T. Rutkowski

Comparison of peptide cancer signatures identified by mass spectrometry in serum of patients with head and neck, lung and colorectal cancers: Association with tumor progression

Mass spectrometry-based analyses of the low-molecular-weight fraction of serum proteome allow identifying proteome profiles (signatures) that are potentially useful in detection and diagnostics of cancer. Here we compared serum proteome profiles of healthy donors and patients with three different types of cancer aiming to identify peptide signatures that were either common for all cancer samples or specific for cancer type. Blood samples were collected before start of the therapy from patients with head and neck squamous cell cancer, colorectal adenocarcinoma and non-small cell lung cancer, and from a corresponding group of healthy volunteers. Mass profiles of the serum proteome were recorded in the range between 2 and 13 kDa using MALDI-ToF spectrometry and 131 identified peptide ions were used for statistical analyses. Similar degrees of overall similarities were observed in all intra-group and inter-group analyses when general features of serum proteome profiles were compared between individual samples. However, classifiers built of selected spectral components allowed differentiation between healthy donors and three groups of cancer patients with 69-74% sensitivity and 82-84% specificity. There were two common peptide species (3766 and 5867 Da) with increased levels in all cancer samples. Several spectral components permitted differentiation between lung cancer samples and either head and neck cancer or colorectal cancer samples, but two latter types of samples could not be properly discriminated. Abundance of spectral components that putatively corresponded to fragments of serum amyloid A (11511 and 11667 Da) was highest in lung cancer samples, yet increased levels of these peptides appeared to generally associate with more advanced cancer cases. We concluded that certain components of serum peptide signatures are common for different cancer signatures and putatively reflect general response of organism to the disease, yet other components of such signatures are more specific and most likely correspond to clinical stage of the malignancy.

Intraoperative Radiotherapy (IORT) with Low-Energy Photons as a Boost in Patients with Early-StageOral Cancer with the Indications for Postoperative Radiotherapy

Purpose:To evaluate the feasibility and preliminary results of intraoperative radiotherapy (IORT) with low-energy photons as a boost in patients with early-stage oral cancer with the indications for postoperative radiotherapy.Patients and Methods:Between 2003 and 2006, 16 patients with early-stage cancer of mobile tongue (n = 10 [63%]) or floor of the mouth (n = 6 [37%]) treated at Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Poland, were evaluated for IORT boost with the INTRABEAM®System (Carl Zeiss Surgical GmbH; IORT-PRS) because of the high risk of local recurrence due to positive margins on frozen pathologic section. After tumor resection, the applicator was positioned in the tumor bed. The applicator’s diameter (range: 1.5–5 cm) was selected to encompass high-risk area of tumor recurrence. The dose (5 Gy, 7 Gy, or 7.5 Gy) was applied according to tumor volume and bone proximity. External-beam radiotherapy (EBRT) was provided to the tumor bed in all patients (50 Gy) and to the nodal area, when needed. Toxicity and local tumor control were assessed.Results:Median follow-up was 36 months. IORT did not increase acute mucosal reaction. Local tumor control was found in all cases. Early mucosal reaction did not exceed 3 according to the RTOG scale and healed in median time of 35 days after completion of EBRT. No late adverse effects were observed.Conclusion:This preliminary report has demonstrated the feasibility of IORT-PRS for patients with early oral cancer with the indications for postoperative radiotherapy. This method may be considered an alternative boost technique, although additional studies are needed to establish long-term results in a larger group of patients.ZusammenfassungZiel:Auswertung der Anwendbarkeit und ersten Ergebnisse der intraoperativen Radiotherapie (IORT) mit niedrigenergetischen Photonen als Boost bei Patienten mit Mundhöhlenkarzinom in einem frühen klinischen Stadium, bei denen Indikationen zur postoperativen Radiotherapie bestehen.Patienten und Methodik:In den Jahren 2003–2006 wurden im Onkologischen Zentrum am Maria-Skłodowska-Curie-Institut in Gliwice, Polen, 16 Patienten mit Mundhöhlenkarzinom in einem frühen klinischen Stadium behandelt. Bei zehn Patienten befand sich das Karzinom im beweglichen Zungenteil, und bei sechs Patienten lag ein Mundbodenkarzinom vor. Aufgrund des hohen Risikos für ein Lokalrezidiv kam die IORT mit dem INTRABEAM®System (Carl Zeiss Surgical GmbH; IORT-PRS) zur Anwendung. Risiko- faktoren für ein Lokalrezidiv wurden anhand der pathologischen Beurteilung im Gefrierschnitt ermittelt. Als Risikofaktoren wurden schmale oder positive Absetzungsränder, Infiltration der Lymphgefäße und/oder der Blutgefäße durch das Karzinom, perineurale Karzinominfiltration, ungünstiger Infiltrationstyp, Tiefe der Infiltration und geringgradige histopathologische Differenzierung des Karzinoms eingestuft. Nach chirurgischer Entfernung des Karzinoms wurde der Applikator auf das Tumorbett aufgesetzt. Der Durchmesser des Applikators (Bereich 1,5–5 cm) wurde so gewählt, dass der Applikator das gesamte Gebiet mit Risiko eines Rezidivs bedeckte. Die Strahlendosis (5 Gy, 7 Gy, 7,5 Gy) richtete sich nach dem Tumorvolumen und dem Zustand der Absetzungsränder. Bei allen Patienten kam eine externe Strahlentherapie (EBRT) auf das Tumorbett (50 Gy) und, wenn indiziert, auf die lokalen Lymphknoten zum Einsatz. Ausgewertet wurden die Therapieverträglichkeit und das Ausmaß der lokalen Ausheilung.Ergebnisse:Die mediane Beobachtungszeit lag bei 36 Monaten. Die IORT führte zu keiner Zunahme akuter Reaktionen der Mundschleimhaut auf die Strahlentherapie. Bei allen Patienten wurde eine gute Wundheilung beobachtet. Die Frühreaktionen der Mundschleimhaut auf die Strahlentherapie wurden nicht höher als Grad 3 nach dem RTOG/EORTC-Score eingestuft. Die mediane Zeit bis zur kompletten Ausheilung von Frühreaktionen lag bei 35 Tagen. Spätreaktionen nach Strahlentherapie wurden nicht beobachtet.Schlussfolgerung:Die Ergebnisse der Studie bestätigen die Anwendbarkeit von IORT-PRS bei Patienten mit einem frühen Mundhöhlenkarzinom, bei denen Indikationen zur postoperativen Strahlentherapie bestehen. Diese Methode kann als alternative Technik für eine Boostapplikation in Erwägung gezogen werden. Weitere Studien sind jedoch erforderlich, damit die Langzeitergebnisse in einer größeren Patientenpopulation ausgewertet werden könnten.

Intraoperative radiotherapy (IORT) with low-energy photons as a boost in patients with early-stage oral cancer with the indications for postoperative radiotherapy : treatment feasibility and preliminary results.

Purpose:To evaluate the feasibility and preliminary results of intraoperative radiotherapy (IORT) with low-energy photons as a boost in patients with early-stage oral cancer with the indications for postoperative radiotherapy.Patients and Methods:Between 2003 and 2006, 16 patients with early-stage cancer of mobile tongue (n = 10 [63%]) or floor of the mouth (n = 6 [37%]) treated at Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Poland, were evaluated for IORT boost with the INTRABEAM®System (Carl Zeiss Surgical GmbH; IORT-PRS) because of the high risk of local recurrence due to positive margins on frozen pathologic section. After tumor resection, the applicator was positioned in the tumor bed. The applicator’s diameter (range: 1.5–5 cm) was selected to encompass high-risk area of tumor recurrence. The dose (5 Gy, 7 Gy, or 7.5 Gy) was applied according to tumor volume and bone proximity. External-beam radiotherapy (EBRT) was provided to the tumor bed in all patients (50 Gy) and to the nodal area, when needed. Toxicity and local tumor control were assessed.Results:Median follow-up was 36 months. IORT did not increase acute mucosal reaction. Local tumor control was found in all cases. Early mucosal reaction did not exceed 3 according to the RTOG scale and healed in median time of 35 days after completion of EBRT. No late adverse effects were observed.Conclusion:This preliminary report has demonstrated the feasibility of IORT-PRS for patients with early oral cancer with the indications for postoperative radiotherapy. This method may be considered an alternative boost technique, although additional studies are needed to establish long-term results in a larger group of patients.ZusammenfassungZiel:Auswertung der Anwendbarkeit und ersten Ergebnisse der intraoperativen Radiotherapie (IORT) mit niedrigenergetischen Photonen als Boost bei Patienten mit Mundhöhlenkarzinom in einem frühen klinischen Stadium, bei denen Indikationen zur postoperativen Radiotherapie bestehen.Patienten und Methodik:In den Jahren 2003–2006 wurden im Onkologischen Zentrum am Maria-Skłodowska-Curie-Institut in Gliwice, Polen, 16 Patienten mit Mundhöhlenkarzinom in einem frühen klinischen Stadium behandelt. Bei zehn Patienten befand sich das Karzinom im beweglichen Zungenteil, und bei sechs Patienten lag ein Mundbodenkarzinom vor. Aufgrund des hohen Risikos für ein Lokalrezidiv kam die IORT mit dem INTRABEAM®System (Carl Zeiss Surgical GmbH; IORT-PRS) zur Anwendung. Risiko- faktoren für ein Lokalrezidiv wurden anhand der pathologischen Beurteilung im Gefrierschnitt ermittelt. Als Risikofaktoren wurden schmale oder positive Absetzungsränder, Infiltration der Lymphgefäße und/oder der Blutgefäße durch das Karzinom, perineurale Karzinominfiltration, ungünstiger Infiltrationstyp, Tiefe der Infiltration und geringgradige histopathologische Differenzierung des Karzinoms eingestuft. Nach chirurgischer Entfernung des Karzinoms wurde der Applikator auf das Tumorbett aufgesetzt. Der Durchmesser des Applikators (Bereich 1,5–5 cm) wurde so gewählt, dass der Applikator das gesamte Gebiet mit Risiko eines Rezidivs bedeckte. Die Strahlendosis (5 Gy, 7 Gy, 7,5 Gy) richtete sich nach dem Tumorvolumen und dem Zustand der Absetzungsränder. Bei allen Patienten kam eine externe Strahlentherapie (EBRT) auf das Tumorbett (50 Gy) und, wenn indiziert, auf die lokalen Lymphknoten zum Einsatz. Ausgewertet wurden die Therapieverträglichkeit und das Ausmaß der lokalen Ausheilung.Ergebnisse:Die mediane Beobachtungszeit lag bei 36 Monaten. Die IORT führte zu keiner Zunahme akuter Reaktionen der Mundschleimhaut auf die Strahlentherapie. Bei allen Patienten wurde eine gute Wundheilung beobachtet. Die Frühreaktionen der Mundschleimhaut auf die Strahlentherapie wurden nicht höher als Grad 3 nach dem RTOG/EORTC-Score eingestuft. Die mediane Zeit bis zur kompletten Ausheilung von Frühreaktionen lag bei 35 Tagen. Spätreaktionen nach Strahlentherapie wurden nicht beobachtet.Schlussfolgerung:Die Ergebnisse der Studie bestätigen die Anwendbarkeit von IORT-PRS bei Patienten mit einem frühen Mundhöhlenkarzinom, bei denen Indikationen zur postoperativen Strahlentherapie bestehen. Diese Methode kann als alternative Technik für eine Boostapplikation in Erwägung gezogen werden. Weitere Studien sind jedoch erforderlich, damit die Langzeitergebnisse in einer größeren Patientenpopulation ausgewertet werden könnten.

Optimization of circulating cell-free DNA recovery for KRAS mutation and HPV detection in plasma

BACKGROUND The precise analysis of tumour markers in blood such as circulating cell-free DNA (cfDNA) could have a significant impact in facilitating monitoring of patients after initial therapy. Although high levels of total cfDNA in plasma of cancer patients are consistently demonstrated, a low sensitivity of DNA alterations is reported. OBJECTIVE The major question regards the recovery of tumour-specific cfDNA such as KRAS mutated DNA and cancer-associated type 16 of human papillomavirus (HPV16). METHODS TaqMan technology was used for detection of KRAS mutation, HPV16 and to quantify cfDNA in blood plasma. RESULTS Comparison of four different column-based commercial kits shows that the cfDNA purification carried out by the Genomic Mini AX Body Fluids kit and the QIAamp Circulating Nucleic Acid kit gave us the possibility to improve the sensitivity of detection of KRAS mutation and HPV16. The optimized method was used to follow the reduction in cancer-specific cfDNA after therapy. We found that large volume extractions with low volume of DNA eluate enabled trace amounts of tumour-specific cfDNA from cancer patients to be effectively identified. CONCLUSIONS Data presented in this study facilitate detection of tumour-specific cfDNA and improve standards needed for the implementation of cfDNA technology into routine clinical practice.

Association between plasma proteome profiles analysed by mass spectrometry, a lymphocyte-based DNA-break repair assay and radiotherapy-induced acute mucosal reaction in head and neck cancer patients.

Purpose: The plasma proteome was analysed as a potential source of markers of radiosensitivity in patients treated with definitive radiotherapy for head and neck cancer. Materials and methods: Acute mucosal reactions that developed during radiotherapy were assessed in 55 patients. Blood samples were collected from each patient before the treatment and also from 50 healthy donors. The low-molecular-weight fraction of the plasma proteome (2,000–10,000 Da range) was analysed by the Matrix-Assisted Laser Desorption Ionisation mass spectrometry. The capacity for DNA break repair was assessed by the comet assay using lymphocytes irradiated in vitro. Results: Spectral components registered in plasma samples were used to build classifiers that discriminated patients from healthy individuals with about 90% specificity and sensitivity (components of 4469, 6929 and 8937 Da were the most essential for cancer classification). Four spectral components were identified (2219, 2454, 3431 and 5308 Da) whose abundances correlated with a maximal intensity of the acute reaction. Several spectral components whose abundances correlated with the rate of DNA repair in irradiated lymphocytes were also detected. Additionally, a more rapid escalation of an acute reaction was correlated with a higher level of unrepaired damage assessed by the comet assay. Conclusions: The plasma proteome could be considered as a potential source of predictive markers of acute reaction in patients with head and neck cancer treated with radiotherapy.

Radiation-induced changes in serum lipidome of head and neck cancer patients

Cancer radiotherapy (RT) induces response of the whole patient’s body that could be detected at the blood level. We aimed to identify changes induced in serum lipidome during RT and characterize their association with doses and volumes of irradiated tissue. Sixty-six patients treated with conformal RT because of head and neck cancer were enrolled in the study. Blood samples were collected before, during and about one month after the end of RT. Lipid extracts were analyzed using MALDI-oa-ToF mass spectrometry in positive ionization mode. The major changes were observed when pre-treatment and within-treatment samples were compared. Levels of several identified phosphatidylcholines, including (PC34), (PC36) and (PC38) variants, and lysophosphatidylcholines, including (LPC16) and (LPC18) variants, were first significantly decreased and then increased in post-treatment samples. Intensities of changes were correlated with doses of radiation received by patients. Of note, such correlations were more frequent when low-to-medium doses of radiation delivered during conformal RT to large volumes of normal tissues were analyzed. Additionally, some radiation-induced changes in serum lipidome were associated with toxicity of the treatment. Obtained results indicated the involvement of choline-related signaling and potential biological importance of exposure to clinically low/medium doses of radiation in patient’s body response to radiation.

Radiation-free weekend rescued! Continuous accelerated irradiation of 7-days per week is equal to accelerated fractionation with concomitant boost of 7 fractions in 5-days per week: report on phase 3 clinical trial in head-and-neck cancer patients.

PURPOSE To report long-term results of randomized trial comparing 2 accelerated fractionations of definitive radiation therapy assessing the need to irradiate during weekend in patients with head and neck squamous cell carcinoma. METHODS AND MATERIALS A total of 345 patients with SCC of the oral cavity, larynx, and oro- or hypo-pharynx, stage T2-4N0-1M0, were randomized to receive continuous accelerated irradiation (CAIR: once per day, 7 days per week) or concomitant accelerated boost (CB: once per day, 3 days per week, and twice per day, 2 days per week). Total dose ranged from 66.6-72 Gy, dose per fraction was 1.8 Gy, number of fractions ranged from 37-40 fractions, and overall treatment time ranged from 37-40 days. RESULTS No differences for all trial end-points were noted. At 5 and 10 years, the actuarial rates of local-regional control were 63% and 60% for CAIR vs 65% and 60% for CB, and the corresponding overall survival were 40% and 25% vs 44% and 25%, respectively. Confluent mucositis was the main acute toxicity, with an incidence of 89% in CAIR and 86% in CB patients. The 5-year rate of grade 3-4 late radiation morbidity was 6% for both regimens. CONCLUSIONS Results of this trial indicate that the effects of accelerated fractionation can be achieve by delivering twice-per-day irradiation on weekday(s). This trial has also confirmed that an accelerated, 6-weeks schedule is a reasonable option for patients with intermediate-stage head-and-neck squamous cell carcinoma because of the associated high cure rate and minimal severe late toxicity.

Ionizing radiation affects profile of serum metabolites: increased level of 3-hydroxybutyric acid in serum of cancer patients treated with radiotherapy

Radiotherapy causes molecular changes observed at the level of body fluids, which are potential biomarker candidates for assessment of radiation exposure. Here we analyzed radiotherapy-induced changes in a profile of small metabolites detected in sera of head and neck cancer patients using the gas chromatography coupled with mass spectrometry approach. There were about 20 compounds, including carboxylic acids, sugars, amines and amino acids, whose levels significantly differed between pre-treatment and post-treatment samples. Among metabolites upregulated by radiotherapy there was 3-hydroxybutyric acid, whose level increased about three times in post-treatment samples. Moreover, compounds affected by irradiation were associated with several metabolic pathways, including protein biosynthesis and amino acid metabolism.

Low dose radiotherapy as an effective treatment in a patient with solitary Wegener’s granulomatosis resistant to systemic treatment – case report

Wegeners granulomatosis is a rare disease (10 per 1 000 000 new cases per year), etiologically connected with necrotizing vasculitis of small to medium-size vessels. The disease occurs predominantly in the upper respiratory tract, lungs and kidneys, but any organ may be affected during the course of the illness. It may be difficult to diagnose, especially when c-ACNA antibodies (serologic symptom of Wegeners granulomatosis) are undetectable and chest X-ray is normal. Early diagnosis is crucial for treatment results. Untreated disease may lead to death. Cyclophosphamide used simultaneously with prednisone is the treatment of choice as the first line procedure. Resistance to standard systemic treatment may be a significant problem. New drugs (rituximab, infliximab) are still under clinical investigation, with promising results. Very limited data concerning effectiveness of radiation therapy exist. We present a report of a female patient with solitary form of Wegeners granulomatosis located in the facial region, who underwent successful radiation therapy with a complete response.

The effect of tumor volume on radiotherapy outcome and correlation with other prognostic factors in patients with T2 supraglottic cancer

The retrospective chart review of 110 patients with T2 supraglottic cancer who underwent radiotherapy was performed to correlate tumor volume with other prognostic factors and to analyze its impact on treatment results. Patients with involved nodes, poor histopathological tumor differentiation, or hemoglobin concentration ≤ 14.3 g/dl had significantly larger tumors. Patients with large tumors had significantly lower 5-year local control rate, overall survival rate and presented significantly higher risk of nodal involvement and the ratio of poor histopathological differentiation of the tumor. Tumor volume significantly impacts radiotherapy outcome and should be considered to optimize treatment strategy for patients with T2 supraglottic cancer.