosarcoma[17, 18] (Table 2, Fig. 3).
Table 2. Small Round Blue Cell Tumors, Their Associated Chromosomal Changes, and FISH Probes
|Tumor(s)||Chromosomal Aberration(s)||FISH Probe(s)|
|Ewing sarcoma||22q12 rearrangement||EWSR1|
|Ewing-like sarcoma||t(4;19)(q3;q13.1); t(10;19)(q26.3;q13.1); inv(X)(p11.4p11.22)||NA|
|Rhabdomyosarcoma (alveolar)||t(2;13)(q35;q14); t(1;13)(q36;q14)||FOXO1|
|Neuroblastoma||del(1p); dmin, HSR||LSI1p36/LSI1q15; MYCN|
|Desmoplastic small round cell tumor||t(11;22)(p13;q12)||EWSR1|
|Non-Hodgkin lymphoma||Various translocations||BCL2-IGH, MYC, BCL6, MALT1|
Figure 3. (A) In Ewing sarcoma, a fine-needle aspiration smear reveals numerous, uniform, small cells with scant cytoplasm (Romanowsky stain; original magnification, ×400). An immunostain for O13 (Ewing sarcoma monoclonal antibody) was only focally positive. (B) Fluorescence in situ hybridization (FISH) on this nucleus reveals an (EWSR1) rearrangement on 22q12, as indicated by the split-apart centromeric (c,red) and telomeric (t, green) probes. (A normal Ewing locus, with adjacent red and green probes, also is present.)
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Most pleomorphic tumors have a complex and nonspecific karyotype; therefore, cytogenetic studies, especially a conventional karyotype, would provide little diagnostic information. One of the exceptions is DDLPS, which can have a complex karyotype in addition to the characteristic supernumerary ring and/or giant chromosomes.
A precise lymphoma diagnosis involves the integration of clinical information, morphology, immunohistochemistry, cytogenetics, and molecular biology. FISH analysis can be performed on cytologic material, allowing maximum use of small diagnostic samples. It requires a priori knowledge of a suspected aberration, however, and depends on the availability of probes[3, 19, 20] (Table 3). Cytogenetic studies, especially FISH, are particularly helpful in distinguishing between diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma (BL), and the so-called “double-hit” or “triple-hit” lymphoma, named “B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL” in the 2008 World Health Organization classification (Fig. 4).4
Table 3. Lymphomas, Their Associated Chromosomal Changes, and FISH Probes
|Lymphoma||Chromosomal Aberration(s)||FISH Probe(s)|
|Anaplastic large cell lymphoma||2p23 abnormalities||ALK|
|Hepatosplenic T-cell lymphoma||i(7)(q10)||D7S486, 7cen|
|Follicular lymphoma and diffuse large B-cell lymphoma||t(14;18)(q32;q21) and 3q37 abnormalities||IGH-BCL2 and BCL6|
|Burkitt lymphoma||t(8;14)(q24;q32); t(8;22)(q24;q11); t(2;8)(p12;q24)||MYC|
|Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue||t(11;18)(q21;q21); t(14;18)(q32;q21)||MALT1|
|Mantle cell lymphoma||t(11;14)(q13;q32)||CCND1/IGH|
|Splenic marginal zone lymphoma||del(7)(q21q31)||D7S486, 7cen|
|Double/triple hit lymphoma||8q24 rearrangement and t(14;18)(q32;q21) or 3q27 rearrangement||MYC, IGH-BCL2, BCL6|
Table 4. Kidney Tumors, Their Associated Chromosomal Changes, and FISH Probes
|Tumor||Chromosomal Aberration(s)||FISH Probe(s)|
|Clear cell RCC||Loss of 3p||3pter/3qter|
|Xp11.2 translocation-associated RCC||t(X;1)(p11.2;q21); t(X;1)(p11.2;p34); inv(X)(p11.2q12); t(X;17)(p11.2;q25.3); t(X;17)(p11.2;q23)||TFE3|
|Papillary RCC||Combination of trisomies 3, 7, 12, 16, 17, and 20||1cen, 7cen, 17cen|
|Chromophobe RCC||Combination of monosomies 1, 2, 3, 6, 10, 13, 17, and 21||1cen, 7cen, 17cen|
|Clear cell sarcoma of kidney||t(10;17)(q22;p13)||YWHAE|
|Congenital mesoblastic nephroma||t(12;15)(p13;q26)||ETV6|
|Oncocytoma||11q13 rearrangement: −Y or −X,−1||NA|
Figure 4. (A) In double-hit lymphoma, a fine-needle aspiration smear reveals intermediate to large lymphoid cells with a background of lymphoglandular bodies (Romanowsky stain; original magnification, ×400). Irregular nuclear contours, deeply cleaved nuclei, and occasional small cytoplasmic vacuoles are common. (B) This composite image reveals the results of sequential fluorescence in situ hybridization on Cytospin slides. The cell on the left reveals a MYC rearrangement, as demonstrated by the split between the centromeric (c, red) and the telomeric (t, green) probes. The cell on the right reveals a t(14;18) translocation, as demonstrated by the coming together of both red and green probe pairs to create 2 yellow signals (corresponding to an IGH-BCL2 fusion.
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Renal cell carcinoma (RCC) represents a group of clinical and genetically diverse diseases (Table 4). Specific chromosomal abnormalities have been described in clear cell RCC (deletion of the short arm of chromosome 3 [3p]), papillary RCC (a combination of trisomies) (Fig. 5), and chromophobe RCC (a combination of monosomies). In benign tumors like angiomyolipoma, metanephric adenoma, and oncocytoma, these abnormalities are not observed. Subtyping an RCC is of clinical relevance, because it stratifies the tumor into more (eg, clear cell RCC) or less (eg, papillary RCC) aggressive tumors, and immunohistochemistry is not always reliable for these distinctions. For example, FISH with the TFE3 probe is more precise than immunohistochemistry for the diagnosis of Xp11.2-translocation RCC.
Figure 5. (A) In papillary renal cell carcinoma, fine-needle aspiration smears reveals a cellular sample comprised of cuboidal cells with focal anisonucleosis (Romanowsky stain; original magnification, ×200). (B) A karyotype obtained from fresh needle-rinse fluid reveals trisomies of chromosomes 7 and 17, which are characteristic of papillary renal cell carcinoma.
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A cocktail of centromeric probes to chromosomes 1, 7, and 17 is useful to screen for papillary and chromophobe RCC in 1 assay. Until recently, there were no useful FISH probes for the rapid detection of clear cell RCC, because the multiple deletions involving 3p encompass a relatively large portion of the chromosome, and identification of a single critical region has proven difficult. Secondary to investigations of copy number alterations in clear cell RCCs (in both primary tumors and cell lines), however, it was noted that the subtelomeric region of the 3p terminal (3pter) was the most frequently deleted region. Therefore, a clinically useful FISH test for clear cell RCC has been developed using the subtelomeric 3pter and 3qter probes.
The differential diagnosis of benign and malignant mesothelial proliferations can be difficult using cytology alone (Fig. 6A). Chromosomal analysis of exfoliated cells from pleural and other body cavity fluids can help in this distinction if they demonstrate consistent chromosomal changes, because consistent changes are not observed in benign/reactive mesothelial proliferations. The characteristic chromosomal aberrations of malignant mesothelioma have been described. Although no specific chromosomal abnormalities distinguish mesothelioma from other malignancies, chromosome regions that are consistently involved in mesothelioma include 1p, 3p, 6q, 9p, and 22q, suggesting the involvement of tumor suppressor genes. Because a normal or unsuccessful karyotype can result from the lack of proliferation of malignant mesothelial cells, a FISH test for deletions of 9p21 and 22q can be helpful (Fig. 6B).
Figure 6. (A) In malignant mesothelioma, a liquid-based preparation of pleural fluid reveals numerous isolated, variably sized mesothelial cells. A definitive diagnosis of malignancy is not possible by cytology alone in this case (Papanicolaou stain; original magnification, ×400). (B) This fluorescence in situ hybridization image includes a normal cell on the right and a malignant mesothelioma cell on the left. Both cells have 2 green signals, corresponding to centromeric probes for chromosome 9 (CEP9), but only 1 red, locus-specific probe for 9p21, indicating a heterozygous deletion in the malignant cell.
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Historically, the difficulty in culturing bladder tumors has adversely impacted the cytogenetic study of these specimens. Although no single cytogenetic anomaly is considered diagnostic of transitional cell carcinoma, a combination of chromosomal aberrations can be exploited for diagnostic purposes. Early publications[26, 27] suggested the application of molecular cytogenetics to specimens obtained through relatively noninvasive methods (eg, voided urine and bladder washings). Subsequently, a commercial multitarget FISH assay was developed with several loci interrogated simultaneously—trisomy (or tetrasomy) for chromosomes 3, 7, and 17, along with loss of 9p—and is now in fairly widespread use (UroVysion; Abbott Molecular, Des Plaines, IL). Several comparative studies have demonstrated that the sensitivity of FISH is superior to that of Cytology alone.
According to published reports, the happy couple has settled into a mutually satisfying relationship, each helping to complete the other. The future holds additional opportunities for spousal collaboration: many genetic events, originally identified in hematologic and mesenchymal lesions, are being discovered in a variety of carcinomas as well, particularly salivary gland carcinomas. No doubt we'll be hearing more from Cytology and Cytogenetics.
Who ever doubted that opposites attract?