Dual-color fluorescence in situ hybridization (FISH) image illustrating MDM2 gene amplification (red) with the chromosome 12 centromere reference probe CEP12 (green) in a soft tissue lipomatous tumor. Nuclei display multiple red signals indicating MDM2 copy number gain on chromosome 12q15, characteristic of atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) and its dedifferentiated counterpart. The green CEP12 signals provide a stable chromosomal reference, enabling robust interpretation of amplification relative to centromeric control. This pattern supports the diagnosis of liposarcoma rather than benign lipomatous lesions, which typically lack MDM2 amplification. Compared with MDM2 immunohistochemistry, FISH offers higher sensitivity and specificity, reducing false positives that can occur with protein-based assays. A small minority of spindle cell or pleomorphic lipomas may exhibit MDM2 positivity due to polysomy of 12q; however, the amplification signal in ALT/WDL is usually robust and well‑defined. Clinically, MDM2 FISH is particularly valuable in older patients (>50 years), in deep-seated or large lesions (>10 cm), retroperitoneal or intraabdominal lipomatous tumors lacking overt atypia, and in recurrent tumors where accurate classification guides management. This image illustrates the decisive genetic alteration underlying a liposarcoma spectrum and informs diagnostic, prognostic, and therapeutic considerations. Credit: Dr. Runjan Chetty.

Imaging modality and technique: Fluorescence in situ hybridization (FISH) on interphase nuclei reveals MDM2 gene amplification (red) with a centromeric 12 reference signal CEP12 (green). In this case, the dual-color assay demonstrates a focal, high MDM2-to-CEP12 signal ratio consistent with amplification rather than mere polysomy; controls show intact reference signals. The specimen derives from soft tissue lipomatous tumor, most compatible with atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL). A normal adipocytic lipoma would typically lack MDM2 amplification. Pathology: MDM2 amplification is a defining molecular hallmark that helps separate ALT/WDL and dedifferentiated liposarcoma from benign lipomatous tumors and other soft tissue sarcomas. Signal pattern: multiple red MDM2 signals co-localize with blue/green nuclei, with green CEP12 signals serving as a centromeric control. The image context emphasizes the diagnostic significance of MDM2 FISH in older patients (>50 years) with deep-seated, large, or recurrent lipomatous lesions or retroperitoneal tumors without peculiar cytology. Clinical relevance: when MDM2 amplification is demonstrated, management typically involves wide surgical excision with clear margins and surveillance for recurrence, since ALT/WDL may recur and DDLS risk rises with dedifferentiation. This assay option enhances diagnostic confidence and guides therapy decisions. Note: image credits and caption indicate courtesy of Dr. Runjan Chetty. used with permission.

Fluorescence in situ hybridization (FISH) image showing MYCN gene status in neuroblastoma cells. The left panel depicts tumor cells with multiple green signals, indicating MYCN amplification and elevated copy number relative to normal. The right panel shows a control with normal diploid signals to demonstrate baseline copy number. This dual-color DNA FISH assay uses MYCN-specific probes (green) and a reference probe (red) for copy-number assessment. The specimen comprises neuroblastic tumor cells derived from a suspected adrenal or sympathetic chain lesion; histology is frequently undifferentiated or poorly differentiated with high mitotic activity and prominent nucleoli in MYCN-amplified tumors. MYCN amplification, defined as greater than 10 copies per diploid genome, is detectable by FISH, PCR, or immunohistochemistry and correlates with rapid tumor progression and poor prognosis; it often co-occurs with 1p deletion and 17q gain. Clinically, MYCN status is a critical prognostic biomarker that informs risk stratification, treatment intensity, and surveillance in neuroblastoma. The image demonstrates how copy-number gain translates into aggressive biology and aids in distinguishing high-risk from nonamplified tumors. This information supports diagnostic confirmation, prognostication, therapeutic decision-making, and research into targeted therapies. Correlative molecular testing across specimens enhances accuracy and guides enrollment in high-risk neuroblastoma trials and informs long-term management.

Fluorescence in situ hybridization (FISH) image using a red FOXO1 region probe and a green FOXO1 region probe (break-apart configuration) was performed on interphase tumor cells from a case of alveolar rhabdomyosarcoma. The predominant pattern shows a PAX7-FOXO1 rearrangement with translocation t(1;13)(p36;q14), as indicated by separation of red and green signals at the fusion locus; amplification of the fusion gene is present, a feature more often associated with PAX7-FOXO1 than PAX3-FOXO1. The bottom cell is a normal interphase nucleus that lacks translocation; it displays two co-localized red-green signal pairs corresponding to two intact FOXO1 loci at 13q14.11. The arrows highlight the rearranged signals, consistent with oncogenic PAX7-FOXO1 fusion driving alveolar rhabdomyosarcoma biology. Observed signal patterns include break-apart separation and signal amplification in tumor cells, with normal control patterns in non-neoplastic cells. This assay provides definitive cytogenetic evidence of the FOXO1 rearrangement, correlating with molecular subtype, prognosis, and potential therapeutic stratification. These findings support a diagnosis of alveolar rhabdomyosarcoma and underscore the clinical value of PAX7-FOXO1 testing for diagnostic confirmation, risk stratification, and targeted therapy planning. This image is annotated with arrows and uses red-green color coding to distinguish rearranged from non-rearranged loci and to illustrate interphase chromosomal dynamics for education.

This medical diagram illustrates the molecular mechanism of a sequential Brightfield Double In Situ Hybridization (BDISH) method for detecting HER2 gene amplification and Chromosome 17 centromere (CEN17) status. The process is divided into two phases. Phase 1 (left) details HER2 gene detection: a DNP-labeled HER2 DNA probe hybridizes to the target gene, followed by binding of a Rabbit Anti-DNP primary antibody. A secondary HRP-conjugated Goat Anti-Rabbit antibody then facilitates a silver precipitation reaction (using silver acetate, hydroquinone, and H2O2), resulting in a black/grey signal. Phase 2 (right) depicts CEN17 detection on the same chromosome: a DNP-labeled CEN17 oligoprobe hybridizes to the centromere, followed by the same Rabbit Anti-DNP primary antibody. A different secondary antibody, AP-conjugated Goat Anti-Rabbit, is applied to catalyze a Fast Red and Naphthol Phosphate reaction, producing a red/pink signal. This dual-color sequential technique allows for simultaneous visual assessment of gene-to-chromosome ratios under a standard brightfield microscope, which is critical for breast cancer diagnostics and HER2 status determination.

Dual-color fluorescence in situ hybridization (FISH) image illustrating MDM2 gene amplification (red) with the chromosome 12 centromere reference probe CEP12 (green) in a soft tissue lipomatous tumor. Nuclei display multiple red signals indicating MDM2 copy number gain on chromosome 12q15, characteristic of atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) and its dedifferentiated counterpart. The green CEP12 signals provide a stable chromosomal reference, enabling robust interpretation of amplification relative to centromeric control. This pattern supports the diagnosis of liposarcoma rather than benign lipomatous lesions, which typically lack MDM2 amplification. Compared with MDM2 immunohistochemistry, FISH offers higher sensitivity and specificity, reducing false positives that can occur with protein-based assays. A small minority of spindle cell or pleomorphic lipomas may exhibit MDM2 positivity due to polysomy of 12q; however, the amplification signal in ALT/WDL is usually robust and well‑defined. Clinically, MDM2 FISH is particularly valuable in older patients (>50 years), in deep-seated or large lesions (>10 cm), retroperitoneal or intraabdominal lipomatous tumors lacking overt atypia, and in recurrent tumors where accurate classification guides management. This image illustrates the decisive genetic alteration underlying a liposarcoma spectrum and informs diagnostic, prognostic, and therapeutic considerations. Credit: Dr. Runjan Chetty.

This molecular pathology image is a dual-color fluorescence in situ hybridization (FISH) study performed on FFPE soft tissue tumor tissue from a dedifferentiated liposarcoma (DLPS). The primary target probes label the MDM2 gene locus on chromosome 12q15 in red, while the reference CEP12 probe specific for the chromosome 12 centromere is shown in green. Nuclei are counterstained blue (DAPI). The image demonstrates high-level amplification of MDM2, evidenced by multiple red signals per nucleus in a background of predominantly two green CEP12 signals; some nuclei display clustered red signals, consistent with focal gene amplification typical of DLPS. The inset highlights a representative cell with prominent red MDM2 signals adjacent to green centromeric signals, illustrating signal co-localization and increased copy number. This amplification profile aligns with known oncogenic drivers of liposarcoma, particularly the 12q13-15 amplicon that also harbors CDK4 and HMGA2; GLI1 and DDIT3 may be variably overexpressed. MDM2 amplification aids diagnostic discrimination from benign lipomas and other sarcomas, and supports a diagnosis of DLPS in morphologically ambiguous cases. Potential pitfalls include signal overlap, copy-number heterogeneity, and FFPE-related artifacts; accurate interpretation requires counting multiple nuclei and cross-referencing with histology and immunohistochemistry. This image, derived from Gambella et al., demonstrates the utility and limitations of FISH for detecting MDM2 amplification in liposarcoma.

This composite educational image illustrates the clinical application of ALK-rearranged Circulating Tumor Cells (CTCs) as a biomarker for monitoring treatment response in Non-Small Cell Lung Cancer (NSCLC). Panel A presents a series of coronal CT scans of the chest and abdomen at three intervals: baseline (0 months), partial response (3 months), and progression (5 months). Red arrowheads indicate metastatic liver lesions, showing a reduction in size at 3 months followed by significant enlargement at 5 months. Below the scans, longitudinal data tracks the CTC count per 1.88 mL and identifies specific ALK rearrangement patterns. Panel B provides representative Fluorescence In Situ Hybridization (FISH) images of CTCs demonstrating different ALK break-apart signal patterns: 1F1R1G (one fusion, one orange, and one green signal), 2R2G (two orange, two green), and 1F1R (one fusion, one orange). Colored arrows (yellow for fusion, red for orange/R, and green for G) highlight these genetic signatures. The figure demonstrates the correlation between increased CTC counts, diversifying rearrangement patterns, and clinical disease progression during targeted therapy.

This composite figure presents clinical imaging and microscopy findings comparing ALK-rearranged (A-D) and ALK wildtype (E-H) lung adenocarcinoma. (A-B) Axial PET-CT fusion images show a large, hypermetabolic mass in the right lower lobe with associated FDG-avid ipsilateral mediastinal lymphadenopathy, characteristic of metastatic ALK-rearranged lung cancer. (C) Cytological preparation (May-Grünwald stain, 400x) displays clusters of atypical adenocarcinoma cells with high nuclear-to-cytoplasmic ratios. (D) ALK fluorescence in situ hybridization (FISH) demonstrates positivity via split red and green signals, indicating gene rearrangement. (E-F) Axial PET-CT and CT images of an ALK wildtype patient reveal a solitary hypermetabolic pulmonary nodule in the right upper lobe without mediastinal involvement. (G) Histopathological section (H&E stain, 200x) shows adenocarcinoma cells arranged in solid and glandular patterns. (H) ALK FISH negativity is indicated by predominantly fused (yellow) signals, representing an intact ALK gene. The collection highlights the correlation between molecular subtypes, radiographic presentation (centrality/lymphadenopathy), and diagnostic cytopathology/FISH testing.

Fluorescence in situ hybridization (FISH) on formalin-fixed paraffin-embedded tissue was performed using a dual-color SS18 (SYT) break-apart probe to detect the characteristic rearrangement seen in synovial sarcoma. The probe targets the 18q11.2 region harboring the SS18 gene. In a normal, non-rearranged cell, the green proximal and orange distal signals overlap to create a fused signal, representing two intact SS18 loci. In cells carrying the translocation t(X;18)(p11.2;q11.2), the probes separate, yielding distinct green and orange signals with loss of fusion; the image panels show examples where one fusion signal coexists with separate green and orange signals, as indicated by arrows. This break-apart pattern is pathognomonic for SS18 rearrangement when interpreted in the appropriate histologic and immunophenotypic context and supports a diagnosis of synovial sarcoma. The assay can be performed directly on FFPE material and complements routine histology and immunohistochemistry, particularly when conventional karyotyping is limited by specimen quality. Interpretation requires adequate tumor cell content and careful counting of nuclei to avoid artifactual signals. Clinically, detection of SS18 rearrangement informs differential diagnosis, influences treatment planning, and has prognostic relevance in soft tissue sarcomas with SYT-SSX fusion. This pattern complements histology and immunohistochemistry, supporting biopsy-based diagnosis and targeted systemic therapy decisions in practice.