Serum microRNAs as xerostomia biomarkers in oropharyngeal cancer patients undergoing radiotherapy.

PURPOSE
Severe xerostomia is noted in the majority of patients irradiated for oropharyngeal cancer (OPC). Extracellular microRNAs (miRNAs) may serve as effective tools allowing prediction of radiation-related toxicity. The aim of this study was to create an efficient prognostic miRNA-based test for severe, patient-rated xerostomia 3 months after primary treatment.


METHODS AND MATERIALS
This prospective study enrolled OPC patients treated between 2016 and 2018 in three centers in XXX. The primary endpoint was severe (grade ≥3) xerostomia as assessed by the EORTC H&N-35 questionnaires. Initially, a group of 10 patients with severe xerostomia was randomly selected and matched with a comparative group of 10 patients without severe xerostomia. Samples were collected before RT, after receiving 20 Gy and within 24 hours after treatment completion. qPCR arrays (QIAGEN, Hilden, Germany) were used to quantify expression levels of 752 miRNAs in the serum at all timepoints. The resulting logistic-regression based model was validated in additional 60 patients: 30 with grade >3 xerostomia and 30 without.


RESULTS
Of 152 eligible patients, we successfully recruited 111 patients. Severe xerostomia 3 months after treatment was reported by 63 patients (56.8%). Mean dose delivered to parotid glands was higher in both exploratory and validation cohort. The model based on miR-185-5p and miR-425-5p expression levels measured before the start of radiotherapy had AUC 0.96 (95% CI: 0.88-1.00). The model based on the same miRNAs remained robust when parameters were measured after 20 Gy (AUC 0.90 (95% CI: 0.75-1.00)). These results were confirmed in the validation group. In the validation group pre-radiotherapy model application yielded 73.3% sensitivity and 80.0% specificity. In the samples taken after 20 Gy, the same two miRNAs yielded 67.7% sensitivity and 72.4% specificity. The model including pretreatment miR-185-5p and miR-425-5p levels together with mean parotid dose, yielded 90.0% sensitivity and 80.0% specificity. In the validation cohort, this model yielded 80.6 % sensitivity and 55.2 % specificity. The model based on miRNA levels measured after 20 Gy and mean parotid dose had 80.0 % sensitivity and 100% specificity in the exploratory group. In the validation cohort its performance fell to 71.0 % sensitivity and 58.6 % specificity.


CONCLUSIONS
Serum expression levels of miR-425-5p and miR-185-5p measured before the start of radiotherapy or during therapy (after 20 Gy) had a significant prognostic value for the occurrence of severe xerostomia 3 months after treatment completion. The variability explained by miRNAs appears to be, at least partially, independent from the one related to the dosimetric data.


Purpose or Objective
Severe xerostomia is noted in up to 75% of patients irradiated for oropharyngeal cancer (OPC). Currently, we do not possess effective tools for measuring radiation sensitivity that would allow tailored therapy. Preliminary literature reports indicate that extracellular microRNAs (miRNAs) may be a new class of biomarkers that will pave the way for further personalization of radiotherapy (RT). Hence, the aim of this study was to analyze temporal changes in expression levels of miRNAs circulating in the serum and to create an efficient test for patient-rated xerostomia 3 months after primary treatment with IMRT with or without chemotherapy for OPC.

Material and Methods
The study was designed as a prospective cohort study that enrolled OPC patients treated with IMRT (total dose of 70 Gy or equivalent) from June 2016 to December 2018 in four oncological centers in Poland. One hundred fifty-two patients with OPC diagnosis were assessed for eligibility and 111 patients were finally qualified for the study. Side effects were prospectively assessed using EORTC QLQ-C30 and EORTC H&N-35 questionnaires. We randomly selected a group of 10 patients with severe (grade ≥3) xerostomia and matched a comparative group of 10 patients without severe xerostomia. We collected serum samples before RT, after receiving 20 Gy and within 24 hours after the end of the treatment. qPCR arrays (miRCURY LNA, Human panels I II, Exiqon, Copenhagen, Denmark) were used to quantify miRNA expression levels. Data were normalized toward the average expression of miRNAs detectable in all samples. MiRNAs were shortlisted on the basis of univariate, Benjamini-Hochberg adjusted, p values. The classifier for xerostomia was created using a stepwise, 5-fold crossvalidated, logistic regression model. The results were validated in a group of 60 patients (30 patients with grade ≥3 xerostomia and 30 patients without severe xerostomia).

Results
Severe xerostomia 3 months after the end of RT was reported by 63 patients (56.8%). The model based on miR-185-5p and miR-425-5p expression levels measured before the start of RT had a very good discriminatory ability -AUC 0.96 (95% CI: 0.88-1.00). The model based on the expression of the same miRNAs maintained a very high discriminatory power when parameters were measured after 20 Gy (AUC 0.90 (95% CI: 0.75-1.00)). Changes in the expression levels of miR-185-5p and miR-425-5p in the samples from the validation group were also confirmed to be significant. The model based on the expression levels of these two miRNAs measured before radiotherapy was characterized by an AUC 0.80 (95% CI: 0.70 -0.91). In samples taken after 20 Gy, the use of expression levels of these two miRNAs resulted in AUC 0.83 (95% CI: 0.73-0.94).

Conclusion
The expression levels of miR-425-5p and miR-185-5p measured in the serum of patients with OPC before the start of RT and during therapy (after 20 Gy) have a significant prognostic value for the occurrence of severe xerostomia 90 days after the end of RT.  Purpose or Objective Prefoldin (PFDN) is a co-chaperone that contributes to both cytoplasmic and nuclear biological processes. Canonical PFDN has a heterohexameric jellyfish-like structure. Four ß-type subunits (PFDN1, 2, 4 and 6) form two dimers onto two subunits of the α type (PFDN3 and 5). PFDN2 and 6 are also components of the URI-prefoldin-like complex, which has been described to promote cancer. It has been shown that PFDN1 overexpresion promotes epithelial-mesenchymal transition (EMT) and lung cancer (LC) progression in different LC cell lines and murine models whereas cyclin A knockdown alone induces EMT and increases cell migration and invasion ability. We investigated whether this putative involvement of canonical PFDN in LC translates into the clinic.

Material and Methods
58 non-small cell LC patients with available tumor tissue samples (59% squamous and 41% adenocarcinoma) were assessed. The stages were as follows: 24% I, 7% II, 61% III, and 8% IV. 90% of patients were primarily treated with surgery and 69% received chemotherapy. 86% underwent thoracic radiotherapy either primarily (41%) or after locorregional recurrence (45%). The levels of PFDN1, 3, 5 were examined by immunoblotting. Additionally, the mRNA expression of 518 LC cases from The Cancer Genome Atlas (TCGA) database was evaluated. To assess the risk of mortality and recurrences we used Kaplan-Meier and Cox proportional hazards analyses.

Conclusion
Overexpression of canonical PFDN associates with the risk of mortality and metastasis in non-small cell LC. These response markers may be usefull biomarkers for guiding therapy intensity in an individualized therapy.  NEJM (1997) andLancet (1999). We hereby present 30-year long-term follow-up of the cancer therapeutic effect as well as a focus on the potential late cardiac and secondary cancer risk.

Material and Methods
Between 1982 and 1990, a total of 3,083 patients with pathological stage II and stage III breast cancer were after mastectomy randomly assigned to receive adjuvant systemic therapy and postoperative irradiation to the chestwall and regional lymph nodes (1,538 pts), or adjuvant systemic therapy alone (1,545 pts). Pre-and menopausal patients received 8-9 cycles of CMF with an interval of 4 weeks, whereas postmenopausal patients received tamoxifen 30 mg daily for one year. The median potential follow-up time was 33 (range 29-37) years. The endpoints were loco-regional control, freedom for distant metastases, overall survival and irradiation related late morbidity.

Results
Overall the 30-year cumulative incidence of loco-regional recurrence was 10% in irradiated patients vs 38%

Conclusion
The study definitely demonstrate that optimal long-term treatment benefit of high-risk breast cancer can only be achieved if both loco-regional and systemic tumor control are aimed for. Therefore, radiotherapy has an important role in the multidisciplinary treatment of breast cancer. The RT treatment did not result in excess ischemic heart damage, nor in other non-cancer related death.