In-situ Hybridization Research Articles

Abstract A007: Transient elevation of ctHPVDNA in human papilloma virus-mediated oropharynx squamous cell carcinoma patients following diagnostic surgical procedures

Abstract Background: Circulating tumor DNA (ctDNA) is now widely used across a variety of malignancies as a viable “liquid biopsy” method, with applications in early detection, treatment response evaluation, prognostication, and recurrence monitoring. Recently, ctDNA derived from the tumor-integrated HPV (ctHPVDNA) has borne promising results in HPV-associated cancers, including HPV+ oropharyngeal squamous cell carcinoma (OPSCC). Studies indicate that undetectable ctHPVDNA after complete surgical excision in HPV+ OPSCC corresponds with improved prognosis. However, ctHPVDNA kinetics after diagnostic procedures such as transoral robotic surgery (TORS) in head neck squamous cell carcinoma from an unknown primary (HNSCCUP) or unknown primary has not been extensively studied. The purpose of this study is to show an unexpected trend in NavDx levels following these procedures. Methods: Patients undergoing TORS at Stanford University (September 2023 and August 2024) were prospectively screened and enrolled. A commercially available ctHPVDNA assay (NavDx) was collected at baseline, immediate postoperative (24-48 hours), 7-14 days following surgery, 3 months post- treatment and every 3 months thereafter up to 12 months. Results: Eight patients presented with HNSCCUP, 87.5% (7/8) had detectable baseline ctHPVDNA levels ranging from 2 to 1237. 75% (6/8) of the patients were confirmed p16+ by immunohistochemistry (IHC), while 25% (2/8) tested HPV+ by in situ hybridization (ISH). Diagnostic TORS was performed in 75% (6/8), while 25% (2/8) underwent direct laryngoscopy, with all primary tumors found in the oropharynx, 100% (8/8). Notably, 85.7% (6/7) of patients showed an unexpected elevation in total tumor mutational volume (TTMV) scores post-diagnostic procedure compared to baseline, with ctHPVDNA levels ranging from 30 to 2466, including one case where levels nearly quintupled. One patient had no post-diagnostic procedure TTMV score due to a negative baseline. However, in one other case, there was a negative post-procedure level. Among those with elevated post ctHPVDNA, 71.4% (5/7) underwent complete TORS excision, resulting in decreased postoperative ctHPVDNA levels in all patients, consistent with recent studies. Conclusion: This study provides insight that diagnostic procedures, such as TORS or direct laryngoscopy, in patients with p16+ HPV-mediated oropharyngeal cancer may lead to transient and unexpected elevations in ctHPVDNA levels, measured by TTMV scores. Although most patients showed a subsequent decrease in ctHPVDNA levels following a complete tumor resection, the observed post-diagnostic elevation emphasizes the importance of considering the timing of ctHPVDNA measurements. These findings suggest surgical manipulation may temporarily influence ctHPVDNA kinetics that could impact clinical decision-making and postoperative monitoring. Further research is needed to understand the implications of these transient elevations and optimizing the timing of ctHPVDNA assessments in clinical practice. Citation Format: Hlu Vang, Joanne Soo, Nikita Bedi, Andrea Garofalo, Michelle M Chen, Andrey Finegersh, Floyd Christopher Holsinger. Transient elevation of ctHPVDNA in human papilloma virus-mediated oropharynx squamous cell carcinoma patients following diagnostic surgical procedures [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A007.

Immunohistochemical Status Predicts Pathologic Complete Response to Neoadjuvant Therapy in HER2-Overexpressing Breast Cancers

Abstract Background Human epidermal growth factor receptor 2 (HER2) overexpression (HER2+) is defined by immunohistochemistry (IHC) and in situ hybridization (ISH) as IHC3+ or IHC2+/ISH+. Response differences to neoadjuvant anti-HER2 therapy (NT) in IHC3+ versus IHC2+/ISH+ breast cancer patients are poorly characterized. We explored whether pathologic complete response (pCR) varies by HER2 IHC status. Methods Patients with stage I–III HER2+ breast cancer undergoing NT and surgery between 2013 and 2020 were identified from the National Cancer Database and stratified by IHC status. Breast and nodal pCR were analyzed. Results Of 40,711 HER2+ patients, 83% were IHC3+ and 17% were IHC2+/ISH+. IHC3+ patients were more likely to be hormone receptor (HR)-negative (33 vs. 21%), have cT3/4 tumors (24 vs. 21%), and be cN+ (52 vs. 47%; all p < 0.0001). Breast conservation rates were similar (each 43%, p = 0.32), although IHC3+ axillary lymph node dissection rates were lower (41 vs. 45%, p < 0.0001). Among all patients, breast pCR was 49%, while nodal pCR was 64%. Compared with IHC2+/ISH+, IHC3+ had higher unadjusted breast (54 vs. 22%, p < 0.0001) and nodal (69 vs. 37%, p < 0.0001) pCR rates. When stratified by HR status, pCR was lower for HR+ disease but remained higher among IHC3+ patients. Analysis of T1cN0 primaries mirrored these trends. In multivariable analysis, IHC3+ remained an independent predictor of breast (odds ratio [OR] 3.91, confidence interval [CI] 3.65–4.19, p < 0.0001) and nodal (OR 3.40, CI 3.12–3.71, p < 0.0001) pCR. Conclusion HER2 IHC status predicts pCR and may help select breast cancer patients who derive the greatest benefit from NT. These findings provide further evidence that revision of HER2 classification may improve clinical management.

Detection of Gram-positive and Gram-negative bacteria in brain abscesses by 16S rRNA in situ hybridization.

In situ hybridization (ISH) staining of bacterial 16S ribosomal RNA (rRNA) is an alternative to standard histological stains (eg, Gram, Warthin-Starry), and may improve the diagnosis of bacterial brain abscesses. To evaluate the utility of 16S rRNA ISH, a 10-year retrospective cohort was assembled from a large academic medical center. Results of histological stains, cultures, and 16S rRNA sequencing were extracted from reports, and new Gram and 16S rRNA ISH stains were performed. Histologically identifiable bacteria were present in 40/63 (63%) cases and 38/57 (67%) were associated with positive cultures. Overall, 16S rRNA ISH was positive in 18/63 (29%) cases, including 16/37 (43%) with positive Gram stains, 12/38 (32%) positive by culture, and 4/8 (50%) positive by sequencing. 16S rRNA ISH highlighted bacteria in 14/40 (35%) cases with Gram-positive organisms and 9/17 (53%) with Gram-negative organisms (including 6 polymicrobial cases). Compared to a composite gold standard of Gram stain and culture, the sensitivity and specificity of 16S rRNA ISH were 35% and 93%, respectively. While sensitivity is relatively low, 16S rRNA ISH may be useful for distinguishing real organisms from artifacts and for identifying brain abscess cases suitable for 16S rRNA sequencing.

High-grade squamous intraepithelial lesions and adenocarcinoma insitu with a negative HPV cervical screening test. The role of HPV-ISH testing: A retrospective review.

Australia has transitioned to primary Human Papillomavirus (HPV) screening; however, high-risk HPV (hrHPV)-negative high-grade squamous intraepithelial lesions and adenocarcinoma insitu have been reported. HPV insitu hybridisation (ISH) testing has been proposed to reclassify these cases. This study identified hrHPV-negative lesions and assessed HPV-ISH. A total of 89 of 1468 patients (6.06%) had hrHPV-negative lesions, and HPV-ISH revealed five (5.75%) reclassified positive cases. No demographical differences were found between groups. Current population-level screening is effective. HPV-ISH was not effective for reclassification. This small, significant population requires further study to assess the phenomenon and augment detection given the implications of misidentification.

A powerful and versatile new fixation protocol for immunostaining and in situ hybridization that preserves delicate tissues

BackgroundUnderstanding how genes function to heal wounds and restore lost tissue is essential for studying regeneration. Whole-mount in situ hybridization (WISH) is a powerful and widely used technique to visualize the expression patterns of genes in different biological systems. Yet, existing methods to permeabilize samples for WISH can damage or destroy fragile regenerating tissues, thereby preventing such experiments.ResultsHere, we describe a new protocol for in situ hybridization (ISH) and immunostaining in the highly regenerative planarian Schmidtea mediterranea. This new Nitric Acid/Formic Acid (NAFA) protocol is compatible with both the assays and prevents degradation of the epidermis and regeneration blastema. The NAFA protocol achieves this without the use of proteinase K digestion which likely leads to better preservation of antigen epitopes. We show that the NAFA protocol successfully permits development of chromogenic and fluorescent signals in situ, while preserving the anatomy of the animal. Furthermore, the immunostaining of different proteins was compatible with the NAFA protocol following fluorescent in situ hybridization. Additionally, the tissue fixation protocol was easily adapted for regenerating killifish tail fin, which yielded better ISH signal with minimal background.ConclusionsThus, the NAFA protocol robustly preserves the delicate wounded tissues while also facilitating probe and antibody penetration into internal tissues. Furthermore, the fixation protocol is compatible for WISH on regenerating teleost fins suggesting that it will be a valuable technique for studying the processes of wounding response and regeneration in multiple species.

Evidence for Unified Assessment Criteria of HER2 IHC in Colorectal Carcinoma

HER2 expression is an important biomarker for the management of RAS wild-type metastatic colorectal carcinoma (CRC). Immunohistochemistry (IHC) with reflex in situ hybridization (ISH) is accepted as a standard method of assessment, yet there are currently two sets of criteria used to interpret results: the HERACLES criteria, and the My Pathway criteria. The HERACLES criteria require ISH confirmation when IHC staining is 3+ in 10-49% of cells, while the My Pathway criteria mirror those for gastric HER2 assessment and do not recommend ISH confirmation in the previously referenced scenario. We aimed to assess the prevalence of HER2 3+ heterogeneity and its association with ERBB2 copy number amplification (CNA) to evaluate the necessity of ISH testing when IHC staining is 3+ in <50% of cells.Next-generation sequencing (NGS) of DNA (592-gene panel or whole exome sequencing; WES) was performed for 13 208 colorectal carcinoma (CRC) tumors submitted to Caris Life Sciences (Phoenix, AZ). HER2 (4B5) expression was tested by IHC. A subset of tumors was tested for ERBB2 amplification via chromogenic ISH and/or via NGS (copy number amplification [CNA]). X2/Fisher-Exact tests were applied where appropriate, with p-values adjusted for multiple comparisons (p< 0.05).Of 13 208 CRCs with HER2 IHC, 87.4% (11 541/13 208) were negative for HER2 expression (intensity ≤3+ and < 10% tumor-cell staining) and 11.2% (1 473/13 208) demonstrated at least low HER2 expression (1-2+ and ≥10%). Only 1.5% (194/13 208) of all tested tumors were either positive or heterogeneously positive for HER2 overexpression (3+, ≥10%). Of these, 14% (28/194) had heterogenous HER2 overexpression (3+ staining of 10-49% of cells). Among 22 HER2-positive/heterogenous cases with successful ISH testing, 100% (22/22) demonstrated amplification via ISH.Because the classification of tumors as HER2-positive/heterogenous by IHC correlated very closely with ISH positivity, our results suggest that ISH is likely unnecessary for CRCs with 3+ HER2 overexpression in 10-49% of neoplastic cells.

Characterization of the wheat-tetraploid Thinopyrum elongatum 7E(7D) substitution line with Fusarium head blight resistance

BackgroundFusarium head blight (FHB), a devastating disease of wheat production, is predominantly elicited by Fusarium graminearum (Fg). The tetraploid Thinopyrum elongatum is a tertiary gene resource of common wheat that possesses high affinity and displays high resistance traits against multiple biotic and abiotic stress. We aim to employ and utilize the novel FHB resistance resources from the wild germplasm of common wheat for breeding.ResultsDurum wheat-tetraploid Th. elongatum amphiploid 8801 was hybridized with common wheat cultivars SM482 and SM51, and the F5 generation was generated. We conducted cytogenetically in situ hybridization (ISH) technologies to select and confirm a genetically stable 7E(7D) substitution line K17-1069-5, which showed FHB expansion resistance in both field and greenhouse infection experiments and displayed no significant disadvantage in agronomic traits compared to their common wheat parents in the field. The F2 segregation populations (K17-1069-5 × SM830) showed that the 7E chromosome conferred dominant FHB resistance with dosage effect. We developed 19 SSR molecular markers specific to chromosome 7E, which could be conducted for genetic mapping and large breeding populations marker-assisted selection (MAS) during selection procedures in the future. We isolated a novel Fhb7 allele from the tetraploid Th. elongatum chromosome 7E (Chr7E) using homology-based cloning, which was designated as TTE7E-Fhb7.ConclusionsIn summary, our study developed a novel wheat-tetraploid Thinopyrum elongatum 7E(7D) K17-1069-5 substitution line which contains stable FHB resistance.

Interleukin‐6 transcripts up‐regulation in lymph nodes from unicentric and multicentric Castleman disease

AbstractIntroductionCastleman disease (CD) represents a spectrum of heterogeneous lymphoproliferative disorders sharing peculiar histopathological features, clinically subdivided into unicentric CD (UCD) and multicentric CD (MCD) and presenting with variable inflammatory symptoms. Interleukin (IL)‐6 and other cytokines play a major role in mediating CD inflammatory manifestations. Although the local microenvironment seems to be among the major sources of hypercytokinemia, the precise cellular origin of IL‐6 production in CD is still debated.MethodsA series of five nodal CD of different subtypes (one UCD, two idiopathic MCDs [iMCDs], one HIV‐negative human herpesvirus 8 (HHV8)‐associated MCD, and one HIV‐positive HHV8‐associated MCD) and a non‐CD reactive control were tested using RNAscope analysis and a dual in situ hybridization (ISH)/immunohistochemistry technique, in order to quantify IL‐6 expression and its spatial distribution. Quantitative analyses of in situ mRNA were performed on digitalized slides using the HISTOQUANT software (3DHISTECH) and differences between cases were evaluated by the Kruskal‐Wallis test.ResultsRNA‐ISH documented increased IL‐6 expression in all CD lymph nodes, independently from clinical and pathological subtypes, however, the highest levels were found in HHV8+ cases and statistically significant differences in IL‐6 expression were found only between HHV8+ MCD and control case. Dual RNA‐ISH for IL6 coupled with immunohistochemistry analysis showed that IL‐6 was overexpressed in CD31‐positive endothelial cells in 5/5 CD tested cases but not in the control case.ConclusionOur findings suggest that nodal IL‐6 expression seems to be significantly upregulated in HHV8+ MCD, but a trend toward increased nodal IL‐6 expression was noticed also in UCD and iMCD‐not otherwise specified. CD31+ endothelial cells probably represent one of the major sources of IL‐6 production in the nodal microenvironment.

Uncommon and Challenging Phenotypes of High-Risk Human Papillomavirus-Associated Head and Neck Carcinomas Revealed by High-Throughput Studies.

HPV- associated squamous cell carcinoma (SCC) is uncommon in non-oropharynx sites and not well characterized. This study aims to investigate uncommon phenotypes of HPV-associated head and neck carcinoma, the prevalence and morphologic spectrum of HPV-associated SCC in the oral cavity, larynx and hypopharynx. P16 immunostaining and HPV E6/7 in situ hybridization (ISH) were performed on tissue microarrays comprised of SCCs from different anatomic sites: oropharynx (n = 270), hypopharynx (n = 52), oral cavity (n = 95) and larynx (n = 123). Tumors were classified as HPV-associated based on a positive E6/7 ISH testing. RNA sequencing was performed on several selected cases. 66% oropharynx SCCs (OPSCCs) were HPV-associated; all were p16/HPV testing concordant except one which was p16 negative. The p16-/HPV + OPSCC resembled similar gene expression signature with p16+/HPV + OPSCCs by transcriptome analysis. 6/95 (6%) oral cavity SCCs were HPV-associated, all from male patients and 5/6 (83%) arose from the floor of mouth. Morphologically, 3/6 (50%) showed keratinizing SCC and 5/6 (83%) demonstrated HPV-associated squamous dysplasia in adjacent mucosa. 1/123 (less than 1%) larynx SCCs and 0/52 hypopharynx SCCs were HPV-associated. Although uncommon, p16 negative HPV-associated OPSCC can occur, emphasizing the importance of judicious HPV testing. The morphology of HPV-associated oral cavity SCCs may deviate from prototypic nonkeratinizing SCC, making them difficult to recognize. Presence of HPV-associated squamous dysplasia could serve as a morphologic clue.

Mapping RANKL- and OPG-expressing cells in bone tissue: the bone surface cells as activators of osteoclastogenesis and promoters of the denosumab rebound effect.

Denosumab is a monoclonal anti-RANKL antibody that inhibits bone resorption, increases bone mass, and reduces fracture risk. Denosumab discontinuation causes an extensive wave of rebound resorption, but the cellular mechanisms remain poorly characterized. We utilized in situ hybridization (ISH) as a direct approach to identify the cells that activate osteoclastogenesis through the RANKL/OPG pathway. ISH was performed across species, skeletal sites, and following recombinant OPG (OPG:Fc) and parathyroid hormone 1-34 (PTH) treatment of mice. OPG:Fc treatment in mice induced an increased expression of RANKL mRNA mainly in trabecular, but not endocortical bone surface cells. Additionally, a decreased expression of OPG mRNA was detected in bone surface cells and osteocytes of both compartments. A similar but more pronounced effect on RANKL and OPG expression was seen one hour after PTH treatment. These findings suggest that bone surface cells and osteocytes conjointly regulate the activation of osteoclastogenesis, and that OPG:Fc treatment induces a local accumulation of osteoclastogenic activation sites, ready to recruit and activate osteoclasts upon treatment discontinuation. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data from murine bone marrow stromal cells revealed that Tnfsf11+ cells expressed high levels of Mmp13, Limch1, and Wif1, confirming their osteoprogenitor status. ISH confirmed co-expression of Mmp13 and Tnfsf11 in bone surface cells of both vehicle- and OPG:Fc-treated mice. Under physiological conditions of human/mouse bone, RANKL is expressed mainly by osteoprogenitors proximate to the osteoclasts, while OPG is expressed mainly by osteocytes and bone-forming osteoblasts.

Discordance of human epidermal growth factor receptor 2-low status between breast primary and distant metastases with clinical-pathological correlation.

Breast cancer with human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) 1+ or 2+ with negative in-situ hybridisation (ISH) (HER2-low) can now be targeted by HER2 antibody drug conjugates. We set out to compare HER2 status between matched primary invasive breast carcinoma (IBC) and distant metastases (DM) with clinical-pathological correlation, with specific interest in HER2-low. Biomarker studies and clinical-pathological features of primary IBC with matched DM diagnosed between 2021 and 2022 were retrospectively analysed. HER2 status was assessed per 2023 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines for IHC (4B5) and ISH (IQFISH pharmDX). Bilateral breast primaries were excluded. HER2 IHC 0 to 1+ were reassessed. One hundred and forty-seven cases of primary IBC with matched DM were identified. Biomarkers were performed on core biopsy (n = 74) and resection (n = 73). One hundred and twenty-six (86%) were initially classified as 'HER2-negative'; of these, 67 (46%) were reclassified as HER2-low. Patients with HER2-positive primaries were younger (P = 0.01) and had an increased incidence of micropapillary carcinoma (P = 0.02). HER2-low primaries also had an increased incidence of micropapillary carcinoma (P = 0.02) and oestrogen receptor (ER) positivity (P = 0.02) compared to HER2 0. One hundred and sixty-nine matched DM cases excluding bone metastasis were identified (range = one to seven metastases per IBC). The most common sites of metastases were liver (50 of 169, 30%), lung (36 of 169; 21%), distant lymph node (26 of 169, 15%); 138 DM cases (82%) were previously classified as 'HER2-negative', and 62 (37%) were reclassified as HER2-low. Like HER2-low primaries, HER2-low metastases were frequently ER-positive (52 of 62; 84%) (P = 0.02). Brain metastases were more frequently HER2-positive (five of 32; 16%) (P = 0.04). Comparing HER2 status in matched primaries and DM, HER2 status was discordant in 62 cases (37%). Most changes occurred from HER2-low to HER2 0 (33 of 169, 20%), HER2 0 to HER2-low (17 of 169, 10%) and HER2-low to positive (10 of 169, 6%). All HER2-low to HER2 0 changes were HER2 1+ to 0. In 30 patients with multiple DM sites (47 cases), HER2 status among different DM samples was discordant in 16 patients (53%), mainly from HER2-low to HER2 0 (16 of 47, 34%). A significant proportion of previous 'HER2-negative' primaries and DM cases were reclassified as HER2-low. Discordant HER2 status between IBC primary and metastasis and between different DM sites demonstrated tumour heterogeneity and highlights the need for HER2 retesting in distant metastasis.

Evaluation of Automatic Signal Detection of In Situ Hybridization for Detecting HPV DNA in Cervical Tissue Derived from Patients with Cervical Intraepithelial Neoplasia

Background: Cervical cancer is fourth the most common cancer in women worldwide. Due to the prevalence of human papillomavirus (HPV) in the population (80–90%), scientists are likely to discover even more associations of this pathogen with other diseases in the future. In recent years, In Situ Hybridization (ISH) assays that use automated signal-detecting methods in formalin-fixed, paraffin-embedded (FFPE) cervical tissue, such as the enzyme-categorized signal-detecting system, have shown a higher sensitivity. Objectives and Methods: To evaluate automatic signal detection of ISH assay for detecting HPV DNA, we compared the ability of an ISH probe, Inform HPV II and III (Ventana Medical Systems, Tucson, AZ), to that of PCR assays to detect HPV DNA in cervical tissue specimens with cervical intraepithelial neoplasia (CIN; CIN 1, 28 cases; CIN 2, 22 cases; and CIN 3, 20 cases) and normal cervix (2 cases). Results: Our findings showed a significant relation was confirmed between ISH III level and HPV outcome (positive/negative). Patients with positive HPV outcomes had significantly lower ISH III levels, MD = −7961.82 CI95 [−17,230.00; −199.21], p = 0.005. Conclusions: Automatic signal detection of ISH assay is not particularly applicable to cervical tissue material. A more useful method of confirming the presence of HPV in the cervix is the HPV test with genotyping, as it allows for collecting a larger amount of material from the cervical disc and canal. The interpretation of a positive or negative ISH test must be guided in the context of clinical history and morphology.

The distribution of Hypocretin/Orexin receptor mRNA in the mouse and human brain

Hypocretin (Hcrtr, HCRTR) / orexin (OX) receptors modulate a range of neurobiological functions and are drug targets for several disorders. Mapping the distribution of receptors in the brain can inform their function and guide targeting of specific disorders. Although studied in rodents, orexin receptor distribution has remained relatively unexplored in humans, and thus there is also a paucity of comparative anatomy. The aim of this study was therefore to map the distribution of hypocretin/orexin receptor mRNA in selected regions of the mouse and human brain by non-radioactive in situ hybridization (ISH) using digoxigenin (DIG)-labelled cRNA anti-sense probes. Data revealed both distinct and overlapping patterns of distributions of Hcrtr1/HCRTR1 and Hcrtr2/HCRTR2 mRNA suggesting that the functions of the orexin system are mediated differently by each receptor. In the mouse brain, the highest expression of Hcrtr1 mRNA was in the locus coeruleus (LC) whereas Hcrtr2 mRNA was most abundant in the lateral hypothalamus (LH). The human caudate nuclei showed significant expression of both HCRTR1 and HCRTR2 mRNA, whereas the mouse predominantly expressed Hcrtr2 mRNA. The noradrenergic neurons of the human LC showed high signals for both HCRTR1 (71.7%) and HCRTR2 (81.5%) mRNA. Expression of HCRTR2 mRNA in non-noradrenergic human LC cells was also notable. The distribution pattern in mouse and human brains is consistent with the involvement of the orexin system in arousal and the sleep/wake cycle in both species, however, variations in receptor subtype expression profiles suggests that species differences in responses to orexin receptor ligands may be expected.

TLR4 Downregulation Identifies High-Risk HPV Infection and Integration in H-SIL and Squamous Cell Carcinomas of the Uterine Cervix.

Growing scientific evidence suggests a link between the expression of toll-like receptor 4 (TLR4) and cervical cancer carcinogenesis. Specifically, a close relation between TLR4 expression and FIGO stage, lymph node metastases, and tumor size has been reported in cervical cancer. In the present study, we aimed to evaluate the relationship between TLR4 expression levels and human papillomavirus (HPV) infection and/or high-risk (hr) HPV integration status in patients with a histological diagnosis of high-grade squamous intraepithelial lesion (H-SIL), and squamous cell carcinoma (SCC) of the uterine cervix. Sixty biopsies of cervical neoplasia, comprising H-SIL (n = 20) and SCC (n = 40), were evaluated for TLR4 expression by immunohistochemistry. All samples were positive for high-risk HPV as confirmed by in situ hybridization (ISH) and broad-spectrum PCR followed by Sanger sequencing analysis. The intensity of TLR4 staining was higher in tissues negative for intraepithelial lesion or malignancy (NILM) than in H-SIL, and further reduced in SCC. Moreover, statistically significant differences have been observed in the percentage of TLR4 expression between NILM and H-SIL and between H-SIL and SCC, with higher percentages of expression in H-SIL than in SCC. Our results showed a significant downregulation of TLR4 in HPV-related H-SIL and SCC, compared to NILM. These data support the hypothesis that TLR4 expression is suppressed in HPV-driven oncogenesis.

Clinical characterization, prognostic, and predictive values of HER2-low in patients with early breast cancer in the PALLAS trial (ABCSG-42/AFT-05/BIG-14–13/PrE0109)

BackgroundBidirectional crosstalk between HER2 and estrogen receptor (ER) pathways may influence outcomes and the efficacy of endocrine therapy (ET). Low HER2 expression levels (HER2-low) have emerged as a predictive biomarker in patients with breast cancer (BC).MethodsPALLAS is an open, international, phase 3 study evaluating the addition of palbociclib for 2 years to adjuvant ET in patients with stage II-III ER-positive/HER2-negative BC. To assess the impact of HER2 expression on patient outcomes in the phase III PALLAS trial, we analyzed (1) the association between rate of HER2-low with demographic and clinicopathological parameters, (2) the prognostic value of HER2-low status on invasive disease-free survival (iDFS), distant relapse-free survival (DRFS), and overall survival (OS) and (3) HER2 expression’s value as a predictive biomarker of response to palbociclib. HER2-low was defined as HER2 immunohistochemistry (IHC) 1 + or IHC 2 + with negative in situ hybridization (ISH). All pathologic evaluation was performed locally. Prognostic and predictive power of HER2 were assessed with Cox models.ResultsFrom the original PALLAS intention-to-treat population (N = 5753), 5304 patients (92.2%) were included in this analysis. Among these, 2254 patients (42.5%) were classified as having HER2 IHC 0 (HER2-0), and 3050 (57.5%) as having HER2-low disease (1838 with IHC 1 + and 1212 with IHC 2 +). Median follow-up was 59.8 months. HER2-low prevalence varied significantly across 21 participating countries (range 16.7% to 75.6%; p < 0.001) and was more frequent in patients enrolled in North America (63.1%) than in Europe (53.4%) or other regions (53.4%) (p < 0.001). HER2 status was not significantly associated with iDFS in a multivariable Cox model (hazard ratio 0.93, 95% confidence interval 0.81 – 1.06). No significant interaction was observed between treatment arm and HER2 status for iDFS (p = 0.43). Similar results were obtained for DRFS and OS.ConclusionsIn this large, prospective, global patient cohort, no differences were observed in clinical parameters, prognosis, or differential benefit from palbociclib between HER2-0 and HER2-low tumors. Significant geographic variability was observed in the prevalence of HER2-low status, suggesting a high degree of variation in pathologic assessment of HER2 expression without impact on outcomes.

Detecting Cholangiocarcinoma in the Setting of Primary Sclerosing Cholangitis: Is Biliary Tract Fluorescence InSitu Hybridization Helpful?

Biliary brushing cytology (BB) to detect cholangiocarcinoma (CCA) is integral in the surveillance of patients with primary sclerosing cholangitis (PSC). Since reactive changes can mimic carcinoma, indeterminant results are frequent. Fluorescence insitu hybridization (FISH) using the UroVysion probe set has been advocated to enhance the detection of CCA. This study evaluates the performance of FISH for detecting CCA in patients with and without PSC. A query of our pathology database for atypical and suspicious BB with concurrent FISH results was performed from 2014 to 2021. FISH (using UroVysion probe set containing centromere enumeration probes to chromosomes 3, 7, and 17) was positive if at least 5 cells demonstrated polysomy. Electronic medical records were reviewed to identify patients with PSC and CCA. CCA was confirmed by pathology or clinical impression. Of the 65 patients (103 BB) in the PSC cohort, 59 patients (94 BB) without CCA and 6 patients (9 BB) with CCA were identified. 33 non-PSC patients (41 BB) with CCA were included for comparison. Positive FISH was highest in non-PSC patients with CCA (10/41 BB, 24%). Positive FISH was seen in both PSC with (1/9 BB, 11%) and without (2/94 BB, 2%) CCA. FISH positivity was lower than expected and was positive in PSC patients without CCA. These results question the clinical utility of FISH for CCA surveillance in PSC patients.

Clinicopathologic Features and Digital Imaging Analysis of HER2 Protein in Breast Carcinomas With Different HER2 Fluorescence in Situ Hybridization Patterns

BackgroundHER2-targeted therapies have significantly improved outcomes for patients with HER2-positive breast cancer (BC), which represents 15% to 20% of all BC cases. HER2 status is assessed via immunohistochemistry (IHC) and/or in situ hybridization (ISH), dividing BCs into five groups (G1-G5). Patients and methodsIn a study of 2,702 primary BC cases, comprising 12.7% G1, 0.2% G2, 2.8% G3, 8.5% G4, and 75.9% G5, we analyzed clinicopathologic features and HER2 protein expression digitally for each ISH group. ResultsNotably, G5 cases had a higher proportion of lobular carcinoma (13.9%) compared to other groups. G3 cases showed the highest percentage of grade 3 tumors (56.9%), while G5 cases had the lowest (21.4%). Additionally, G5 cases had the highest rate of estrogen receptor (ER) positivity (84.6%), while G1-HC (high copy number) cases had the lowest (70.4%). Most G1-HC cases were HER2 IHC 3+ (76.1%), while most G5 cases were IHC 0/1+ (75.7%). IHC 2+ was most common in G1-LC (low copy number) and G3 cases (83.8% and 90.7%, respectively), with G4 cases predominantly IHC 2+ (56.3%) and IHC 1+ (30.1%). Discordant HER2 IHC and ISH results were observed in 12 cases (0.4%), including 7 G1-HC (2.3%), 4 G1-LC (10.8%), and 1 G5 case (0.1%). Digital quantification of HER2 IHC levels in all groups except G5 revealed that G1-HC tumors had the highest HER2 protein expression, followed by G3, with G4 showing the lowest. ConclusionThese findings offer valuable insights into the clinicopathologic characteristics and future management for different HER2 ISH groups.

A Novel HER2 Protein Identification Methodology in Breast Cancer Cells Using Raman Spectroscopy and Raman Imaging: An Analytical Validation Study.

Conventional assays such as immunohistochemistry (IHC) and in situ hybridization (ISH) used in clinical procedures for quantification of the human epidermal growth factor receptor-2 (HER2) status in breast cancer have many limitations. In the current study, we have used HER2 expression in a broad range of breast cancer phenotypes to explore the potential utility of a novel immunodetection technique using Raman spectroscopy and Raman imaging combined with artificial intelligence models. The correlations between the Raman method and conventional HER2 testing methodologies (IHC and ISH) have been tested. Raman measurements showed a strong linear correlation (p = 0.05, R2=0,9816) with IHC analysis in the studied breast cell lines: MCF-10A, MCF-7, MDA-MB-231, HTB-30 (SK-BR-3), and AU-565 represent normal, nontumorigenic epithelial cells, triple-positive breast carcinoma, and triple-negative breast cancer cell lines. Analytic testing of Raman spectroscopy and Raman imaging demonstrated that this method may offer advantages over the currently used diagnostic methodologies.

Review of In Situ Hybridization (ISH) Stain Images Using Computational Techniques.

Recent advancements in medical imaging have greatly enhanced the application of computational techniques in digital pathology, particularly for the classification of breast cancer using in situ hybridization (ISH) imaging. HER2 amplification, a key prognostic marker in 20-25% of breast cancers, can be assessed through alterations in gene copy number or protein expression. However, challenges persist due to the heterogeneity of nuclear regions and complexities in cancer biomarker detection. This review examines semi-automated and fully automated computational methods for analyzing ISH images with a focus on HER2 gene amplification. Literature from 1997 to 2023 is analyzed, emphasizing silver-enhanced in situ hybridization (SISH) and its integration with image processing and machine learning techniques. Both conventional machine learning approaches and recent advances in deep learning are compared. The review reveals that automated ISH analysis in combination with bright-field microscopy provides a cost-effective and scalable solution for routine pathology. The integration of deep learning techniques shows promise in improving accuracy over conventional methods, although there are limitations related to data variability and computational demands. Automated ISH analysis can reduce manual labor and increase diagnostic accuracy. Future research should focus on refining these computational methods, particularly in handling the complex nature of HER2 status evaluation, and integrate best practices to further enhance clinical adoption of these techniques.

Multi-marker analysis of circulating tumor cells in localized intermediate/high-risk and metastatic prostate cancer

AbstractCirculating tumor cells (CTCs) are an established prognostic marker in metastatic prostate cancer (PrC) but have received little attention in localized high-risk disease. Peripheral blood was obtained from patients with early intermediate and high-risk PrC (n = 15) at baseline, after radiotherapy, and during follow-up, as well as from metastatic PrC patients (n = 23). CTCs were enriched using the microfluidic Parsortix® technology. CTC-related marker were quantified with qPCR and RNA in-situ hybridization (ISH). Positivity and associations to clinical parameters were assessed using McNemar test, Fisher Exact test or log-rank test. The overall positivity was high in both cohorts (87.0% metastatic vs. 66.7% early at baseline). A high concordance of qPCR and RNA ISH was achieved. In metastatic PrC, PSA and PSMA were prognostic for shorter overall survival. In early PrC patients, an increase of positive transcripts per blood sample was observed from before to after radiation therapy, while a decrease of positive markers was observed during follow-up. CTC analysis using the investigated qPCR marker panel serves as tool for achieving high detection rates of PrC patient samples even in localized disease. RNA ISH offers the advantage of confirming these markers at the single cell level. Employing the clinically relevant marker PSMA, our CTC approach can be used for diagnostic purposes to screen patients profiting from PSMA-directed PET-CT or PSMA-targeted therapy.