Antitumoral effect of PLK-1-inhibitor BI2536 in combination with cisplatin and docetaxel in squamous cell carcinoma cell lines of the head and neck.

Inhibition of the polo-like-kinase-1 (PLK-1) has been shown to be effective in several haematological and solid tumor models. In this systemic in vitro study, the antitumor effect of BI2536, a small molecule inhibitor of PLK-1, in combination with cisplatin and docetaxel was examined in nine squamous cell carcinoma cell lines, most of which had a head and neck origin (SCCHN). Dose escalation studies were conducted with nine SCCHN cell lines using BI2536, cisplatin and docetaxel in cell line-specific concentrations. Growth inhibitory and proapoptotic effects were measured quantitatively using cytohistology and a Human Apoptose Array kit. BI2536 in combination with cisplatin and docetaxel showed a markedly higher antiproliferative and apoptotic activity in the SCCHN cell lines investigated (P≤0.008), compared with single agent cisplatin or docetaxel alone. The findings of this study showed that the addition of PLK-1-inhibitor BI2536 to conventional chemotherapeutic drugs led to a statistically higher antiproliferative and apoptotic effect in SCCHN cell lines compared with cisplatin or docetaxel alone. Inaugurating BI2536 in the clinical setting might enhance the antitumoral activity of conventional drugs, possibly leading to less toxic side effects of cancer therapy.

The role of recombinant epidermal growth factor and serotonin in the stimulation of tumor growth in a SCCHN xenograft model.

One challenge of squamous cell carcinoma of the head and neck (SCCHN) chemotherapy is a small percentage of tumor cells that arrest in the G0 phase of the cell cycle and are thus not affected by chemotherapy. This could be one reason for tumor recurrence at a later date. The recruitment of these G0-arresting cells into the active cell cycle and thus, proliferation, may increase the efficacy of chemotherapeutic agents. The aim of this study was to investigate whether stimulation with recombinant epidermal growth factor (EGF) or serotonin leads to an increased tumor cell proliferation in xenografts. Detroit 562 cells were injected into NMRI-Foxn1nu mice. Treatment was performed with 15 µg murine or human EGF, or 200 µg serotonin. The control mice were treated with Lactated Ringer's solution (5 mice/group). Tumor size was measured on days 4, 8 and 12 after tumor cell injection. The EGF stimulated mice showed a significantly higher tumor growth compared to the serotonin-stimulated mice and the untreated controls. In the present study, we show that it is possible to stimulate tumor cells in xenografts by EGF and thus, enhance cell proliferation, resulting in a higher tumor growth compared to the untreated control group. In our future investigations, we plan to include a higher number of mice, an adjustment of the EGF dosage and cell subanalysis, considering the heterogeneity of SCCHN tumors.

Effects of the Polo-like-kinase-1-inhibitor BI2536 in squamous cell carcinoma cell lines of the head and neck.

Inhibition of the Polo-like-kinase-1 (PLK1) has been shown to be effective in a number of solid tumor models. In this in vitro study, we examined the antitumor effect of BI2536, a small molecule inhibitor of PLK1, in squamous cell carcinoma of the head and neck (SCCHN) cell lines. Dose escalation studies were performed with nine SCCHN cell lines using BI2536. Growth inhibitory and proapoptotic effects were measured quantitatively using cytohistology and a Human Apoptosis Array Kit. BI2536 demonstrated a significant antiproliferative and apoptotic activity in all nine SCCHN cell lines investigated (p<0.009). Our results indicate that inhibition of PLK1 by BI2536 leads to an antiproliferative and apoptotic effect in SCCHN cell lines. In vivo and in the clinical setting, the application of BI2536 may support the antitumoral activity of conventional drugs that are in current use and could decrease the systemic toxicity of these drugs.

The challenge of tumor heterogeneity--different phenotypes of cancer stem cells in a head and neck squamous cell carcinoma xenograft mouse model.

BACKGROUND/AIM: Besides late diagnosis, tumor metastasis and cancer relapse are the main reasons for the poor prognosis of patients with head and neck cancer. Several investigations have shown that tumor is of heterogeneous molecularity consisting of several subpopulations, with a broad range of biological behaviors. The ability and potential of tumor to infiltrate into vessels and into neighbouring organs, as well as the resistance to chemotherapeutical cancer therapy may be caused by cancer stem cells (CSCs). The aim of the present study was to illuminate the role and behaviour of (CD44) and (ALDH1A1) as tumor stem cell markers in a xenograft mouse model of squamous cell carcinoma. MATERIALS AND METHODS: Five female NMRI-Foxn1nu mice were injected with five million Detroit 562 cells (100 µl). After sacrifice of the mice, tumors were excised. Then ALDH1A1, CD44, (EGFR), CD31 and Ki 67 were detected as molecular markers for tumor stem cells by immunohistopathology and immunofluorescence. RESULTS: The amount of putative CSC marker proteins CD44 and ALDH1A1 vary. ALDH1A1high tumor cells express low levels of CD44 and EGFR. The CD44+high expressers also exhibit expression of high levels of the EGFR. CSCs must be sub-classified depending on their expression of marker proteins. CONCLUSION: We assume that CSCs can also be sub-classified into migratory and stationary CSCs. ALDH1A1high/CD44low/EGFRlow tumor cells may be stationary and quiescent, whereas ALDH1A1-/CD44high/EGFRhigh expressers have a migratory, invasive nature. It is likely that a regulatory mechanism, as yet unknown, controls this conversion, from quiescent to active cancer stem cells.

Reirradiation with cetuximab in locoregional recurrent and inoperable squamous cell carcinoma of the head and neck: feasibility and first efficacy results.

PURPOSE: To report our experience with a prospective protocol of external beam reirradiation (Re-RT) combined with cetuximab for patients with inoperable, recurrent squamous cell carcinoma of the head and neck (SCCHN). PATIENTS AND METHODS: Between August 2008 and June 2010, 18 patients with inoperable recurrence of SCCHN after adjuvant or definitive radiotherapy (RT) and simultaneous or sequential cisplatin-based chemotherapy for primary SCCHN were enrolled. Acute and late toxicity from the experimental regimen were recorded every week during RT and every 3 months thereafter. Efficacy was assessed with repeated imaging using response evaluation criteria in solid tumors and clinical examinations 8-12 weeks after completion of the treatment and every 3 months thereafter. RESULTS: Median follow-up time for all patients was 9.4 (range: 3.85-31.7) months and for patients alive 30.4 (range: 15.7-31.7) months. Acute toxicity was generally mild or moderate. Five patients developed a grade 3 acneiform rash related to cetuximab. Late toxicity occurred as grade 3 trismus in five and as grade 3 abacterial salivary gland inflammation in one patient, respectively. Overall response rate was 47%. Median overall and progression-free survival for all patients was 8.38 months and 7.33 months, respectively. The overall survival rate was 44% at 1 year, with a 1 year local control rate of 33%. CONCLUSION: Notwithstanding the limitations of our preliminary data Re-RT combined with cetuximab for recurrent and inoperable SCCHN is feasible and the integration of newer targeted agents seems to be less toxic compared to conventional chemotherapy with encouraging response rates at least for a subset of patients.

Heterogeneity of primary site biopsies in head and neck squamous cell carcinoma.

AIM: There is no common standard defining how biopsies for translational research purposes should be performed. In our study, the impact of two different biopsy methods on the results of immunohistochemical staining of the samples for epidermal growth factor receptor (EGFR) and the proliferation antigen Ki-67 were evaluated. PATIENTS AND METHODS: Twenty-four patients who underwent surgical treatment of their HNSCC tumour were included. From each surgically resected tumour, one superficial biopsy and one core-needle biopsy through the cross-section of the tumour were taken. As a positive control, a tissue slide through the primary tumour was made. RESULTS: The analysis showed that neither the superficial nor the core biopsy expression of EGFR correlated significantly with that of the tumour. The analysis showed that the superficial biopsy expression of Ki-67 correlated significantly with that of tumour. CONCLUSION: Translational research projects based on biopsy tissues should be using whole surgical resection specimens of a tumour.

Low-dose computed tomography of the paranasal sinus and facial skull using a high-pitch dual-source system--first clinical results.

OBJECTIVE: Computed tomography (CT) of the paranasal sinus is the standard diagnostic tool for a wide range of indications in mostly younger patients. This study aims to assess the image quality of CT of the sinus by using a high-pitch dual-source technique with special regard to the radiation dose. METHODS: Examinations were performed on a second-generation dual-source CT with a pitch factor of 3.0 (dual-source mode). Images were compared with those with a pitch factor of 0.9 on the same system (single-source mode) and with those of 16-slice CT. Image quality was evaluated by four blinded readers using a 5-point scale (1=poor, 5=excellent). Comparison of the dose length product (DLP) was used to estimate radiation exposure. RESULTS: Seventy-three consecutive patients underwent imaging with the proposed CT protocols. The viewers rated the image quality of the dual-source image sets as nearly as good (3.62) as the single-source images on the same device (4.18) and those on 16-slice CT (3.7). DLP was cut to half of the dose [51 mGycm vs. 97.8 mGycm vs. 116.9 mGycm (p<0.01)]. CONCLUSIONS: Using the proposed dual-source mode when examining the paranasal sinus, diagnostic image quality can be achieved while drastically lowering the patient's radiation exposure.

AdOnco database - six years' experience with the documentation of clinical and scientific data on patients with head and neck cancer.

PURPOSE: To report the experience with AdOnco, a computerized database for head and neck cancer patients. PATIENTS AND METHODS: AdOnco is a Filemaker Pro 6.0 based database integrated into the local network of the host ENT department. It is used by the physicians as a clinical and scientific documentation system to store and retrieve information about all patients with head and neck cancer referred to the host oncology center. This study reviews the achievements to date of AdOnco and, as an example of its enormous data evaluation potential, presents survival curves of patients with laryngeal cancer undergoing laser resection. RESULTS: Over a period of six years, the data of 881 patients with head and neck cancer were entered into the AdOnco database. CONCLUSION: AdOnco has proven to be a useful patient database and documentation system which has become an integral and essential part of daily clinical routine and also a valuable research tool.

Does dexamethasone inhibit the antineoplastic effect of cisplatin and docetaxel in head and neck cancer cells?

BACKGROUND/AIM: Comedication with glucocorticoids such as dexamethasone is frequently given to head and neck cancer patients treated with chemotherapy. However, an increasing body of evidence suggests that dexamethasone may induce resistance to antineoplastic agents. The present study was the first to investigate the effect of dexamethasone on the antiproliferative activity of cisplatin and docetaxel in vitro in squamous cell carcinoma of the head and neck (SCCHN) cell lines. MATERIALS AND METHODS: The cytotoxic effect of cisplatin and docetaxel on eight SCCHN cell lines was determined for each drug alone or with increasing concentrations of dexamethasone. Cell growth inhibition and viability were measured quantitatively after 24, 48, 72 hours of treatment using water-soluble-tetrazolium-test and lactate dehydrogenase assays. Absolute tumor cell numbers were determined by cell counting in a Rosenthal chamber. RESULTS: Cisplatin and docetaxel alone inhibited the growth of all eight SCCHN cell lines significantly (p=0.012). The antiproliferative activity of both agents was not decreased by the addition of dexamethasone in any of the cell lines (p>0.05). CONCLUSION: Dexamethasone does not interfere with the cytotoxic action of cisplatin or docetaxel in the investigated SCCHN cell lines.

Combined cetuximab and reirradiation for locoregional recurrent and inoperable squamous cell carcinoma of the head and neck.

PURPOSE: To investigate the feasibility, toxicity, and efficacy of external-beam reirradiation (Re-RT) combined with cetuximab for patients with inoperable and recurrent squamous cell carcinoma of the head and neck (SCCHN). PATIENTS AND METHODS: Seven patients with inoperable recurrence of SCCHN after adjuvant or definitive radiotherapy (RT) and simultaneous or sequential cisplatin-based chemotherapy for primary SCCHN were treated between August and December 2008 with Re-RT (1.8 Gy/fraction to 50.4 Gy) and cetuximab (400 mg/m(2) initial dose in the 1st week, and then 250 mg/m(2) once weekly). Recurrence had to be located at least > or = 50% in the preirradiated field. Long term toxicity from previous treatment was recorded before Re-RT as a baseline value. Acute and late toxicity derived from the experimental regimen were recorded every week during RT, and then every 3 months. Efficacy was assessed with repeated imaging using response evaluation criteria in solid tumors (RECIST) and clinical examinations 8-12 weeks after end of the treatment and every 3 months thereafter (Tables 1 and 2). RESULTS: Only mild localized mucositis occurred in all patients. Two patients developed a grade 3 acneiform rash related to cetuximab. After treatment one patient developed a grade 2 trismus, another showed grade 3 abacterial salivary gland inflammation with severe pain requiring opioid medication. Two patients achieved a complete response after 7 months, one remained stable, three progressed, and one died from pneumonia without having restaging magnetic resonance imaging. CONCLUSION: A second course of RT combined with cetuximab in patients with inoperable, recurrent HNSCC proved to be feasible with mild or moderate toxicity and encouraging response to treatment.

Does dexamethasone inhibit anticancer activity of cetuximab in squamous cell carcinoma cell lines of the head and neck?

Glucocorticoids such as dexamethasone are widely used as comedication in the treatment of head and neck cancer, e.g., to improve appetite and decrease weight loss and fatigue in patients with advanced disease or as antiallergic and antiemetic prophylaxis during anti-EGFR therapy. However, the literature suggests that dexamethasone induces resistance to antineoplastic agents in many solid tumor models in vitro and in vivo. Since this phenomenon has never been investigated in head and neck cancer, the present study was conducted to investigate the effect of dexamethasone on the antiproliferative activity of cetuximab in vitro in squamous cell carcinoma of the head and neck (SCCHN) cell lines. The antiproliferative effect of the anti-EGFR agent cetuximab alone and in combination with increasing concentrations of dexamethasone was examined in eight SCCHN cell lines at three different time-points (24, 48 and 72 h). Cell growth inhibition and viability were measured quantitatively using WST and LDH assays. Absolute tumor cell numbers were determined by cell counting in a Rosenthal chamber. Cetuximab alone inhibited the growth of all eight SCCHN cell lines significantly (p=0.008). In some cases the addition of dexamethasone reduced the antiproliferative activity of cetuximab (p

Cetuximab enhances the efficacy of bortezomib in squamous cell carcinoma cell lines.

PURPOSE: Proteasome inhibition has been shown to be effective in multiple myeloma and solid tumor models. In this in vitro study, we investigated the antitumor effect of bortezomib (Velcade) in combination with cetuximab in squamous cell carcinoma cell lines (SCC). METHODS: Dose-escalation studies were performed in five squamous cell carcinoma cell lines using bortezomib or cetuximab alone or in combination. Cell survival and growth inhibition were measured quantitatively using an MTT and LDH assay. RESULTS: Bortezomib alone showed a significant antiproliferative activity in all SCC cell lines (P < 0.042), and the activity was further significantly enhanced by the addition of cetuximab (P < 0.043). CONCLUSIONS: Our results indicate that cetuximab increases the cytotoxic activity of bortezomib in SCC cell lines. Combination therapy of SCC with bortezomib and cetuximab might be less toxic than conventional drug regimens used in the treatment of these tumors.

Effects of combination treatment of bortezomib and dexamethasone in SCCHN cell lines depend on tumor cell specificity.

Bortezomib has recently become the new treatment standard for relapsed or refractory multiple myeloma. We previously demonstrated that bortezomib also had a significant growth-inhibiting and apoptotic effect on squamous cell carcinoma of the head and neck (SCCHN) cells in vitro. Preclinical evidence has provided a rationale for combining bortezomib with dexamethasone in multiple myeloma, suggesting that the therapeutic effects of the two agents might be additive. These findings are in contrast with the results achieved in solid tumor models where the addition of dexamethasone reduced the efficacy of other antineoplastic drugs. In the present study, we investigated the effect of dexamethasone in combination with bortezomib in SCCHN cell lines for the first time. The antiproliferative effect of bortezomib alone or in combination with increasing concentrations of dexamethasone was investigated in four SCCHN cell lines. Cell growth inhibition and viability were measured quantitatively using WST and LDH assays. Bortezomib alone inhibited the growth of all four SCCHN cell lines significantly (p<0.047). The addition of dexamethasone leads to a clear tumor cell decline and showed a trend in enhancing the growth-inhibitory effect of bortezomib although the difference failed to reach statistical significance (p>0.05). Our first results show that dexamethasone increased the cytotoxic activity of bortezomib in most SCCHN cell lines investigated. These findings might be dependent on molecular factors such as the degree of tumor cell differentiation and proliferation rate. Therefore, further studies will be required to elucidate these molecular factors to substantiate our findings from a cancer biological point of view.

Effect of bortezomib and cetuximab in EGF-stimulated HNSCC.

BACKGROUND: Proteasome inhibition has been shown to be effective in multiple myeloma and solid tumor models. In this in vitro study, the antitumor effect of bortezomib (Velcade) in combination with cetuximab was investigated in epidermal growth factor (EGF)-stimulated head and neck squamous cell carcinoma cell lines (HNSCC). MATERIALS AND METHODS: Dose escalation studies were performed with five EGF-stimulated squamous cell carcinoma cell lines using bortezomib alone or in combination with cetuximab. Growth inhibitory and cell decline effects were measured quantitatively using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and lactate dehydrogenase (LDH) assay. RESULTS: Bortezomib alone showed no significant antiproliferative activity in any EGF-stimulated HNSCC cell line (p > 0.05), whereas the combination of bortezomib and cetuximab had highly significant antitumoral activity (p < 0.043). CONCLUSION: Our results indicate that cetuximab increases the cytotoxic activity of bortezomib in EGF-stimulated HNSCC cell lines. A combination treatment of HNSCC with bortezomib and cetuximab may allow a therapeutical regimen to be developed that is less toxic than the conventional drugs used for these tumors.

Craniofacial reconstructions with bone-anchored epithesis in head and neck cancer patients--a valid way back to self-perception and social reintegration.

BACKGROUND: Patients with advanced head and neck cancer often require radical and mutilating surgery resulting in severe impairment of their aesthetic self-perception and social life. Cosmetically satisfying results associated with high aesthetic self-perception and social reintegration are possible with bone-anchored epithesis representing a serious alternative to craniofacial reconstructive techniques using regional and free tissue transfer. PATIENTS AND METHODS: Five head and neck cancer patients treated in our Ear, Nose and Throat Department in the years 2003-2004 were evaluated after epithesial reconstruction. RESULTS: Three out of the five patients scored self-perception after epithesial reconstruction as "very good", while social integration was scored as "very good" by three and as "satisfactory" by two patients. Daily getting along was scored as "very good" by four and as satisfactory by one patient. One patient had a very good acceptance of the epithesis as a part of the body and for four patients it was satisfactory. CONCLUSION: For the first time, the high degree of satisfaction in head and neck cancer patients receiving epithesial reconstruction in the maxillofacial region is demonstrated.

Effect of bortezomib on EGFR expression in head and neck squamous cell carcinoma cell lines.

BACKGROUND: Overexpression of epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinoma (HNSCC) has often been correlated with poor prognosis. Recent investigations have shown that the proteasome inhibitor bortezomib exhibits a high antiproliferative and apoptotic activity in HNSCC cell lines. The present study investigated whether bortezomib has an effect on EGFR expression in different squamous cell carcinoma cell lines. MATERIALS AND METHODS: Six EGF-stimulated or non-stimulated squamous carcinoma cell lines were treated with bortezomib. Western blots were performed to determine EGFR expression. For statistical analysis, a Wilcoxon test for matched pairs (dependent samples) was performed using SPSS 13.0 software for Windows. RESULTS: Changes in EGFR expression after bortezomib treatment in EGF non-stimulated and EGF-stimulated squamous carcinoma cell lines failed to reach statistical significance in either experimental group (p > 0.05). CONCLUSION: Given the high expression of EGFR in head and neck tumors, further investigations should address the question whether the apoptotic activity of bortezomib can be enhanced by adding an anti-EGFR antibody.

Antiproliferative activity of bortezomib alone and in combination with cisplatin or docetaxel in head and neck squamous cell carcinoma cell lines.

PURPOSE: Proteasome inhibition has been shown to be effective in multiple myeloma and solid tumor models. In this in vitro study, we investigated the antitumor effect of bortezomib (Velcade((R))) in squamous cell carcinoma of the head and neck (SCCHN) cell lines and examined the interaction of the drug with docetaxel (TAX) and cisplatin (CDDP). METHODS: Dose escalation studies were performed with eight squamous cell carcinoma cell lines using bortezomib alone or in combination with TAX or CDDP. Growth inhibitory and proapoptotic effects were measured quantitatively using cytohistology and western blot analysis. RESULTS: Bortezomib alone showed a significant antiproliferative activity in all SCCHN cell lines (P = 0.012), and the activity was further enhanced by the addition of TAX or CDDP (P

Iron oxide particle-enhanced magnetic resonance imaging for detection of benign lymph nodes in the head and neck: how reliable are the results?

AIM: To evaluate the accuracy of ultrasmall paramagnetic iron oxide (USPIO: Sinerem)-enhanced MRI in patients with head and neck cancer and enlarged lymph nodes compared with current staging examinations using histology as a gold standard. PATIENTS AND METHODS: Seventeen patients with a histologically proven squamous cell cancer of the head and neck (SCCHN) and different N-stages underwent a non-enhanced and a USPIO-enhanced MRI examination. Signal intensity (SI) was measured in a region of interest evaluation. Pathohistological examination was used as a reference. RESULTS: On a patient basis, USPIO-enhanced MRI showed a higher specificity and diagnostic accuracy (94%) compared with non-enhanced MRI (53%). One patient showed a lymph node of 6 mm in the short axial diameter which was suggested as being metastatic in Sinerem-enhanced MRI according to the enhancement pattern of Sinerem. This patient was staged as N1 with Sinerem-enhanced MRI. The histopathological examination did not confirm the Sinerem-enhanced MRI result. CONCLUSION: The high values for the specifity and diagnostic accuracy of Sinerem- enhanced MRI justifies further investigations with this contrast agent. USPIO-enhanced MRI could be advantageous in avoiding surgical overtreatment.

Enhancement of docetaxel efficacy in head and neck cancer treatment by G0 cell stimulation.

OBJECTIVES: Docetaxel has recently taken part in new chemotherapy regimens with promising activity especially in the first line therapy (induction chemotherapy) of head and neck cancer (SCCHN). Nevertheless a major problem concerning the response of SCCHN to chemotherapy is the high percentage of resting cells (G0-phase cells) being resistant to chemotherapy. To overcome this phenomenon we have investigated the capacity of several cytokines to switch on cells into division cycle and progress to the chemosensitive phases (S, M-phase). METHODS: Il-6, Serotonin, G-CSF and EGF were used to stimulate G0-phase squamous cell cancer cells (Detroit 562, A431, UM-SCC 10B) for reentry in the cell cycle to enhance the response to docetaxel. The proportion of G0-phase cells was detected through multicolor FACS analysis and Ki67 staining. RESULTS: Cell cycle reentering was most effective after combination treatment with Serotonin+EGF. The proportion of G0 phase cells was significantly reduced after stimulation with Serotonin+EGF (p<0.05). Corresponding to cell cycle reentry the cytotoxic effect of docetaxel was significantly (p<0.04) enhanced in the prestimulated cells compared to the control (docetaxel monotreatment). CONCLUSIONS: Our investigations demonstrate for the first time that sensitizing G0 phase squamous cell carcinoma cells for docetaxel treatment is possible by prestimulation with target cytokines. Considering that up to 95% of tumor cells are in the resting (G0) phase of the cell cycle at the initiation of chemotherapy, prestimulation with EGF and serotonin could contribute to a synchronization of cancer cells. This would clearly enhance the cytotoxic effect.

Demands on caring relatives of head and neck cancer patients.

BACKGROUND: Relatives of cancer patients experience high levels of stress that influence the quality of life of these individuals. To investigate whether there is a necessity for simultaneous supportive care of patient relatives, we performed for the first time a study asking the closest relatives of head and neck cancer patients about their needs during and after the treatment to consider how to optimize the situation for such patient groups. MATERIAL AND METHODS: Patients' relatives were assessed using an anonymous self-report questionnaire that was established in our department by expanding on a questionnaire for cancer patients' relatives from the psycho-oncologic society in Switzerland. The evaluation was multidimensional, cancer specific, and relative based. RESULTS: Relatives feel confronted themselves with cancer, although indirectly. The majority of the respondents were of the opinion that simultaneous psychological care of the patients and for the caring relatives would be helpful to cope with the situation. CONCLUSION: This study shows the significant impact of cancer on caring relatives of head and neck cancer patients. In our opinion, health services should become more aware of this potential to ensure that the needs of the involved patient relatives are met as well as those of the patients.