Positive Fluorescence In Situ Hybridization Research Articles

Human Epidermal Growth Factor Receptor 2-Low Breast Cancer Brain Metastases: An Opportunity for Targeted Systemic Therapies in a High-Need Patient Population.

Trastuzumab deruxtecan is a new treatment option for patients with advanced human epidermal growth factor receptor 2 (HER2)-low breast cancer (BC). Although HER2-low status has been characterized in early and advanced BC, it has yet to be fully characterized in brain metastases (BrM). Patients who underwent surgery for BC BrM at Sunnybrook Health Sciences Centre and for whom HER2 status was available on resected BrM were studied. Estrogen receptor, progesterone receptor, and HER2 status were assessed on the basis of ASCO/College of American Pathologists (CAP) guidelines. HER2-zero was defined as immunohistochemistry (IHC) 0; HER2-low was defined as IHC 1+ or IHC 2+ with fluorescence in situ hybridization (FISH)-negative status. HER2-positive (HER2+) was defined as IHC 3+ or IHC 2+ with positive FISH. Clinicopathologic features were recorded. We also assessed the prognostic association between extent of HER2 expression and (1) brain-specific progression-free survival (bsPFS), as well as (2) overall survival (OS). In this retrospective cohort of 102 patients with resected BC BrM, 53% (n = 54) were HER2+, 29.4% (n = 30) were HER2-low, and 17.6% (n = 18) had HER2-zero status. Among BrM that were triple-negative on the basis of ASCO/CAP guidelines, 63.6% (n = 14/22) were reclassified as being HER2-low. Sixty percent (n = 15/25) of BrM that were hormone receptor-positive/HER2-negative (HR+/HER2-) were reclassified as being HER2-low. In total, 51 patients had matched primary breast and BrM tissue available; results of HER2 status when categorized as HER2-zero, HER2-low, and HER2+ were concordant in 82.3% (n = 42/51) of cases (Cohen's kappa, 0.58; P = .07). There was no significant association between HER2-zero, HER2-low, and HER2+ status in BrM and either bsPFS or OS. Among patients with surgically resected BrM, a high proportion of those with metastatic triple-negative BC and HR+/HER2- disease have HER2-low BrM with potential to benefit from HER2-targeted therapy.

Нyperdiploid мultiple мyeloma-cytogenetic and clinical aspects

In the current research the attention is focused on the possibilities of identifying a hyperdiploidy myeloma clone (HdMC) by using a triple-color fluorescence “in situ” hybridization (FISH) probe. The cytogenetic results from the bone marrow aspirates of 26 patients with newly diagnosed multiple myeloma admitted in the Hematology Department of our hospital during the period from March to September 2023 have been analyzed. The group consists of 12 female and 14 male patients with an average age of 67 years. A FISH probe for establishing hyperdiploidy myeloma clone was used as well as the most common methods for detecting genetic aberrations affecting the long and short arms of the 1 and 14q32 chromosome rearrangements. According to the results, two subgroups of patients have been established. The first subgroup consists of the patients with positive FISH probes for hyperdiploidy myeloma and/or 14q34 rearrangements and 1q25/1p36, while the second one consists of patients, negative for all the three probes listed above. A comparison between the demographic, laboratory data and the ISS (International Staging System) stage of the two subgroups has been made. The collected data suggests that the use of the triple-color FISH probe, as well as some other factors in the analyzed information, increases the probability of detecting a HdMC by 23%.

UroVysion™ fluorescence in situ hybridization (FISH) possibly has a high positive rate in carcinoma of non-urothelial lineages.

Background: Positive UroVysion™ fluorescence in situ hybridization (FISH) is generally considered as urothelial carcinoma (UC). We clarify if UroVysion™ FISH can be positive in carcinoma of non-urothelial lineages (CNUL), and verify the consistency of urine FISH and histological FISH in CNUL. Methods: All CNUL subjects detected by urine FISH assay due to haematuria from Tongji Hospital were screened. Meanwhile, 2 glandular cystitis and 2 urothelial carcinoma were served as negative or positive control. Paraffin-embedded tissue sections of all subjects were sent to the pathology department for histological FISH detection. Results: A total of 27 patients were included in this study, including 9 with adenocarcinomas, 11 with squamous cell carcinomas, and 7 with other tumour types. The overall positive rate in urine FISH was 64.00% (16/25) in patients with CNUL, 77.78% (7/9) in those with adenocarcinoma and 54.55% (6/11) in those with squamous carcinoma. There was a significant difference in the GLP p16 gene deletion rate between UC and CNUL (100% vs. 8.00%, p = 0.017). Histological FISH results showed that the histological results of 19 patients were consistent with their urine FISH results, and only one patient with stage Ⅲa urachal carcinoma had inconsistent histological FISH results (positive) and urine FISH (negative) results. Conclusion: We demonstrated for the first time the application value of FISH in CNUL on urine samples. Positive urine FISH tests indicate not only UC, but also CNUL. UroVysion™ FISH possibly has a high positive rate in CNUL. CNUL and UC have different genetic changes shown by FISH.

Preoperative fluorescence in situ hybridization analysis as a predictor of tumor recurrence in patients with non-muscle invasive bladder cancer: a bi-institutional study

BackgroundNon-muscle invasive bladder cancer (NMIBC) is known for its elevated recurrence rate, necessitating an enhancement in the current risk stratification for recurrence. The urine-based fluorescence in situ hybridization (FISH) assay has emerged as a noninvasive auxiliary tool for detecting bladder cancer. The aim of this study was to explore the potential relationship between the preoperative FISH assay and recurrence, and to develop a FISH-clinical nomogram for predicting the recurrence-free survival (RFS) in NMIBC patients.MethodsIn total, 332 eligible patients were enrolled from two hospitals. The SYSMH cohort was randomly assigned to the training set (n = 168) and the validation set I (n = 72) at a ratio of 7:3, while the SYSUTH cohort was allocated to the validation set II (n = 92). The correlation between the preoperative FISH assay and recurrence was determined through the Cox regression analysis. The least absolute shrinkage and selection operator (LASSO) Cox regression algorithm was used for model construction. The performance of the model was assessed by its discrimination, calibration, and clinical usefulness.ResultsWe uncovered that chromosome 7 aneuploidy, p16 locus loss, number of the positive FISH sites, and the FISH test result were significantly associated with tumor recurrence. Then, a FISH-clinical nomogram incorporating the FISH test result, T stage, associated CIS, tumor grade, and tumor status was developed. It showed favorable calibration and discrimination with a C-index of 0.683 (95%CI, 0.611–0.756) in the training set, which was confirmed in the validation set I and validation set II with C-indexes of 0.665 (95%CI, 0.565–0.765) and 0.778 (95%CI, 0.665–0.891), respectively. Decision curve analysis revealed the clinical usefulness of the nomogram. Moreover, our proposed nomogram significantly outperformed the guideline-recommended EORTC and CUETO scoring models.ConclusionOur study confirmed the prognostic value of the preoperative FISH assay and proposed a FISH-clinical nomogram to predict RFS in NMIBC patients. Our nomogram can serve as a more precise tool for recurrence risk stratification, which may optimize disease management in bladder cancer and improve patient prognosis.

Evaluation of the diagnostic efficiency of voided urine fluorescence in situ hybridization for predicting the pathology of preoperative "low-risk" upper tract urothelial carcinoma.

To evaluate the clinical utility of voided urine fluorescence in situ hybridization (FISH) for predicting the pathology of preoperative "low-risk" upper tract urothelial carcinoma (UTUC). Information of patients preoperatively diagnosed with "low-risk" UTUC receiving radical nephroureterectomy (RNU) between May 2014 and October 2019 were retrospectively collected. All of the patients accepted the FISH test and then were divided into two groups according to the results of FISH. The diagnostic value of FISH was assessed through the receiver operating characteristics (ROC) curve and area under the curve. Logistic regression analysis was applied to examine FISH as a predictive factor of tumor final stage and grade of preoperative "low-risk" UTUC. In total, 129 patients were included. Of them, 70 (54.2%) were marked with positive FISH result. The difference at final pathology in tumor stage and tumor grade between these two groups of FISH (-) and FISH (+) had significantly statistical significance (p<0.001). Regarding to the tumor stage at final pathology, the sensitivity, specificity, positive predictive value and negative predictive value of FISH were 70.7 (58.9-80.3)68.5 (54.3-80.1)75.7 (63.7-84.8) and 62.7 (49.1-74.7), respectively. Regarding to the tumor grade at final pathology, the sensitivity, specificity, positive predictive value and negative predictive value of FISH were 64.7 (53.5-74.6), 65.9 (50.0-79.1), 78.6 (66.8-87.1) and 49.1 (36.5-62.3), respectively. The results of logistic regression analysis indicated that FISH could predict the pathologic characteristics of preoperative "low-risk" UTUC independently. FISH was qualified with relatively high diagnostic estimates for predicting tumor stage and grade of preoperative "low-risk" UTUC, and could be an independent predictive factor in clinical practice. For preoperative "low-risk" UTUC patients but with positive FISH result, choosing nephron-sparing surgery may require special caution.

The diagnostic value of fluorescence in situ hybridization in secondary electroresection of bladder cancer.

To investigate the utility of fluorescence in situ hybridization (FISH) in secondary electroresection of bladder cancer. From January 2016 to April 2022, bladder cancer patients who had undergone secondary electroresection in Tongji Hospital and had preoperative urine FISH were recruited, and the positive rate, accuracy, sensitivity, specificity, genetic material changes and predictive power on malignancy degree of FISH in the secondary electroresection of bladder cancer were examined. Twenty-six patients with bladder cancer were included in this study, and 8 were confirmed by secondary electroresection, including 6 cases positive for FISH positive and 2 negative for FISH. Besides, among the subjects, 18 were without tumor recurrence, including 1 case with positive FISH results and 17 with negative FISH results. Tumor recurrence was diagnosed in 85.71% (6/7) of FISH-positive patients in secondary electroresection while only 10.53% (2/19) of FISH-negative patients were found to develop tumor recurrence in the secondary electroresection. The sensitivity of FISH for the detection of bladder cancer before secondary electroresection was 75%, with a specificity of 94.44%, and an accuracy of 88.46%. A 6-month follow-up revealed that 2 of the 8 recurrent patients underwent radical resection of bladder cancer, and the remaining 6 patients had no recurrence, as confirmed by regular bladder perfusion and microscopy. In the 18 non-recurrent patients during secondary electroresection, no recurrence developed. Urine FISH can achieve a high detection rate and specificity for secondary electroresection of bladder cancer. If a bladder cancer patient who are indicated for secondary electroresection is negative for urine FISH, the recurrence rate after secondary electroresection will be low, and the cystoscopy can be performed before deciding whether to perform secondary electroresection.

HER2-LOW BREAST CANCER BRAIN METASTASES: INCIDENCE AND TREATMENT IMPLICATIONS

Abstract Brain metastases (BrM) are a major cause of morbidity and mortality among women with breast cancer (BC). Central nervous system (CNS)-penetrating systemic therapies for patients with HER2-negative BrM are lacking; given CNS activity of trastuzumab deruxtecan, efficacy for patients with HER2-low BrM is of interest. METHODS: A retrospective study of two cohorts of consecutive patients who underwent surgery for BC BrM at Sunnybrook Health Sciences Centre were identified. BrM subtype was assessed based on 2018 ASCO/CAP guidelines. HER2-zero was defined as immunohistochemistry (IHC) 0; HER2-low was defined as IHC 1+ or 2+ and fluorescence in situ hybridization (FISH) negative status. HER2-positive was defined as IHC 3+ or IHC 2+ with positive FISH. We also assessed the prognostic association between extent of HER2 expression and i) brain-specific progression free survival (bsPFS), as well as ii) overall survival (OS). RESULTS: Out of 137 patients with resected BrM, tissue for HER2 assessment was available in 74.5% (n=102) of cases. In this cohort, the median age at BrM diagnosis was 53.5 (range, 32- 85). 68.6% (n=70) had extracranial disease. 53% (n=54) of the BrM were HER2-positive; 29.4% (n=30) were HER2-low and 17.6% (n=18) had HER2-zero status. Among BrM that were triple negative based on ASCO/CAP guidelines, 14 out of 22 cases (63.6%) were re-classified as being HER2-low. 15/25 (60%) BrM that were hormone receptor positive/HER2 negative (HR+/HER2-) based on ASCO/CAP guidelines were re-classified as being HER2-low. There was no significant association between the extent of HER2 expression (HER2-zero, HER2-low versus HER2-positive status) in BrM and either bsPFS or OS. CONCLUSION: Among patients with surgically resected BrM, a high proportion of those with metastatic TNBC and HR+/HER2-negative disease have “HER2-low” BrM with potential to benefit from HER2-targeted therapy.

HER2-low breast cancer brain metastases: Incidence and treatment implications.

2035 Background: Brain metastases (BrM) are a major cause of morbidity and mortality among women with breast cancer (BC). Central nervous system (CNS)-penetrating systemic therapies for patients with HER2-negative BrM are lacking; this is particularly problematic for patients with triple negative disease (TNBC) who have a high likelihood of developing BrM. Given CNS activity of trastuzumab deruxtecan, efficacy for patients with HER2-low BrM is of interest. Methods: A retrospective study of two cohorts of patients who underwent surgery for BC BrM at Sunnybrook Health Sciences Centre between 1999-2013 and 2008-2018 were identified. Estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 (HER2) status were assessed based on 2018 ASCO/CAP guidelines. HER2-zero was defined as immunohistochemistry (IHC) 0; HER2-low was defined as IHC 1+ or IHC 2+ with fluorescence in situ hybridization (FISH) negative status. HER2-positive was defined as IHC 3+ or IHC 2+ with positive FISH. Clinicopathological features were recorded. We also assessed the prognostic association between extent of HER2 expression and i) brain-specific progression free survival (bsPFS), as well as ii) overall survival (OS). Results: Out of 137 patients with resected BrM, tissue for HER2 assessment was available in 74.5% (n=102) of cases. In this cohort, the median age at BrM diagnosis was 53.5 (range, 32-85). 18.6% (n=19) had leptomeningeal disease and 68.6% (n=70) had extracranial disease. 53% (n=54) of the BrM were HER2-positive; 29.4% (n=30) were HER2-low and 17.6% (n=18) had HER2-zero status. Among BrM that were triple negative based on ASCO/CAP guidelines, 14 out of 22 cases (63.6%) were re-classified as being HER2-low. 15/25 (60%) BrM that were hormone receptor positive/HER2 negative (HR+/HER2-) based on ASCO/CAP guidelines were re-classified as being HER2-low. In total, 51 patients had matched primary breast and BrM tissue available; results of HER2 status when categorized as HER2-zero, HER2-low and HER2-positive were concordant in 82.3% (n= 42/51) of cases (Cohen’s kappa 0.58, p=0.0719). In 7 cases, the primary breast tissue was HER2-zero whereas the BrM was either HER2-low (n=5) or HER2-positive (n=2). In only one case (2%), expression of HER2 was lower in the BrM (HER2-low) compared to the primary BC (HER2-positive). Median time from primary BC to the development of BrM was 35 months (IQR, 14-69) in the overall cohort; patients with HER2-zero BrM had a numerically longer time to development of BrM (45.8 months), compared to those with HER2-low (35 months) and HER2-positive (35 months) BrM (p=0.948). There was no significant association between HER2-zero, HER2-low and HER2-positive status in BrM and either bsPFS or OS. Conclusions: Among patients with surgically resected BrM, a high proportion of those with metastatic TNBC and HR+/HER2-negative disease have “HER2-low” BrM with potential to benefit from HER2-targeted therapy.

PRAME expression by Immunohistochemistry and Reverse Transcription Quantitative PCR in conjunctival melanocytic lesions -A comprehensive clinicopathologic study of 202 cases and correlation of cytogenetics with PRAME expression in challenging conjunctival melanocytic lesions.

This study examined PRAME expression by immunohistochemistry and reverse transcription quantitative PCR (RT-qPCR) in 202 histologically unequivocal conjunctival melanocytic lesions: 76 nevi, 29 benign melanoses, 25 low-grade conjunctival intraepithelial melanocytic lesions (LGCMIL), 26 high-grade conjunctival melanocytic intraepithelial lesions/in-situ melanoma (HGCMIL) and 46 invasive melanomas. PRAME score 0 was seen in 96% of nevi (73/76), 96% of benign melanoses (28/29) and 88% of LGCMIL (22/25). PRAME score 4 was seen in 50% HGCMIL (13/26) and 76% invasive melanomas (35/46). PRAME score 4 had sensitivity of 50% and specificity of 100% in differentiating between HGCMIL and benign melanosis/LGCMIL. PRAME score 4 had sensitivity of 76% and specificity of 100% in differentiating between melanoma and nevi. Relative quantification of PRAME mRNA expression by RT-qPCR was performed on 49 (24%) cases: 17 nevi, 3 benign melanoses, 5 LGCMIL, 9 HGCMIL, and 15 invasive melanomas. The analysis generated two distinct groupings with 'high' relative PRAME expression for the HGCMIL and invasive melanoma and 'low/zero' expression for nevi, benign melanosis and LGCMIL. Thirty-three challenging conjunctival melanocytic lesions that had previous FISH analysis were studied: 18 nevi, 12 melanomas in a nevus, 2 nevoid melanomas, 1 in-situ melanoma. All nevi (100%) showed concordance between negative FISH and PRAME (scores 0-3). 4/13 (31%) melanomas (in-situ, invasive, isolated, and in association with nevus) showed concordance between positive FISH and PRAME score 4. In conclusion, PRAME score 4 has 100% specificity for diagnosis of HGCMIL and melanoma. PRAME is limited in its sensitivity in the evaluation of challenging melanocytic lesions.

False positivity in break apart fluorescence in-situ hybridization due to polyploidy.

In-situ hybridization (ISH) is a diagnostic tool in the detection of chromosomal anomalies, which has important implications for diagnosis, classification and prediction of cancer therapy in various diseases. Certain thresholds of number of cells showing an aberrant pattern are commonly used to declare a sample as positive for genomic rearrangements. The phenomenon of polyploidy can be misleading in the interpretation of break apart fluorescence in-situ hybridization (FISH). The aim of this study is to investigate the impact of cell size and ploidy on FISH results. In sections of varying thickness of control liver tissue and non-small cell lung cancer cases, nuclear size was measured and the number of MET chromogenic ISH and ALK FISH (liver) or ALK and ROS1 FISH (lung cancer) signals was manually counted and quantified. In liver cell nuclei the number of FISH/chromogenic ISH signals increases with nuclear size related to physiological polyploidy and is related to section thickness. In non-small cell lung cancer cases tumour cells with higher ploidy levels and nuclear size have an increased chance of single signals. Furthermore, additional lung cancer samples with borderline ALK FISH results were examined with a commercial kit for rearrangements. No rearrangements could be demonstrated, proving a false positive ALK FISH result. In case of polyploidy there is an increased likelihood of false positivity when using break apart FISH probes. Therefore, we state that prescribing one single cut-off in FISH is inappropriate. In polyploidy, the currently proposed cut-off should only be used with caution and the result should be confirmed by an additional technique.

Abstract HER2-16: HER2-16 Inter-lesion heterogeneity of HER2-status in metastatic breast cancer: possible implications for treatment with anti-HER2 antibody-drug conjugates

Abstract Background. Trastuzumab deruxtecan (T-DXd) has shown promising activity in patients with HER2-low metastatic breast cancer. As the HER2-status can vary between the primary and its corresponding metastases, treatment decisions should ideally be based on HER2 assessment of a recent biopsy. However, limited data is available on intra-patient inter-metastatic heterogeneity in HER2-status, affecting representability of a single biopsy and potential therapeutic options and outcome. We therefore assessed HER2 status on multiple metastases from patients with primary ER-positive/HER2-non-amplified breast cancer in our prospective post-mortem tissue donation program UPTIDER (NCT04531696). Methods. Ninety-one metastatic samples retrieved during the autopsies of 6 patients (range: 13–16/patient) and their respective primary tumours were immunohistochemically (IHC) stained for HER2 (HercepTestTM, RTU, ISO-15189 accredited) in our institution. Consensus scoring was performed between two pathologists according to ASCO/CAP 2018 guidelines. The observers were blinded for patient ID. Reflex fluorescence in situ hybridization (FISH) testing was performed for samples with IHC score of 2+. HER2 status was categorized as HER2-zero (IHC 0), HER2-low (IHC 1+ or IHC 2+ with negative FISH), or HER2-positive (IHC 3+ or IHC 2+ with positive FISH). To assess stability of the performance of IHC scoring in the post-mortem setting, an additional 13 samples taken from 3 metastases at regular (every 1.5h) time intervals during the autopsy underwent HER2 IHC scoring. Results. Evaluation of HER2-status in the primary tumour showed 2 patients with HER2-zero disease and 4 with HER2-low disease. A discordance between HER2 status of the metastases and their respective primary was seen in all patients. Not a single lesion was found to be HER2-positive. For every patient, at least one HER2-low metastasis was observed, with the percentage being highly variable between patients and ranging between 7 and 100%. No association was observed between HER2 status and organ site: HER2-low as well as HER2-zero lesions were found in all organs evaluated in at least 4 patients (liver, bone, pleura, lymph nodes). For 5 patients, multiple lesions within the liver were evaluated: while HER2-zero versus HER2-low status was concordant in those lesions in 4 patients, a mix of HER2 IHC scores was seen in 3 of them. IHC scores were stable over time for tumour lesions assessed repeatedly. Discussion. Important inter-lesion heterogeneity in terms of HER2-low status was observed in patients with primary ER-positive/HER2-non-amplified breast cancer participating to our post-mortem tissue donation program. This observed heterogeneity is unlikely to be due to post-mortem changes in HER2 expression. HER2-low status was found in at least one distant lesion in all patients, complicating therapeutic decision-making based on a single biopsy. Of note, IHC 1+ and 2+ scores varied between metastases of each patient too, making assessment on a single biopsy less reliable for stratification in clinical trials. Further assessment on samples from UPTIDER-patients with ER-negative disease is currently ongoing and results will be available to be presented. Citation Format: Tatjana Geukens, Maxim De Schepper, François Richard, Marion Maetens, Karen Van Baelen, Amena Mahdami, Ha-Linh Nguyen, Edoardo Isnaldi, Sophia Leduc, Anirudh Pabba, Imane Bachir, Freya Mertens, Sara Vander Borght, Ann Smeets, Ines Nevelsteen, Kevin Punie, Patrick Neven, Hans Wildiers, Wouter Van Den Bogaert, Giuseppe Floris, Christine Desmedt. HER2-16 Inter-lesion heterogeneity of HER2-status in metastatic breast cancer: possible implications for treatment with anti-HER2 antibody-drug conjugates. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr HER2-16.

Preoperative accuracy of diagnostic evaluation of urachal carcinoma.

We analyzed the clinical data of patients with urachal carcinoma (UrC) in order to strengthen urologists' understanding of UrC and improve preoperative diagnosis. The clinical data of 37 patients with UrC admitted to our hospital from October 2005 to April 2022 were retrospectively analyzed, and 40 patients with urothelial carcinoma (UCa) of bladder were enrolled as the control group. We compared and analyzed the imaging, cystoscopy and immunohistochemistry, serum tumor markers, fluorescence in situ hybridization (FISH) of UrC and bladder UCa for early diagnosis and evaluation of diagnostic accuracy. A total of 37 patients with UrC were enrolled in this study, including 30 males and seven females, with a median age of 52.00 (44.50-63.50) years. Imaging and cystoscopy suggest that UrC grows primarily outside the bladder cavity and is found in the middle line of the dome or anterior wall of the bladder. There was a significant difference in tumor location between the UrC group and the UCa group (10.13 mm vs. -7.06 mm, p < 0.001). Immunohistochemistry revealed that CK20 and CDX-2 were both diffusely and strongly positive. β-catenin was strongly positive in cytoplasm and membrane, but negative in nuclear staining. Carcinoembryonic antigen (CEA) and carbohydrate antigen 72-4 (CA724) expression levels were significantly higher in the UrC group than in the UCa group (p < 0.05). In the diagnosis of UrC, the area under the curve (AUC) of CEA combined with CA724 was the greatest. FISH's sensitivity in diagnosing UrC (5/7, 71.43%) was not significantly different from that of UCa (71.43% vs. 77.50%, p=0.659). Imaging examination has the highest sensitivity and specificity among the accuracy evaluation of different diagnostic methods. Imaging and cystoscopy are the powerful diagnostic methods for UrC. Serum tumor markers may assist in diagnosis, prognosis, and monitoring. Positive urine FISH can easily misdiagnose UrC as UCa.

USP6-associated soft tissue tumors with bone metaplasia: Clinicopathologic and genetic analysis and the identification of novel USP6 fusion partners.

Among those tumors with consistent USP6 rearrangement, some arise from soft tissue and show bone metaplasia, including myositis ossificans (MO), fibro-osseous pseudotumor of digits (FOPD), soft tissue aneurysmal bone cyst (ST-ABC) and fasciitis ossificans (FO). These lesions are easily confused with malignancies because they show a rapid growth rate and brisk mitoses. Here, we aim to clarify the clinicopathologic and genetic characteristics of this entity and analyze the correlations among the different subtypes in one of the largest cohorts. The clinicopathologic features of 73 cases of MO, FOPD, ST-ABC and FO diagnosed at West China Hospital, Sichuan University from January 2010 to December 2021 were retrospectively analyzed. Forty-three undecalcified samples were analyzed by systematic genetic studies, including fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), Sanger sequencing and next-generation-based sequencing were performed. This series included 40 males and 33 females aged 2 to 80 years old (median: 31 years). FOPD occurred in extremal soft tissue, while lower extremities (38/58, 65.5%) were the most commonly involved lesions in the other three subgroups. Histologically, proliferative myofibroblasts/fibroblasts with varying degrees of osteoid tissue were present. Fluorescence in situ hybridization (FISH) results indicated that 22 cases (22/27, 81.5%) were positive for USP6 rearrangement, and 5 cases were negative. Among those cases with positive FISH results, 18 underwent reverse transcription-polymerase chain reaction (RT-PCR) detection that successfully detected common USP6 fusion types. Thirteen cases showed COL1A1::USP6 fusion, one showed MYH9::USP6 fusion, and 4 were negative for common fusion types. Next-generation-based sequencing technology was performed on two lesions with negative RT-PCR results and novel fusion partners SNHG3 and UBE2G1 were discovered. Our findings revealed that COL1A1 is the most common fusion partner in this entity, unlike primary aneurysmal bone cysts and nodular fasciitis. Notably, we believed that FO may demonstrate more similar clinicopathologic and genetic manifestations with MO/FOPD and ST-ABC instead of nodular fasciitis for involving lower limbs most frequently and showing recurrent COL1A1::USP6 fusion. Additionally, this study also found two novel USP6 fusion partners, which further expanded our knowledge of this neoplastic spectrum.

HER2 amplification by next-generation sequencing to identify HER2-positive invasive breast cancer with negative HER2 immunohistochemistry

BackgroundHuman epidermal growth factor receptor 2 (HER2) positive breast carcinomas due to HER2 amplification are associated with aggressive behavior and a poor prognosis. Anti-HER2-targeted therapies are widely used to treat HER2-positive breast carcinomas with excellent outcomes. Accurate identification of HER2 amplification status in breast carcinomas is of important diagnostic and treatment value. Currently, HER2 amplification status is routinely determined by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) testing. This study will review our past HER2 data to determine and characterize discordant results between HER2 IHC and FISH. It will also determine a potential impact of HER2 amplification status by next-generation sequencing (NGS) on these patients.MethodsWe reviewed a total of 4884 breast carcinomas with coexisting HER2 IHC and HER2 FISH performed at our institution from 2010 to 2022. 57 cases also had a Next-Generation-Sequencing-based (NGS) gene panel performed. Given the advances in biostatic analysis pipelines, NGS methods were utilized to provide results on HER2 amplification status along with somatic mutations.ResultsWhile the majority (ranging from 98.5% with IHC score of 0 and 93.1% with IHC score of 1 +) of 4884 breast carcinomas had concordant results from HER2 IHC and HER2 FISH testing, a small percentage of patients (ranging from 1.5% in those with IHC score of 0, to 6.9% with IHC score of 1 +) had discordant results, with negative HER2 IHC and positive HER2 FISH results. These patients could be reported as HER2-negative breast carcinomas if only HER2 IHC testing has been performed according to a current cost-effective HER2 test strategy. 57 patients had HER2 amplification status determined by NGS, and all patients had concordant results between HER2 NGS and FISH tests. A HER2-amplified breast carcinoma by NGS had a negative IHC and a positive HER2 FISH result. This case was classified as a HER2-positive breast carcinoma, had anti-HER2-targeted therapy, and achieved a complete clinical response.ConclusionsA small percentage of HER2-positive breast carcinomas are unidentified because of a negative HER2 IHC based on our current cost-effective HER2 test strategy. It is not feasible and affordable in routine clinical practice to perform HER2 FISH for the cases with negative HER2 IHC (IHC score 0 and 1 +). Therefore, NGS assays capable of simultaneously detecting both somatic mutations and HER2 amplification could provide a more comprehensive genetic profiling for breast carcinomas in a clinical setting. Identification of HER2 amplification by NGS in HER2-positive breast carcinomas with negative HER2 IHC results is important since these cases are concealed by our current cost-effective HER2 test strategy with IHC first (for all cases) and FISH reflex (only for cases with IHC score of 2 +), and would offer the opportunity for potentially beneficial anti-HER2-targeted therapies for these patients.

Fluorescence in situ hybridization (FISH) to predict the efficacy of Bacillus Calmette-Guérin perfusion in bladder cancer

Transurethral resection of bladder tumor (TURBT) combined with intravesical Bacillus Calmette-Guérin (BCG) perfusion is a widely accepted treatment for moderate or high risk non-muscular-invasive bladder cancer (BCa). Despite its effectiveness, the recurrence and progression rate of tumor are still high. We evaluated the predictive role of fluorescence in situ hybridization (FISH) for the response to BCG perfusion in BCa. Patients with BCa who underwent BCG perfusion and FISH test in our hospital were selected. Logistic regression and Kaplan-Meier methods were used to evaluate the relationship between FISH results and tumor recurrence or progression. COX proportional hazards regression analysis was used to identify risk factors for recurrence or progression. SPSS (version 24.0, IBM Corporation, USA) was used for statistical analysis. Seventy-six patients were included in this study, with a median age of 63.0 (55.0-70.0) years, and a median follow-up time of 19.0 (7.5-29.0) months. Fifteen patients relapsed after BCG perfusion. Before TURBT, 39 patients were positive for FISH and 20 were negative. There was no significant difference in the recurrence rate of BCG after perfusion predicted by preoperative FISH positive (10.3% vs. 10%, P=0.675). No association was found between preoperative FISH and tumor recurrence (P=0.955) or disease progression (P=0.186). After BCG perfusion, 10 patients were FISH positive and 14 patients were FISH negative. There was significant difference in recurrence rate of BCa predicted by positive FISH (100% vs. 7.1%, P<0.001). FISH results were significantly associated with tumor recurrence (P<0.001) and disease progression (P=0.001). Kaplan-Meier and univariate COX proportional hazards regression analysis clarified that FISH positive after BCG perfusion, tumor-node-metastasis (TNM) stage, and multiple tumors were risk factors for tumor recurrence and progression (P<0.05). Tumor TNM stage and FISH positive after BCG perfusion were independent risk factors for recurrence. Positive FISH after BCG perfusion can well predict the risk of recurrence and progression of BCa, and recurrence within six months is more likely. After BCG perfusion, it is better to recommend close follow up in patients with positive FISH.

The Role of Fluorescence In Situ Hybridization in the Surveillance of Non-Muscle Invasive Bladder Cancer: An Updated Systematic Review and Meta-Analysis.

Background: Fluorescence in situ hybridization (FISH) has become a popular biomarker for subsequent monitoring the recurrence of non-muscle invasive bladder cancer (NMIBC), several studies have investigated the ability of FISH to detect recurrence in the surveillance of NMIBC. However, the results were inconsistent. Methods: We conducted a systematic literature search extensively on authenticated databases including PubMed/Medline, Embase, Web of Science, Ovid, and Cochrane Library. Meta-analysis was performed to find out the sensitivity and specificity of FISH in predicting recurrence of NMIBC. Results: 15 studies were ultimately included in this meta-analysis, a total of 2941 FISH evaluations from 2385 NMIBC patients were available. The pooled sensitivity of FISH was 68% (95% CI: 0.58–0.76), and the pooled specificity was 64% (95% CI: 0.53–0.74). Subgroup analyses were performed in 7 studies without Bacillus Calmette–Guerin (BCG) treatment, the pooled sensitivity was 82% (95% CI: 0.68–0.90), and the pooled specificity was 63% (95% CI: 0.37–0.82). And in 9 studies using “UroVysion standard” to define positive FISH results showed a pooled sensitivity of 60% (95% CI: 0.50–0.70) and specificity of 70% (95% CI: 0.61–0.78). Conclusions: The findings of this study indicate that FISH has a satisfactory sensitivity (68%) and specificity (64%) and could be a potential biomarker in the surveillance of NMIBC. Moreover, BCG treatment and different FISH methods may have an impact on the sensitivity and specificity, these factors should be taken into account when making clinical strategy.

A New Multi-Color FISH Assay for Brush Biopsy-Based Detection of Chromosomal Aneuploidy in Oral (Pre)Cancer in Patients with Fanconi Anemia.

Simple SummaryPatients with Fanconi Anemia (FA) are at high risk of developing oral squamous cell carcinoma with poor prognosis. Oral brush biopsy–based cytology represents an attractive non-invasive advantage in the characterization of (pre) malignant oral lesions, but a sufficient number of abnormal cells is mandatory to make the diagnosis. The aim of this 2-phases retrospective study is to introduce a new multi-color FISH assay including a 9p21 FISH assay to determine the oral lesions that require treatment, with just a few cells. We demonstrate a good accuracy of the multicolor FISH assay if applied on oral brush biopsy-based specimens from FA patients, using different algorithms to determine chromosomal aneuploidy. Nevertheless, some false positive results were observed and the detection of a genetically altered field in the oral cavity as a possible reason for what may hamper the application of multi-color FISH is discussed.Background: Fanconi anemia (FA) is a rare inherited DNA instability disorder with a remarkably elevated risk of oral squamous cell carcinoma. These cancers can be detected with oral brush biopsy-based cytology even at early stages. This study aims to determine the diagnostic accuracy of a new multi-color fluorescent in situ hybridization (FISH) assay consisting of probes for CCND1, TERC, MYC and centromere of chromosome 6, as well as a 9p21 FISH assay consisting of probes for CDKN2A and centromere of chromosome 9 for the detection of oral (pre) malignant lesions in FA. Methods: (I) Cutoffs for the dichotomization of positive or negative multi-color FISH results are determined and (II) retrospectively validated by using archived oral brush biopsy specimens from individuals with Fanconi anemia. In addition, the specimens for cutoff determination were re-hybridized with the 9p21 FISH assay. Results: A cutoff of six or more chromosomal aneuploid cells for a positive FISH result was determined in the cutoff study on 160 biopsy specimens. The validating of this cutoff on 152 specimens showed at best a sensitivity of 87% and a specificity of 82.9%. Conclusion: Multi-color FISH is a sufficient tool to detect chromosomal aneuploidy in oral (pre) malignant lesions of individuals with Fanconi anemia. However, some false positive results may hamper the application as an adjuvant method to oral brush biopsy-based cytology in an oral cancer surveillance program.

Proof of an optimized salicylic acid paste-based treatment concept of ulcerative M2-stage digital dermatitis lesions in 21 dairy cows.

The efficacy of salicylic acid paste (SA) in the treatment of ulcerative bovine digital dermatitis (BDD) was assessed by combining clinical and histopathological analyses with molecular biological techniques. The latter were conducted in a blinded manner to reach maximum objectivity. Prior to treatment, M2-stage BDD lesions (n = 26, diagnosed in 21 dairy cows) exhibited ulceration, with severe perivascular, chronic, lymphoplasmacytic dermatitis and extensive keratinolysis being noted in most cases. Pretreatment biopsy samples (n = 12) followed by povidone-iodine ointment under bandage for one week before administration of SA paste were tested positive for Treponema spp. by blinded PCR and fluorescent in situ hybridization (FISH). Subsequent treatment consisted of application of SA and bandaging at weekly intervals until lesions had completely resolved. The treatment duration ranged between 2 and 4 weeks. Complete healing was achieved in 100% of cases, with 2/21 animals requiring a second round of treatment upon disease reoccurrence. Importantly, only 3/26 biopsies taken from previously affected sites still tested positive by Treponema PCR, and in another biopsy, the outermost layers of the stratum corneum scored weakly positive by Treponema-specific FISH. None of these Treponema DNA-positive biopsies showed signs of ulceration. One case exhibited focal keratinolysis. Positive PCR or FISH in these cases may have arisen from DNA traces of dead bacteria or environmental contamination during biopsy harvesting. To our knowledge, this is the first study on blinded molecular biological monitoring of the therapeutic efficacy of SA with respect to treponemal infection, and on complete BDD M2-stage remission in all animals achieved by SA treatment according to an optimized protocol. Although the etiology of BDD is considered as multifactorial, our data further support the concept that treponemes have a decisive role in BDD pathogenesis.

Health Supervision for Children and Adolescents With Down Syndrome.

Health Supervision for Children and Adolescents With Down Syndrome.

Identification of Pneumocystis jirovecii with Fluorescence In-Situ Hybridization (FISH) in Patient Samples-A Proof-of-Principle.

In resource-limited settings, where pneumocystosis in immunocompromised patients is infrequently observed, cost-efficient, reliable, and sensitive approaches for the diagnostic identification of Pneumocystis jirovecii in human tissue samples are desirable. Here, an in-house fluorescence in situ hybridization assay was comparatively evaluated against Grocott’s staining as a reference standard with 30 paraffin-embedded tissue samples as well as against in-house real-time PCR with 30 respiratory secretions from immunocompromised patients with clinical suspicion of pneumocystosis. All pneumocystosis patients included in the study suffered from HIV/AIDS. Compared with Grocott’s staining as the reference standard, sensitivity of the FISH assay was 100% (13/13), specificity was 41% (7/17), and the overall concordance was 66.7% with tissue samples. With respiratory specimens, sensitivity was 83.3% (10/12), specificity was 100% (18/18), and the overall concordance was 93.3% as compared with real-time PCR. It remained unresolved to which proportions sensitivity limitations of Grocott’s staining or autofluorescence phenomena affecting the FISH assay accounted for the recorded reduced specificity with the tissue samples. The assessment confirmed Pneumocystis FISH in lung tissue as a highly sensitive screening approach; however, dissatisfying specificity in paraffin-embedded biopsies calls for confirmatory testing with other techniques in case of positive FISH screening results. In respiratory secretions, acceptable sensitivity and excellent specificity were demonstrated for the diagnostic application of the P. jirovecii-specific FISH assay.