Chromosome Enumeration Research Articles

Effect of the HER-2/CEP17 ratio in IHC 2+/FISH-amplified breast cancer on pathological complete response to neoadjuvant pertuzumab and trastuzumab treatment-a retrospective cohort study.

Human epidermal growth factor receptor-2 (HER-2) protein expression level could serve as a predictor of pathological complete response (pCR) to neoadjuvant trastuzumab-containing regimens. The aim of the present study was to evaluate whether pCR to neoadjuvant trastuzumab and pertuzumab treatment is dependent on the level of the HER-2/CEP17 (chromosome enumeration probe 17) ratio in immunohistochemistry (IHC) 2+/fluorescence in situ hybridization (FISH)-amplified breast cancer. Patients with primary IHC 2+/FISH-amplified breast cancer who underwent neoadjuvant anti-HER-2 dual-targeted therapies were retrospectively included between January 1, 2020 and May 30, 2021. The primary predictive variable was HER-2/CEP17 ratio, and the primary outcome variable was pCR. Other variables consisted of age, menopausal status, tumor-node-metastasis (TNM) stage, estrogen receptor (ER), progesterone receptor (PgR), and Ki-67. Association between clinicopathologic variables and pCR was evaluated using the chi-square test and logistic regression analysis. The median age of the patients was 51.78 years (25-67 years), and 50.7% of the patients were in the premenopausal stage. The clinical stage at diagnosis was Stage III in 38 patients (55.1%). Of all patients, 40.6% patients were estrogen receptor positive, and 75.4% patients had a Ki-67 index of ≥30%. The overall pCR (ypN0/isypN0) rate was 31.9%. Patients with HER-2/CEP17 ratio ≥6.0 had a pCR rate of 55.0%, it was statistically higher than 22.4% in patients with HER-2/CEP17 ratio <6.0. Logistic regression analysis confirmed the independent association between HER-2/CEP17 ratio and pCR (P=0.020, OR: 5.203, 95% CI: 1.302-20.783). A HER-2/CEP17 ratio ≥6.0 might be related to more achievement of pCR in the neoadjuvant anti-HER-2 dual-targeted therapies. Further studies are needed to validate the finding.

AP4 suppresses DNA damage, chromosomal instability and senescence via inducing MDC1/Mediator of DNA damage Checkpoint 1 and repressing MIR22HG/miR-22-3p

BackgroundAP4 (TFAP4) encodes a basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor and is a direct target gene of the oncogenic transcription factor c-MYC. Here, we set out to determine the relevance of AP4 in human colorectal cancer (CRC) cells.MethodsA CRISPR/Cas9 approach was employed to generate AP4-deficient CRC cell lines with inducible expression of c-MYC. Colony formation, β-gal staining, immunofluorescence, comet and homologous recombination (HR) assays and RNA-Seq analysis were used to determine the effects of AP4 inactivation. qPCR and qChIP analyses was performed to validate differentially expressed AP4 targets. Expression data from CRC cohorts was subjected to bioinformatics analyses. Immunohistochemistry was used to evaluate AP4 targets in vivo. Ap4-deficient APCmin/+ mice were analyzed to determine conservation. Immunofluorescence, chromosome and micronuclei enumeration, MTT and colony formation assays were used to determine the effects of AP4 inactivation and target gene regulation on chromosomal instability (CIN) and drug sensitivity.ResultsInactivation of AP4 in CRC cell lines resulted in increased spontaneous and c-MYC-induced DNA damage, chromosomal instability (CIN) and cellular senescence. AP4-deficient cells displayed increased expression of the long non-coding RNA MIR22HG, which encodes miR-22-3p and was directly repressed by AP4. Furthermore, Mediator of DNA damage Checkpoint 1 (MDC1), a central component of the DNA damage response and a known target of miR-22-3p, displayed decreased expression in AP4-deficient cells. Accordingly, MDC1 was directly induced by AP4 and indirectly by AP4-mediated repression of miR-22-3p. Adenomas and organoids from Ap4-deficient APCmin/+ mice displayed conservation of these regulations. Inhibition of miR-22-3p or ectopic MDC1 expression reversed the increased senescence, DNA damage, CIN and defective HR observed in AP4-deficient CRC cells. AP4-deficiency also sensitized CRC cells to 5-FU treatment, whereas ectopic AP4 conferred resistance to 5-FU in a miR-22-3p and MDC1-dependent manner.ConclusionsIn summary, AP4, miR-22-3p and MDC1 form a conserved and coherent, regulatory feed-forward loop to promote DNA repair, which suppresses DNA damage, senescence and CIN, and contributes to 5-FU resistance. These findings explain how elevated AP4 expression contributes to development and chemo-resistance of colorectal cancer after c-MYC activation.Graphical abstract

Comprehensive Atlas of Circulating Rare Cells Detected by SE-iFISH and Image Scanning Platform in Patients With Various Diseases.

ObjectiveCirculating rare cells (CRCs) are known as a crucial nucleated cellular response to pathological conditions, yet the landscape of cell types across a wide variety of diseases lacks comprehensive understanding. This study aimed at detecting and presenting a full spectrum of highly heterogeneous CRCs in clinical practice and further explored the characterization of CRC subtypes in distinct biomarker combinations and aneuploid chromosomes among various disease groups.MethodsPeripheral blood was obtained from 2,360 patients with different cancers and non-neoplastic diseases. CRC capture and identification were accomplished using a novel platform integrating subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) strategy with a high-throughput automated image scanning system, on which hemocyte, tumor, epithelial, endothelial, mesenchymal, and stemness biomarkers were immunostained and displayed simultaneously. Double chromosome enumeration probe (CEP8 and CEP12) co-detection was performed on isolated CRCs from an extended trial for two chromosome ploidy patterns.ResultsA comprehensive atlas categorizing the diverse CRCs into 71 subtypes outlining was mapped out. The presence of epithelial–mesenchymal transition (EMT) or endothelial–mesenchymal transition (EndoMT), the cells with progenitor property, hematologic CRCs expressing multiple biomarkers, CRCs at “naked nuclei” status, and the rarely reported aneuploid mesenchymal epithelial–endothelial fusion cluster were described. Circulating tumor cells (CTCs) were detected in 2,157 (91.4%) patients; the total numbers of CTCs and circulating tumor-derived endothelial cells (CTECs) were relatively higher in several digestive system cancer types and non-neoplastic infectious diseases (p < 0.05). Co-detection combining CEP8 and CEP12 showed a higher diagnostic specificity on account of 57.27% false negativity of CRC detection through a single probe of CEP8.ConclusionsThe alternative biomarkers and chromosomes to be targeted by SE-iFISH and the image scanning platform, along with the comprehensive atlas, offer insight into the heterogeneity of CRCs and reveal potential contributions to specific disease diagnosis and therapeutic target cell discovery.

FISH-Guided Evaluation of Hyperdiploidy and Other Cytogenetic Abnormalities in Childhood Burkitt Lymphoma

Background: Burkitt lymphoma (BL) is the most common lymphoma in childhood. Apart from MYC rearrangement, considered the hallmark of the disease, BL often presents with additional chromosome aberrations, the biological and clinical significance of which is not fully understood. Materials and methods: The study included 72 children (55 boys, 17 girls), aged 2-16 years (median 9), with BL diagnosed on the basis of histopathology and a demonstrable MYC rearrangement. Touch imprints from the diagnostic biopsy samples were investigated with i-FISH for MYC, BCL2, BCL6, IGH, IGK and IGL rearrangements genes and copy number aberrations involving 1q21/1p32, 7cen/7q31, 9cen/9p21, 13q14/13q34 and 17cen/17p13. Deviations from the diploid status were further investigated for aneusomies of the carrier chromosome with the use of appropriate chromosome enumeration probes. Results: MYC gene was demonstrated in all cases with MYC/IGH fusion in 83.3% (60/72). 47 cases (65.3%) were found with least one additional aberration, most commonly with 1q gain (29.2%), 7q gain (19.4%), 13q deletion (19.4%), hemizygous 9p loss (8.3%) and hyperdiploidy (8.3%). Advanced clinical stage (IV), 7q gain (but not trisomy 7) and -9/9p- were significantly associated with shorter overall survival. There was no instance of relapse or death among the hyperdiploid cases. Conclusions: This extensive FISH investigation on imprints of affected tissue provides clinically meaningful information on the genetic profile of BL and may prove valuable in the management of patients with no karyotype available. In particular, the demonstration of hyperdiploidy through the use of chromosome enumeration probes could offer the means for the delineation of clonal evolution pathways and the recognition of a subgroup of children with excellent prognosis who could be cured with low-intensity chemotherapy regimens. DisclosuresKattamis: VIFOR: Consultancy; CRISPR/Vertex: Consultancy, Honoraria; Agios Pharmaceuticals: Consultancy; IONIS: Consultancy; BMS/Celgene: Consultancy, Honoraria, Research Funding; Chiesi: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy.

Fish evaluation of additional cytogenetic aberrations and hyperdiploidy in childhood Burkitt lymphoma

Beyond MYC rearrangement, Burkitt lymphoma (BL) often presents with additional aberrations. Biopsy touch imprints from 72 children with BL were tested with interphase fluorescence in-situ hybridization (i-FISH) for MYC, BCL2, BCL6, IGH, IGK and IGL rearrangements and copy-number aberrations involving 1q21/1p32, 7cen/7q31, 9cen/9p21, 13q14/13q34 and 17cen/17p13. Diploid status deviations were investigated with chromosome enumeration probes. MYC rearrangement was demonstrated in all cases. Additional aberrations included +1q (21/72:29.2%), +7q (14/72:19.4%), 13q- (14/72:19.4%), 9p-(6/72:8.3%) and hyperdiploidy (6/72:8.3%). Advanced clinical stage IV, +7q and 9p- were associated with shorter overall survival, with stage IV and +7q retaining prognostic significance on multivariate analysis. No relapse or death was reported among the hyperdiploid cases. This i-FISH investigation provides information on the genetic profile of BL and may prove valuable for patients with no karyotype analysis. Demonstration of hyperdiploidy could evolve research on clonal evolution pathways and probably identify a subgroup of children with favorable prognosis.

UroVysion fluorescence in situ hybridization in urothelial carcinoma: a narrative review and future perspectives.

The number of patients with urothelial carcinoma (UC) is high, with a corresponding demand for detecting UC easily and non-invasively. Cystoscopy and urine cytology, with widely known diagnostic accuracies, are the gold standards for identifying UC originating from the bladder. However, cystoscopy or other tests, such as ureteroscopy or retrograde pyelography, are uncomfortable for patients. Tests for urinary biomarkers are expected to satisfy the demand for less invasive tests that will benefit patients with anxiety for invasive tests such as cystoscopy or ureteroscopy. Although several urinary biomarkers have been reported to support the diagnosis or follow-up of UC, their use in the clinic is uncommon. The UroVysion test examines urinary biomarkers using a multitarget, multicolor fluorescence in situ hybridization (FISH) assay. The test uses exfoliated cells found in urine and is a mixture of centromeric fluorescent denatured chromosome enumeration probes for chromosomes 3, 7, and 17 (labelled stratum red, spectrum green and spectrum aqua, respectively), and a locus-specific identifier probe for 9p21 (spectrum gold). It is used for the initial diagnosis of patients with hematuria or the monitoring of patients previously diagnosed with bladder cancer. Almost 20 years have passed since UroVysion was approved by the U.S. Food and Drug Administration, and so this is a well-established test. However, room exists for further research, with numerous reports on this test having been recently published. In order to update our knowledge, we herein present a brief overview of UroVysion and its features that follows the latest findings as they relate to UC.

Clinicopathological Features of Breast Cancer with Polysomy 17 and Its Response to Neoadjuvant Chemotherapy.

The interpretation of human epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) results may be challenging in tumors with polysomy 17, which is defined as increased signals of chromosome enumeration probe 17 (CEP17). The effect of polysomy 17 on HER2 protein expression and tumor treatment response has not been established. In this retrospective study, we investigated the clinicopathological features of breast cancer with polysomy 17 and determined the tumors' response to neoadjuvant chemotherapy (NACT). The study included 366 patients with primary breast cancer whose tumors had a CEP17 count of ≥ three/nucleus based on HER2 FISH studies. These cases were categorized according to HER2/CEP17 ratio and HER2 signals/nucleus using the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. We compared the clinicopathological characteristics and tumor response to NACT among different groups. There was a statistically significant difference in patients' age at diagnosis, tumor pathological grade, estrogen and progesterone receptor status, and NACT response among different HER2 FISH groups. Polysomy 17 tumors in group 1 had a higher rate of response (pathological complete response and residual cancer burden class I) to NACT containing anti-HER2 reagent than did those in other groups (p = 0.004), whereas polysomy 17 tumors in group 3 did not show a significant response to anti-HER2 treatment. Polysomy 17 tumors in different HER2 FISH groups have different pathological features and respond to NACT differently. These results may help us identify patients who will benefit from anti-HER2 therapy.

HER2 fluorescent in situ hybridization signal degradation: a 10-year retrospective study.

Fluorescence in situ hybridization (FISH) analysis is recommended for invasive breast carcinomas with equivocal (2+) immunohistochemical expression of human epidermal growth factor receptor 2 (HER2). However, existing guidelines for the retention and storage requirements for HER2 FISH slides vary widely among countries and laboratories. To determine the degradation rateof HER2 FISH signals, and the optimal retention time and storage conditions for HER2 FISH slides. Dual-probe HER2 FISH slides from March 2009 to June 2019 were retrieved from the archive to assess the presence, intensity and quantity of the green chromosome enumeration probe 17 (CEP 17) and orange HER2 signals. Per the institutional policy, FISH slides are placed in slide boxes and stored in -80°C freezers for up to 4years, whereas older slides are stored at room temperature. After excluding HER2 FISH slides that were deemed uninterpretable due to technical issues, a total of 6255 slides were assessed. Slides from 2009 to 2014 were stored at room temperature, while slides from 2015 to 2019 were stored in -80°C freezers. Slides stored in freezers showed retention of both the green and the orange signals. Slide stored at room temperature demonstrated significant decrease in the signal retention rate and the loss of signal did not progress in a linear fashion. The CEP17 signal was quenched much faster than the HER2 signal. Our study is the first to demonstrate HER2 FISH signal degradation with time and slide storage conditions. Storing HER2 FISH slides in a -80°C freezer allows for retention of both HER2 and CEP17 signals. At room temperature, the signals start to degrade with CEP17 signals lost at a faster rate. The results of the study may be used in official guidelines for storage conditions and retention time for HER2 FISH slides.

DeepACEv2: Automated Chromosome Enumeration in Metaphase Cell Images Using Deep Convolutional Neural Networks.

Chromosome enumeration is an essential but tedious procedure in karyotyping analysis. To automate the enumeration process, we develop a chromosome enumeration framework, DeepACEv2, based on the region based object detection scheme. The framework is developed following three steps. Firstly, we take the classical ResNet-101 as the backbone and attach the Feature Pyramid Network (FPN) to the backbone. The FPN takes full advantage of the multiple level features, and we only output the level of feature map that most of the chromosomes are assigned to. Secondly, we enhance the region proposal network's ability by adding a newly proposed Hard Negative Anchors Sampling to extract unapparent but essential information about highly confusing partial chromosomes. Next, to alleviate serious occlusion problems, besides the traditional detection branch, we novelly introduce an isolated Template Module branch to extract unique embeddings of each proposal by utilizing the chromosome's geometric information. The embeddings are further incorporated into the No Maximum Suppression (NMS) procedure to improve the detection of overlapping chromosomes. Finally, we design a Truncated Normalized Repulsion Loss and add it to the loss function to avoid inaccurate localization caused by occlusion. In the newly collected 1375 metaphase images that came from a clinical laboratory, a series of ablation studies validate the effectiveness of each proposed module. Combining them, the proposed DeepACEv2 outperforms all the previous methods, yielding the Whole Correct Ratio(WCR)(%) with respect to images as 71.39, and the Average Error Ratio(AER)(%) with respect to chromosomes as about 1.17.

Abstract A20: Characterizing chromosome instability in chemonaïve, chemosensitive, and chemoresistant high-grade serous ovarian cancer

Abstract High-grade serous ovarian cancer (HGSOC) is the most aggressive epithelial ovarian cancer subtype, and &amp;gt;75% of patients experience tumor recurrence following initial treatment, often with drug-resistant disease. Unfortunately, the aberrant events driving tumor progression, recurrence, and multidrug resistance in HGSOC remain largely unknown. Chromosome instability (CIN) is defined as an increased rate at which whole chromosomes (or large fragments) are gained or lost, and in many cancer types CIN is associated with tumor initiation, disease recurrence, multidrug resistance, and poor patient prognosis. Conceptually, the ongoing chromosome changes associated with CIN promote the production of genetically distinct daughter cells and drive cell-to-cell heterogeneity. As CIN remains largely unstudied in HGSOC, we now seek to characterize the prevalence and dynamics of CIN in chemonaïve, chemosensitive, and chemoresistant HGSOC patient samples, including samples isolated from ascites (metastatic cells) and solid tumors (primary tumor). Using single-cell quantitative imaging techniques, we evaluated CIN by assessing CIN phenotypes, including changes in nuclear areas and chromosome numbers. Changes in nuclear areas and increases in cell-to-cell heterogeneity are suggestive of CIN. Next, we employed fluorescence in situ hybridization and chromosome enumeration probes (CEPs) recognizing pericentric regions of chromosomes 8, 11, and 17 to quantify changes in chromosome numbers. Deviations from the expected number of two CEP foci/chromosome/nucleus (i.e., diploid state) are indicative of CIN. CEP specificity was validated within diploid and aneuploid fallopian tube (FT) secretory epithelial cell lines, a cell type of origin for HGSOC. The diploid FT cell lines show two CEPs/chromosome/nucleus and the aneuploid FT cell lines show an aberrant number of CEPs/chromosome/nucleus. CEPs are now being employed in HGSOC ascites samples and solid tumors within a HGSOC tissue microarray. Preliminary data show that nuclear areas and CEP numbers are heterogeneous and dynamic over time. More specifically, our data show CIN is highly prevalent in HGSOC cells isolated from ascites, increases with disease progression and drug resistance, and decreases in response to treatment. Additionally, we show that solid tumor samples (primary tumor) typically contain more frequent gains and/or losses in CEP foci (i.e., higher level of CIN) compared to the ascites samples (metastatic cells). Importantly, characterizing the prevalence of CIN in HGSOC will help researchers understand the association between CIN and tumor development, progression, response, and acquisition of drug resistance. Citation Format: Claire Morden, Ally Farrell, Mirka Sliwowski, Zelda Lichtensztejn, Mark Nachtigal, Kirk McManus. Characterizing chromosome instability in chemonaïve, chemosensitive, and chemoresistant high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A20.

Automatic quantification of HER2 gene amplification in invasive breast cancer from chromogenic in situ hybridization whole slide images.

Human epidermal growth factor receptor 2 (HER2), a transmembrane tyrosine kinase receptor encoded by the ERBB2 gene on chromosome 17q12, is a predictive and prognostic biomarker in invasive breast cancer (BC). Approximately 20% of BC are HER2-positive as a result of ERBB2 gene amplification and overexpression of the HER2 protein. Quantification of HER2 is performed routinely on all invasive BCs, to assist in clinical decision making for prognosis and treatment for HER2-positive BC patients by manually counting gene signals. We propose an automated system to quantify the HER2 gene status from chromogenic in situ hybridization (CISH) whole slide images (WSI) in invasive BC. The proposed method selects untruncated and nonoverlapped singular nuclei from the cancer regions using color unmixing and machine learning techniques. Then, HER2 and chromosome enumeration probe 17 (CEP17) signals are detected based on the RGB intensity and counted per nucleus. Finally, the HER2-to-CEP17 signal ratio is calculated to determine the HER2 amplification status following the ASCO/CAP 2018 guidelines. The proposed method reduced the labor and time for the quantification. In the experiment, the correlation coefficient between the proposed automatic CISH quantification method and pathologist manual enumeration was 0.98. The -values larger than 0.05 from the one-sided paired -test ensured that the proposed method yields statistically indifferent results to the reference method. The method was established on WSI scanned by two different scanners. Through the experiments, the capability of the proposed system has been demonstrated.

HER2 status in breast cancer: changes in guidelines and complicating factors for interpretation.

Human epidermal growth factor receptor 2 (HER2) protein overexpression and/or HER2 gene amplification is found in about 20% of invasive breast cancers. It is a sole predictive marker for treatment benefits from HER2 targeted therapy and thus, HER2 testing is a routine practice for newly diagnosed breast cancer in pathology. Currently, HER2 immunohistochemistry (IHC) is used for a screening test, and in situ hybridization is used as a confirmation test for HER2 IHC equivocal cases. Since the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines on HER2 testing was first released in 2007, it has been updated to provide clear instructions for HER2 testing and accurate determination of HER2 status in breast cancer. During HER2 interpretation, some pitfalls such as intratumoral HER2 heterogeneity and increase in chromosome enumeration probe 17 signals may lead to inaccurate assessment of HER2 status. Moreover, HER2 status can be altered after neoadjuvant chemotherapy or during metastatic progression, due to biologic or methodologic issues. This review addresses recent updates of ASCO/CAP guidelines and factors complicating in the interpretation of HER2 status in breast cancers.

Clinicopathologic Factors Associated With Response to Neoadjuvant Anti-HER2–Directed Chemotherapy in HER2-Positive Breast Cancer

BackgroundHER2-targeted neoadjuvant therapy has high efficacy in treating HER2-positive breast cancer. Response to neoadjuvant therapy helps clinicians make treatment decisions and make estimates about prognosis. This study examined clinicopathologic features to determine which may be most predictive of response to neoadjuvant therapy in HER2+ breast cancer. Patients and MethodsPatients with HER2+ breast cancer (n = 173) who had an initial biopsy performed between 2010 and 2016 were identified at our institution. Tumor response was evaluated on excisional specimens using the MD Anderson residual cancer burden (RCB) classification. Tumors with pathologic complete response (defined as no residual invasive carcinoma in the breast and lymph nodes) and RCB-I were classified as having response and tumors with RCB-II and -III as having no response. Patient age, tumor size, nuclear grade (1/2 vs. 3), mitosis, Nottingham grade, HER2 immunohistochemistry (1/2+ vs. 3+), HER2/CEP17 (chromosome enumeration probe 17) ratio, HER2 copy number, estrogen receptor, progesterone receptor, Ki-67, and tumor-infiltrating lymphocytes (TIL) were evaluated and correlated with response. TILs were evaluated for an average and also for the hot spot/total tumor stromal ratio. ResultsSmall tumor size, low estrogen receptor and progesterone receptor expression, HER2 immunohistochemistry 3+, high Ki-67, high HER2/CEP17 ratio, and high HER2 copy number were significantly associated with response (all P < .05). TIL hot spot was associated with RCB in univariate (P < .05) but not multivariate analyses. ConclusionClinicopathologic features may help predict HER2+ breast cancer response to neoadjuvant therapy. Larger studies would be useful to confirm these associations, which may have relevance to clinical practice.

Abstract P6-17-38: Clinical and pathological features of breast cancer with 'polysomy' of Chromosome 17

Abstract Background: Anti-HER2 therapy is a standard of care for patients with HER2+ breast cancer. HER2 status is routinely evaluated using immunohistochemical stain and/or florescence in situ hybridization (FISH). Interpretation of FISH results may be challenging in tumors with 'polysomy' of chromosome 17 ('polysomy'17), which is defined by increased chromosome enumeration probe 17 (CEP17) signal number. There is evident that 'polysomy'17 might account for anti-HER2 therapy response in breast cancer (BC) with normal HER2/CEP17 ratio. This study aims to study the clinical and pathological features of BC with 'polysomy'17. Method: Primary BC were selected based on elevated CEP17 count (≥3.0) in HER2 FISH performed at MD Anderson Cancer Center between April 2014 and March 2018 (n=385). Patient charts were reviewed for detailed clinical and pathological features. These cases were further divided into four groups according to HER2/CEP17 and HER2 copy number, based on ASCO-CAP guidelines: group 1 (HER2+) – HER2/CEP17≥2.0, HER2 copies≥4.0; group 3 (HER2+) – ratio&amp;lt;2.0, HER2 copies≥ 6.0; group 4 (HER2 equivocal) – ratio &amp;lt;2.0, HER2 copies ≥4.0 and &amp;lt;6.0; group 5 (HER2-) – ratio&amp;lt;2.0, HER2 copies&amp;lt;4.0. Chi-square tests were performed to study the difference of these characters. Results: In comparison with groups 1 and 3, BC in groups 4 and 5 are more commonly seen in elder patients (p=0.001). Also, these tumors show higher pathological category (p&amp;lt;0.001) and higher rate of lymph nodes metastasis (p&amp;lt;0.05). The clinical and pathological features are summarized in [Table 1]. Conclusion: Understanding the clinical and pathological features of BC with 'polysomy'17 may help clinically to choose patients who might be benefit from anti-HER2 therapy. Further study is to follow up the therapy response of those who received anti-HER2 treatment in this cohort of patients. Clinical and pathological characteristics of breast cancer with 'polysomy' of chromosome 17 Total (N=385)Group 1 ( N=131)Group 3 (N=25)Group 4 (N=69)Group 5 (N=160)P valueAge (year)Mean (range)56.8 (25-94)53.9 (25-83)52.8 (26-75)58 (33-92)59.3 (29-94)0.001Race RaceBlack (%)37 (9.6)14 (10.7)4 (16)5 (7.2)14 (8.8)0.873 Hispanic (%)61 (15.8)23 (17.6)3 (12)12 (17.4)23 (14.4) White (%)251 (65.2)82 (62.6)14 (56)44 (63.8)111 (69.4) Other (%)36 (9.4)12 (9.1)4 (16)8 (11.6)12 (7.4) GenderFemale (%)383 (99.5)131 (100)25 (100)68 (98.6)159 (99.4)0.478 Male (%)2 (0.5)0 (0)0 (0)1 (1.4)1 (0.6) Histology typeIDC, NOS (%)350 (90.9)121 (92.4)20 (80)66 (95.7)143 (89.4)0.471 ILC (%)9 (2.3)3 (2.3)1 (4)0 (0)5 (3.1) Others (%)26 (6.8)7 (5.3)4 (16)3 (4.3)12 (7.5) Nuclear gradeI (%)4 (1.2)2 (1.8)0 (0)1 (1.8)1 (0.7)0.044 II (%)122 (37)30 (26.3)12 (50)19 (33.3)61 (45.5) III (%)203 (61.8)82 (71.9)12 (50)37 (64.9)72 (53.8) NA561711226 Pathological tumor categorypT0+Tis (%)61 (21)36 (36.3)3 (20)5 (10.9)17 (13.1)&amp;lt;0.001 pT1 (%)125 (43.2)39 (39.4)7 (46.7)20 (43.5)59 (45.4) pT2+T3+T4 (%)104 (35.8)24 (24.3)5 (33.3)21 (45.6)54 (41.5) NA9532102330 Pathological lymph node categorypN0 (%)187 (65.4)81 (82.7)11 (73.3)22 (47.8)73 (57.5)0.048 pN1+N2+N3 (%)99 (34.6)17 (17.3)4 (26.7)24 (52.2)54 (42.5) pNx (%)9933102333 Citation Format: Sun H, Chen H, Lim B, Sahin AA. Clinical and pathological features of breast cancer with 'polysomy' of Chromosome 17 [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-17-38.

Equivocal (HER2 IHC 2+) breast carcinomas: gene-protein assay testing reveals association between genetic heterogeneity, individual cell amplification status and potential treatment benefits.

Genetic heterogeneity can pose a challenge to identifying eligible cases for targeted therapy in the human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) 2+ breast carcinoma group. In this study, we characterised this subset of tumours according to clinicopathological parameters. We assessed 1000 tumour cells per case and recorded the number of HER2 and chromosome enumeration probe 17 (CEP17) copies using gene-protein assay slides. HER2 status was determined based on ASCO/CAP 2013 guidelines. Tumours with 5-50% of cancer cells with amplification were considered to be heterogeneous, whereas those with >50% were considered to be non-heterogeneous. In a study cohort of 110 HER2 IHC 2+ carcinomas, 93 (84.5%) were non-amplified, 12 (10.9%) were amplified and five (4.5%) were ISH-equivocal. All the HER2-amplified and two of ISH-equivocal cases (12.7%) corresponded to non-heterogeneous tumours, with highly significant differences evident in the average HER2/CEP17 ratio (P=0.0002) and the proportion of cells with HER2 >6 copies (P<0.0001) compared with heterogeneous lesions. NST grade 3 and HER2-amplified carcinomas average HER2/CEP17 ratio correlated with an increased number of cells with HER2/CEP17 ≥2.0 (P<0.014). Triple-negative CEP17 polysomic carcinomas showed increased metastatic capacity (P=0.003) compared with other tumour types. Non-heterogeneous HER2 IHC 2+ tumours tend to be HER2-amplified. Adding the percentage of cells with HER2 >6 copies to the average HER2/CEP17 ratio may facilitate assessment of amplification status in ISH-equivocal cases. The proportion of cells with HER2/CEP17 ≥2.0 contributes information concerning the actual average HER2/CEP17 ratio, depending on tumour type.

Rapid micro fluorescence in situ hybridization in tissue sections.

This paper describes a micro fluorescence in situ hybridization (μFISH)-based rapid detection of cytogenetic biomarkers on formalin-fixed paraffin embedded (FFPE) tissue sections. We demonstrated this method in the context of detecting human epidermal growth factor 2 (HER2) in breast tissue sections. This method uses a non-contact microfluidic scanning probe (MFP), which localizes FISH probes at the micrometer length-scale to selected cells of the tissue section. The scanning ability of the MFP allows for a versatile implementation of FISH on tissue sections. We demonstrated the use of oligonucleotide FISH probes in ethylene carbonate-based buffer enabling rapid hybridization within <1 min for chromosome enumeration and 10–15 min for assessment of the HER2 status in FFPE sections. We further demonstrated recycling of FISH probes for multiple sequential tests using a defined volume of probes by forming hierarchical hydrodynamic flow confinements. This microscale method is compatible with the standard FISH protocols and with the Instant Quality FISH assay and reduces the FISH probe consumption ∼100-fold and the hybridization time 4-fold, resulting in an assay turnaround time of <3 h. We believe that rapid μFISH has the potential of being used in pathology workflows as a standalone method or in combination with other molecular methods for diagnostic and prognostic analysis of FFPE sections.

Performance evaluation of detecting circulating tumor cells and tumor cells in bronchoalveolar lavage fluid in diagnosis of peripheral lung cancer.

To evaluate the diagnostic performances of detecting circulating tumor cells (CTCs) and tumor cells in bronchoalveolar lavage fluid (BALF) for peripheral lung cancer. A total of 247 patients with lung cancer and 70 cases with benign lung disease were recruited in this study. Peripheral blood and BALF samples were collected, in which the tumor cells were enriched by negative immunomagnetic selection and detected by fluorescence in situ hybridization (FISH) of chromosome enumeration probe 8 (CEP8). The levels of tumor-associated markers (e.g., CEA, CA125, and NSE) in peripheral blood plasma were measured by using electrochemiluminescence. The numbers of CTCs detected in peripheral blood were significantly higher in patients with lung cancer than those with benign lung disease (5.78±0.57 vs. 1.13±0.39, Z=-8.64, P<0.01). Similarly, tumor cells count in BALF of malignancy were higher than that of benign lesions (6.76±0.89 vs. 0.89±0.23, Z=-6.254, P<0.01). However, as for patients with lung cancer and benign lung disease, the numbers of tumor cells in peripheral blood were comparable with those in BALF (both P>0.05). Detecting CTCs and tumor cells in BALF had similar areas under curves (AUC =0.871 and 0.963, respectively; P>0.05) in discriminating benign lesions from lung cancer (sensitivity 83.8% and 92.6%, specificity 86.5% and 99.9%, respectively), both of which were larger than those of NSE, CEA, and CA125 (AUC =0.564, 0.512 and 0.554, respectively; all P<0.05). The diagnostic performances of discriminating benign lesions and lung cancer in BALF and peripheral blood were both in concordance with that of histopathology (kappa values 0.662 and 0.569, respectively; both P<0.001). Detecting tumor cells in peripheral blood and BALF may sensitive to identify benign and malignant peripheral lung lesions and be of value for early diagnosis of lung cancer.

Retrospective analysis of the association between human epidermal growth factor receptor 2 amplification and chromosome enumeration probe 17 status in patients with breast cancer.

The aim of the present study was to identify potential human epidermal growth factor receptor 2 (HER2) amplification, according to American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) 2013 HER2 testing guidelines, in patients previously determined not to possess HER2 amplification, in accordance with previous 2007 guidelines. Potential discrepancies may arise from chromosome enumeration probe 17 (CEP17) amplification, deletion, polysomyor monosomy. HER2, CEP17, tumor protein p53 (TP53) and retinoic acid receptor α (RARA) genes from 67 patient specimens with suspected amplification, polysomy or monosomy of CEP17 were analyzed using fluorescence in situ hybridization. HER2 status was interpreted using 2007 and 2013 ASCO HER2 test guidelines as well as the reference genes TP53 and RARA. According to ASCO/CAP2007 HER2 guidelines, 20 patients exhibited HER2 amplification (29.85%), 41 were without HER2 amplification (including 25 with polysomy, 15 with monosomy and 1 with suspected monosomy plus co-amplification of HER2 and CEP17) and the remaining 6 patients were equivocal. Using ASCO/CAP 2013 HER2 guidelines, 49 patients exhibited HER2 gene amplification (73.1%). The 29-patient increase included 6 originally at equivocal levels but now demonstrating amplification, 22 originally with polysomy but now revealing co-amplification, and 1 with suspected monosomy plus co-amplification of HER2 and CEP17. According to TP53 and RARA, HER2 was amplified in 43 patients (64.1%). Using the revised guidelines, HER2, originally identified as amplified in 6 patients, was not amplified following the introduction of TP53 and RARA control genes. Among these 6, 4 possessed normal TP53 and RARA. The incidence of co-amplification of HER2 and CEP17 was 1.4% (21/1,518). RARA and TP53 are suitable control genes to evaluate HER2 status.

Her2/neu Status Determination in Breast Cancer: A Single Institutional Experience Using a Dual-Testing Approach With Immunohistochemistry and Fluorescence In Situ Hybridization.

According to current guidelines, either immunohistochemistry (IHC) or in situ hybridization (ISH) can be used to determine human epidermal growth factor receptor 2 (Her2/neu) status in breast carcinoma. While the guidelines explicitly delineate result interpretation, there is no consensus on the most appropriate testing algorithm. The Her2/neu statuses of 369 consecutive cases of invasive breast cancer (from 351 patients) were assessed in a dual-testing algorithm that uses both IHC and fluorescence ISH (FISH). FISH was performed using dual-color HER2/ chromosome enumeration probe 17 ( CEP17 ) probes, and if equivocal results were obtained, reflex testing using HER2/lissencephaly gene 1 ( LIS1 ) probes was used. Results from both modalities were scored and reported using American Society of Clinical Oncology/College of American Pathologists 2013 criteria. Sixty-one (16.5%) of the 369 tumors were found to be Her2/neu positive by at least one modality. The overall concordance between IHC and FISH results was 97.6%. Six of the 369 tumors were reclassified as Her2/neu positive after a negative IHC result. FISH was also able to identify significantly more Her2/neu-positive cases than IHC. The commonly used reflex strategy based on IHC results may deny potentially beneficial targeted therapy for a small cohort of patients, which should be considered as testing guidelines are formulated and the cost-benefit analyses of various testing algorithms are assessed.

Prognostic significance of centromere 17 copy number gain in breast cancer depends on breast cancer subtype

Increased copy number of chromosome enumeration probe (CEP) targeting centromere 17 is frequently encountered during HER2 in situ hybridization (ISH) in breast cancer. The aim of this study was to clarify the clinicopathologic significance of CEP17 copy number gain in a relatively large series of breast cancer patients. We analyzed 945 cases of invasive breast cancers whose HER2 fluorescence ISH reports were available from 2004 to 2011 at a single institution and evaluated the association of CEP17 copy number gain with clinicopathologic features of tumors and patient survival. We detected 186 (19.7%) cases of CEP17 copy number gain (CEP17≥3.0) among 945 invasive breast cancers. In survival analysis, CEP17 copy number gain was not associated with disease-free survival of the patients in the whole group. Nonetheless, it was found to be an independent adverse prognostic factor in the HER2-negative group but not in the HER2-positive group. In further subgroup analyses, CEP17 copy number gain was revealed as an independent poor prognostic factor in HER2-negative and hormone receptor-positive breast cancers, and it was associated with aggressive histologic variables including high T stage, high histologic grade, lymphovascular invasion, p53 overexpression, and high Ki-67 proliferative index. In conclusion, we found that elevated CEP17 count can serve as a prognostic marker in luminal/HER2-negative subtype of invasive breast cancer. We advocate the use of the dual-colored fluorescence ISH using CEP17 rather than the single-colored one because it gives additional valuable information on CEP17 copy number alterations.