Probes for INS

The fibroblast growth factor receptor FGFR2 is overexpressed in a variety of solid tumors, including breast, gastric and ovarian tumors, where it offers a potential therapeutic target. In this study, we present evidence of the preclinical efficacy of BAY 1187982, a novel antibody-drug conjugate (ADC). It consists of a fully human FGFR2 monoclonal antibody (mAb BAY 1179470), which is specific for the FGFR2 isoforms FGFR2-IIIb and FGFR2-IIIc, conjugated through a non-cleavable linker to a novel derivative of the microtubule-disrupting cytotoxic drug auristatin (FGFR2-ADC). In FGFR2-expressing cancer cell lines, this FGFR2-ADC exhibited potency in the low nM to sub-nM range and was more than 100-fold selective against FGFR2-negative cell lines. High expression levels of FGFR2 in cells correlated with efficient internalization, efficacy and cytotoxic effects in vitro. Pharmacokinetic analyses in mice bearing FGFR2-positive NCI-H716 tumors indicated that the toxophore metabolite of FGFR2-ADC was enriched more than 30-fold in tumors compared to healthy tissues. Efficacy studies demonstrated that FGFR2-ADC treatment leads to a significant tumor growth inhibition or tumor regression of cell line-based or patient-derived xenograft models of human gastric or breast cancer. Further, FGFR2 amplification or mRNA overexpression predicted high efficacy in both of these types of in vivo model systems. Taken together, our results strongly support the clinical evaluation of BAY 1187982 in cancer patients, and a Phase I study (NCT02368951) has been initiated.

Abstract

Abstract

Purpose

High-risk human papillomavirus (HR-HPV) infections was the causal factor in the development of cervical cancer, but the significance of HPV viral load in the prediction of the response to current therapeutic approaches had not reached consensus. The present study was performed to assess the high risk HPV viral load of cervical cancer patients who underwent radiotherapy alone or in combination with chemotherapy or hyperthermotherapy or both in correlation to long-term survival.

Methods

116 cervical cancer patients were recruited and assigned into four groups of different therapeutic modalities. The prevalent high risk types of HPV 16, 18, 58 were detected by type specific in situ hybridization (ISH), and HPV mRNA was detected by RNA scope assay using RNA scope 2.0 FFPE Reagent Kit. Semi-quantification of the HR-HPV viral load was measured based on the intensity of ISH signal captured from the tumor nests in the grey scale.

Results

The HR-HPV viral load had a significant negative correlation with survival (r_s = −0.368,P = 0.001). The 15-year survival rate of low viral load group was 68.18%, moderate viral load group was 52.17%, and high viral load group was 34.69% (P = 0.001). HPV mRNA expression was strongly consistent with HPV viral load. The 15-year survival rates of different therapeutic groups were 39.29%, 58.62%, 50.00%, 55.17%, respectively (P = 0.545). Combinatorial treatment modalities improved the actual survival, which demonstrated no significant difference among 5,10 and 15 years comparison. Cox regression analysis showed that the relative risk of death was obviously higher in the HPV 18 single positive group and high HPV viral load group.

Conclusions

The semi-quantitive viral load assessment in situ is a feasible approach in clinical practice. The more the HPV viral load was, the worse the survival of patients would be. The combinational treatments were in favor of the disease-stabilization.

Human papillomavirus (HPV) infection modulates several host cytokines contributing to cancer development. Oncostatin M (OSM), an IL-6 family cytokine, acts to promote cell senescence and inhibit growth. Its dysregulation promotes cell survival, cell proliferation and metastasis in various malignancies. The effect of HPV on OSM dysregulation has not been investigated. To elucidate this, immunohistochemistry was used on formalin-fixed, paraffin-embedded oral squamous cell carcinoma (OSCC) tissues: HPV-positive (50) and HPV-negative (50) cases. Immortalized human cervical keratinocytes expressing HPV16E6 (HCK1T, Tet-On system) were used to demonstrate the role of HPV16E6 in OSM expression. In addition, a vector containing HPV16E6/E7 was transiently transfected into oral cancer cell lines. Cell viability, cell-cycle progression and cell migration were evaluated using flow cytometry and a wound healing assay, respectively. The results showed various intensities of OSM expression in OSCC. Interestingly, the median percentages of strongly stained cells were significantly higher in HPV-positive OSCCs than in HPV-negative OSCCs. To explore the role of HPV oncoproteins on OSM expression, the expression of HPV16E6 in the HCK1T Tet-On condition was induced by doxycycline and HPV16E6 was found to significantly upregulate levels of OSM mRNA and protein, with concomitant upregulation of c-Myc. In addition, the levels of OSM mRNA and protein in E6/E7 transiently transfected oral cancer cells also gradually increased in a time-dependent manner and these transfected cells showed greater viability and higher migration rates and cell-cycle progression than controls. This result demonstrates that HPV16 oncoproteins upregulate OSM and play an important role to promote OSCC development.

Persistent high-risk human papillomavirus (HPV) infection is strongly associated with development of high-grade cervical intraepithelial neoplasia or cancer (CIN3+). In single type infections serial type-specific viral-load measurements predict the natural history of the infection. In infections with multiple HPV-types, the individual type-specific viral-load profile could distinguish progressing HPV-infections from regressing infections. A case-cohort natural history study was established using samples from untreated women with multiple HPV-infections who developed CIN3+ (n=57) or cleared infections (n=88). Enriched cell pellet from liquid based cytology samples were subjected to a clinically validated real-time qPCR-assay (18 HPV-types). Using serial type-specific viral-load measurements (≥3) we calculated HPV-specific slopes and coefficient of determination (R2 ) by linear regression. For each woman slopes and R2 were used to calculate which HPV-induced processes were ongoing (progression, regression, serial transient, transient). In transient infections with multiple HPV-types, each single HPV-type generated similar increasing (0.27copies/cell/day) and decreasing (-0.27copies/cell/day) viral-load slopes. In CIN3+ at least one of the HPV-types had a clonal progressive course (R2 ≥0.85;0.0025copies/cell/day). In selected CIN3+ cases (n=6) immunostaining detecting type-specific HPV 16,31,33,58 and 67 RNA showed an even staining in clonal populations (CIN3+), whereas in transient virion-producing infections the RNA-staining was less in the basal layer compared to the upper layer where cells were ready to desquamate and release newly-formed virions. RNA-hybridization patterns matched the calculated ongoing processes measured by R2 and slope in serial type-specific viral-load measurements preceding the biopsy. In women with multiple HPV-types, serial type-specific viral-load measurements predict the natural history of the different HPV-types, and elucidates HPV-genotype attribution. 

Pan-fibroblast growth-factor receptor (FGFR) inhibitors hold promise in FGFR-altered patients, but such alterations are rare in advanced urothelial carcinoma. In order to assess whether we may increase the number of eligible patients by using different molecular techniques for detecting alterations, we pooled the results of the centralised FGFR mutation/translocation assays that were performed in Clinical Laboratory Improvement Amendments-certified laboratories within multiple phase 2 trials. At our centre, the same tissue blocks were used to analyse FGFR1-3 messenger RNA expression through messenger RNA in situ hybridisation (ISH; RNAscope 2.5 assay). From October 2016 to March 2017, 52 cases were analysed. Seventeen patients (32.7%) had an upper tract primary tumour. Ten patients (19.2%) had FGFR DNA alterations. Twenty-nine (55.8%) had positive ISH analysis: N=17 score 3, N=12 score 4. Of note, concordance between the two tests was obtained in seven out of 10 patients. Sixty percent of mutated patients had an upper tract primary tumour versus 31% of ISH-positive patients.

PATIENT SUMMARY:

We found three-fold higher frequency of fibroblast growth-factor receptor alterations at the RNA versus DNA level in advanced urothelial carcinoma, with a different distribution according to the method used and the site of the primary tumour. The evaluation of the therapeutic response to pan-fibroblast growth-factor receptor inhibitors according to the method of assessment is warranted.

Our understanding of adenoid basal tumors of the cervix has evolved over time. Most of the proliferations referred to as adenoid basal carcinoma have a clinically benign course-leading some to suggest the term "adenoid basal epithelioma." However, rarely, these may be associated with invasive carcinomas. These tumors have been etiologically linked with high-risk human papillomavirus (HR-HPV) infection. Here, we investigate the use of p16 immunohistochemistry and HR-HPV RNA in situ hybridization (ISH) in the classification of adenoid basal tumors of the cervix. Seventeen cases of adenoid basal tumors of the cervix were included. The patients' age ranged from 19 to 79 yr (average, 59 yr). p16 immunostain was performed on all cases and RNA ISH was performed in 4 cases with available formalin-fixed paraffin-embedded tissue. There were 11 low-grade tumors, 5 frankly invasive carcinomas, and 1 with histologic features that were intermediate between the former 2 categories. p16 immunostain was negative or showed patchy cytoplasmic staining in the low-grade tumors and was strongly and diffusely positive in the invasive carcinomas. HR-HPV RNA ISH was negative in the 3 low-grade tumors and was positive in 1 case of invasive carcinoma including the adenoid basal component. Distinct p16 immunostaining and HR-HPV RNA ISH patterns exist between low-grade adenoid basal tumors and invasive adenoid basal carcinomas. Our study indicates that p16 immunostaining and HR-HPV RNA ISH can be employed as useful ancillary tools in differentiating between noninvasive and invasive adenoid basal tumors along with careful histopathologic evaluation.

Abstract

Background

Fibroblast growth factor receptor (FGFR2) has been proposed as a target in gastric cancer. However, appropriate methods to select patients for anti-FGFR2 therapies have not yet been established.

Methods

We used in situ techniques to investigate FGFR2 mRNA expression and gene amplification in a large cohort of 1036 Japanese gastric cancer patients. FGFR2 mRNA expression was determined by RNAscope. FGFR2 gene amplification was determined by dual-color in situ hybridization (DISH).

Results

We successfully analyzed 578 and 718 samples by DISH and RNAscope, respectively; 2% (12/578) showed strong FGFR2 gene amplification (FGFR2:CEN10 >10); moderate FGFR2gene amplification (FGFR2:CEN10 <10; ≥2) was detected in 8% (47/578); and high FGFR2 mRNA expression of score 4 (>10 dots/cell and >10% of positive cells with dot clusters under a 20× objective) was seen in 4% (29/718). For 468 samples, both mRNA and DISH data were available. FGFR2 mRNA expression levels were associated with gene amplification; FGFR2 mRNA levels were highest in the highly amplified samples (n = 12). All highly amplified samples showed very strong FGFR2 mRNA expression (dense clusters of the signal visible under a 1× objective). Patients with very strong FGFR2 mRNA expression showed more homogeneous FGFR2 mRNA expression compared to patients with lower FGFGR2 mRNA expression. Gastric cancer patients with tumors that had an FGFR2 mRNA expression score of 4 had shorter RFS compared with score 0–3 patients.

Conclusion

RNAscope and DISH are suitable methods to evaluate FGFR2 status in gastric cancer. Formalin-fixed paraffin-embedded (FFPE) tissue slides allowed evaluation of the intratumor heterogeneity of these FGFR2 biomarkers.