HRD Testing of Ovarian Cancer in Routine Practice: What Are We Dealing With?

Assessment of homologous recombination deficiency (HRD) status is now essential for ovarian cancer patient management. The aim of our study was to analyze the influence of ethnic variations, tumor purity, and neoadjuvant chemotherapy (CT) on the determination of HRD scores as well as to evaluate feasibility of HRD testing with the Amoy HRD Focus Assay in routine clinical practice. The HRD status, including the BRCA status and genomic scar score (GSS), was analyzed in 452 ovarian cancer specimens. The successful rate of HRD testing was 86% (388/452). The BRCA mutational rate was 29% (114/388); 252 samples (65%) were classified as HRD-positive. Our data demonstrate the feasibility of internal HRD testing by the AmoyDx HRD Focus Panel for high-grade serous ovarian cancer (HGSOC), showing results similar to other methods. The HRD rate in the Russian population is very similar to those of other European populations, as is the BRCA mutation frequency. The most substantial contribution to HRD level diversity is testing criteria depending on intrahospital arrangements. The analysis shows that biallelic BRCA alterations had higher GSS compared with those with monoallelic inactivation, consistent with positive HRD status. The study indicates that grades 1-2 of the pathological response caused by chemotherapy affect HRD scores and suggests controlling for tumor purity of 40% or more as a critical factor for GSS measurement.

Live births and maintenance with levonorgestrel IUD improve disease-free survival after fertility-sparing treatment of atypical hyperplasia and early endometrial cancer.

OBJECTIVE: Our objectives were to (1) compare different regimens of hormonal therapy (HT) in young women with atypical endometrial hyperplasia (AEH) and early endometrial cancer (EC), (2) assess reproductive and oncologic outcomes and (3) explore possible predictors of complete response (CR) and disease free survival (DFS). METHODS: Reproductive age women with AEH and Grade 1-2 endometrioid EC with no or minimal myometrial invasion on MRI treated with different regimens of HT were prospectively analyzed. Treatment protocols included levonorgestrel intrauterine device (LNG IUD), gonadotropin-releasing hormone agonist (aGnRH) or high-dose oral medroxyprogesteron acetate (MPA) separately and in combinations. RESULTS: Total of 418 patients with AEH (n = 228) and EC (n = 190) aged 19-46 years received HT. Overall CR rate was 96% in AEH and 88% in EC patients (р < 0.001). None of the regimens used in AEH (LNG IUD + 2 D&C vs. LNG IUD + aGnRH vs. LNG IUD + 3 D&C) was found inferior to the others (CR of 98%, 95%, 100%, respectively, p > 0.05) except for MPA alone (CR 87%, р = 0.009). Out of four HT regimens used in EC LNG IUD + aGnRH+3 D&C was superior to all others (CR 96%, р = 0.026) where 2 D&Cs were performed or oral MPA was prescribed. The median follow-up for 339 patients was 33 months (range: 3-136), 68% of patients (n = 232) attempted conception, 38% (n = 89) of them used ART. The birth rate was 42% (n = 97). The rate of recurrence was 26% (50/196) in AEH group and 36% (51/143) in EC group (p = 0.05). Birth after treatment (HR = 0.24) or LNG IUD maintenance (HR = 0.18) were associated with superior DFS (p < 0.001 for both). ART use did not influence DFS. CONCLUSION: Hormonal therapy of AEH and early EC with LNG IUD is superior to MPA-containing regimens, however still carries high risk of recurrence. Post-treatment pregnancy rates are satisfactory and can be further improved by broader ART use which was proven safe. Initial diagnosis of AEH, post-treatment child birth and LNG IUD maintenance were associated with decreased rates of recurrence.

Agreement between PDL1 immunohistochemistry assays and polymerase chain reaction in non-small cell lung cancer: CLOVER comparison study.

The goal of the CLOVER study was to perform a pairwise comparison of four tests based on the same patient population with non-small cell lung cancer (NSCLC): three validated PDL1 immunohistochemistry (IHC) assays (Ventana SP142, Ventana SP263, Dako 22C3) and one PCR test. Four hundred seventy-three NSCLC samples were obtained from a biobank and were stained using PDL1 IHC assays. Four trained pathologists independently evaluated the percentage of tumor cells (TC) and immune cells (IC) that stained positive at any intensity. PDL1 transcripts were quantified in 437 patients by a standard Taqman RT-PCR assay using SDHA as a reference gene. A concordance analysis was performed to assess (1) the correlation of TC and IC between different assays and (2) the predictive properties of one test for another. "High" RNA expression was detected in 187 of 437 (43%) patients. The percentage of PDL1-positive cells (≥1%) was higher among the IC than the TC in all IHC three assays. The Pearson correlation coefficients (PCC) for TC were 0.71, 0.87, and 0.75 between 22C3/SP142, 22C3/SP263, and SP263/SP142, respectively. The PCC for IC were 0.45, 0.61, and 0.68 for the same pairs. A low correlation was observed between the PCR test and each of the three IHC assays; however, if a patient tested low/negative by PCR, then they were likely to test negative by any single IHC test with a high probability (92-99%). Among patients who tested positive by PCR, only 9-45% tested positive by IHC assays. There was excellent positive and negative agreement (>91%) between 22C3 and SP263 staining using the recommended individual cutoffs for first-line treatment. PCR RNA expression analysis is not equivalent to IHC. However, this method may have some potential for the identification of PDL1-negative tumors. 22C3 could be considered as a substitute for SP263 in first-line treatment.

RUSSCO-RSP comparative study of immunohistochemistry diagnostic assays for PD-L1 expression in urothelial bladder cancer.

In this collaborative study by the Russian Society of Clinical Oncology and the Russian Society of Pathology, we assessed the concordance among three validated, commercially available PD-L1 immunohistochemistry assays for patients with urothelial cancer. Tumors from 100 urothelial cancer patients were stained with the antibody clones 22C3 (Agilent), SP142 (Ventana Medical Systems), and SP263 (Ventana Medical Systems), which are used in clinical trials of second-line therapy with checkpoint inhibitors. Four trained pathologists independently evaluated the percentages of tumor cells (TC) and tumor-infiltrating immune cells (IC) that were stained at any intensity by each of the antibodies. The test-specific cutoffs for the proportions of stained cells in a positive sample were pre-specified as TC + IC ≥ 10% or TC ≥ 10% for 22C3, IC ≥ 5% for SP142, and TC ≥ 25% or IC ≥ 25% for SP263. Three hundred immunohistochemistry slides were scored. The percentages of PD-L1 staining in the three assays without using any cutoff were higher in the IC than in the TC (55% versus 24% for 22C3, 45% versus 8% for SP142, and 72% versus 27% for SP263, respectively). The Pearson correlation coefficients for anti-PD-L1 staining in the IC were 0.5, 0.69, and 0.85 with 22C3/SP142, 22C3/SP263, and SP142/SP263, respectively. The Pearson correlation coefficients for PD-L1 staining in the TC were 0.93, 0.99, and 0.91 for the same pairs. Among the patients who were negative for PD-L1 staining by one test, 91-100% were also negative by the other tests. Among the patients who were positive by one test, 43-100% were also positive by the other tests. Our data indicate that repeated testing can be avoided as a patient with urothelial cancer who is classified as negative for PD-L1 expression by one of the three single tests using the corresponding cutoff rule is highly likely (91-100%) to be classified as negative by either of the other tests.

Enzymatic Nanocomposites with Radio Frequency Field-Modulated Activity.

The control over enzymatic activity by physical stimuli is of interest to many applications in medicine, biotechnology, synthetic biology, and nanobionics. Although the main focus has been on optically responsive systems, alternative strategies to modulate the enzymatic activity of hybrid systems are needed. Here we describe a radiofrequency (RF) field controlled catalytic activity of an enzymatic sol-gel composite. Specifically, the activity of bovine carbonic anhydrase entrapped in sol-gel-derived magnetite (enzyme@ferria) composite was accelerated by a factor of 460% compared to its initial value, by applying the RF field of 937 A/m, with fast response time. This acceleration is reversible and its magnitude controllable. An acceleration mechanism, based on RF-induced heating of the magnetite by the Néel relaxation effect, is proposed and proven. The entrapment within a sol-gel matrix solves the problem of enhancing activity by heating without denaturing the enzyme. RF-controlled enzymatic composites can be potentially applied as biological RF sensors or to control biochemical reactions within living organisms.

The controllable destabilization route for synthesis of low cytotoxic magnetic nanospheres with photonic response.

We present a new approach for obtaining magnetic nanospheres with tunable size and high magnetization. The method is implemented via controllable destabilization of a stable magnetite hydrosol with glycerol, leading to the formation of aggregates followed by their stabilization with the citrate shell. This inexpensive, simple and easily scalable approach required no special equipment. The obtained samples were characterized by high stability and magnetization over 80 emu/g. Effects of synthetic conditions on physicochemical properties of nanospheres were monitored by hydrodynamic size, zeta potential, and polydispersity of magnetite aggregates. The size of the resulting aggregates varied between 650 nm and 40 nm, and the zeta potential from +30 mV to -43 mV by changing the ratio of the reagents. Under optimal conditions the clusters with a diameter of 80 nm were produced with a narrow size distribution ±3 nm. These characteristics allowed for optical response to the external magnetic field, thereby producing a magnetic photon liquid. Due to biocompatibility of the reagents used in the synthesis the nanospheres evoked a negligible cytotoxicity for human non-malignant and tumor cell lines. These results make new materials valuable in photonics and biomedicine.

Up-regulation of expression and lack of 5' CpG island hypermethylation of p16 INK4a in HPV-positive cervical carcinomas.

BACKGROUND: High risk type human papilloma viruses (HR-HPV) induce carcinomas of the uterine cervix by expressing viral oncogenes E6 and E7. Oncogene E7 of HR-HPV disrupts the pRb/E2F interaction, which negatively regulates the S phase entry. Expression of tumor suppressor p16ink4a drastically increases in majority of HR-HPV associated carcinomas due to removal of pRb repression. The p16ink4a overexpression is an indicator of an aberrant expression of viral oncogenes and may serve as a marker for early diagnostic of cervical cancer. On the other hand, in 25-57% of cervical carcinomas hypermethylation of the p16 INK4a promoter has been demonstrated using a methylation-specific PCR, MSP. To evaluate a potential usage of the p16 INK4a 5' CpG island hypermethylation as an indicator of tumor cell along with p16ink4a overexpression, we analyzed the methylation status of p16 INK4a in cervical carcinomas METHODS: Methylation status of p16 INK4a was analyzed by MSP and by bisulfite-modified DNA sequencing. The expression of p16ink4a was analyzed by RT-PCR and by immunohistochemical technique. RESULTS: The extensive methylation within p16 INK4a 5' CpG island was not detected either in 13 primary cervical carcinomas or in 5 cancer cell lines by bisulfite-modified DNA sequencing (including those that were positive by MSP in our hands). The number and distribution of rare partially methylated CpG sites did not differ considerably in tumors and adjacent normal tissues. The levels of the p16 INK4a mRNA were increased in carcinomas compared to the normal tissues independently of the number of partially methylated CpGs within 5'CpG island. The transcriptional activation of p16 INK4a was accompanied by p16ink4a cytoplasmic immunoreactivity in the majority of tumor cells and presence of a varied number of the p16 positive nuclei in different tumors. CONCLUSION: Hypermethylaion of the p16INK4a 5' CpG island is not a frequent event in HR-HPV-positive cervical carcinomas and cannot be an effective marker of cancer cells with up-regulated expression of p16ink4a. Our data confirm other previous studies claiming specific p16INK4a up-regulation in the majority of cervical carcinomas at both the protein and mRNA levels. Cytoplasmic accumulation of p16ink4a is a feature of cervical carcinomas.

Protein p16 as a marker of dysplastic and neoplastic alterations in cervical epithelial cells.

BACKGROUND: Cervical carcinomas are second most frequent type of women cancer. Success in diagnostics of this disease is due to the use of Pap-test (cytological smear analysis). However Pap-test gives significant portion of both false-positive and false-negative conclusions. Amendments of the diagnostic procedure are desirable. Aetiological role of papillomaviruses in cervical cancer is established while the role of cellular gene alterations in the course of tumor progression is less clear. Several research groups including us have recently named the protein p16INK4a as a possible diagnostic marker of cervical cancer. To evaluate whether the specificity of p16INK4a expression in dysplastic and neoplastic cervical epithelium is sufficient for such application we undertook a broader immunochistochemical registration of this protein with a highly p16INK4a-specific monoclonal antibody. METHODS: Paraffin-embedded samples of diagnostic biopsies and surgical materials were used. Control group included vaginal smears of healthy women and biopsy samples from patients with cervical ectopia. We examined 197 samples in total. Monoclonal antibody E6H4 (MTM Laboratories, Germany) was used. RESULTS: In control samples we did not find any p16INK4a-positive cells. Overexpression of p16INK4a was detected in samples of cervical dysplasia (CINs) and carcinomas. The portion of p16INK4a-positive samples increased in the row: CIN I - CIN II - CIN III - invasive carcinoma. For all stages the samples were found to be heterogeneous with respect to p16INK4a-expression. Every third of CINs III and one invasive squamous cell carcinoma (out of 21 analyzed) were negative. CONCLUSIONS: Overexpression of the protein p16INK4a is typical for dysplastic and neoplastic epithelium of cervix uteri. However p16INK4a-negative CINs and carcinomas do exist. All stages of CINs and carcinomas analyzed are heterogeneous with respect to p16INK4a expression. So p16INK4a-negativity is not a sufficient reason to exclude a patient from the high risk group. As far as normal cervical epithelium is p16INK4a-negative and the ratio p16INK4a-positive/ p16INK4a-negative samples increases at the advanced stages application of immunohisto-/cytochemical test for p16INK4a may be regarded as a supplementary test for early diagnostics of cervical cancer.