The pattern of cell survival in the pig liver following one freeze-thaw cryosurgery cycle.

BACKGROUND: It is well established that in cryosurgery some cells can survive one freeze thaw cycle and that surviving cells are found at the margin of the frozen lesion. Numerous techniques are being developed to ensure the survival of frozen cells to the margin of the frozen region. OBJECTIVE: We thought that it would be of fundamental interest to observe the pattern of cell survival in a liver treated with one freeze-thaw cycle. MATERIALS AND METHODS: We performed six ultrasound-guided single freeze-thaw cryosurgery procedures on the liver of four Landrace pigs, using two cryosurgery probes separated by 25 mm inserted in parallel. Treated organs were removed 24 hours after the cryosurgery and processed for histology with hematoxylin and eosin. The tissues were analyzed with a digital slice scanner. RESULTS: We found an unexpected pattern of cell survival; sheets of live cells, about 200 µm in width, that follow the network of interlobular connective tissue septae to a distance of several millimeter from the outer edge of the one freeze-thaw cycle cryosurgery treated lesion. The sheets of live cells surround lobule cores that have undergone complete coagulative necrosis. In addition, larger blood vessels, as far as 5 mm from the outer rim of the treated lesion, have a major and complex effect on cell survival with large areas of completely necrotic and completely alive cells intermixed. CONCLUSION: This study may have value as a baseline for developing new cryosurgery protocols designed to ablate cells to the margin of the frozen lesion.

The role of neuroendocrine cells in prostate cancer: a comprehensive review of current literature and subsequent rationale to broaden and integrate current treatment modalities.

UNLABELLED: Neuroendocrine prostate carcinoma (NE-PCa) is a heterogeneous disease. Due to a high prevalence of NE (neuroendocrine) differentiation in patients who receive prolonged androgen deprivation treatment, the real incidence of NE-PCa remains unknown. Similarly, the biological steps from prostate carcinoma (PCa) toward NE differentiation are far less than definitive and, consequently, there is a lack of evidence to support any of the treatments as the "gold standard". MATERIALS AND METHODS: A systematic literature search was conducted using the PubMed, Scopus, and Embase databases to identify original articles and review articles regarding NE-PCa . Keywords were "prostate cancer" and "neuroendocrine". Articles published between 1995 and 2013, were reviewed and selected with the consensus of all of the authors. RESULTS: Fifty-one articles were selected by the authors for the purpose of this review. The principle findings were reported into some subsections: Epidemiology, Biological steps of NE differentiation (with some principle articles on animal and in vitro, since there is very little in the literature on human studies); for the treatment options, we had to expand the search on PubMed to a larger timeframe and selection since very little was specifically found in the first criteria: surgery, radiotherapy, ablative techniques, immunomodulation and epigenetic therapy were then reviewed. A multidisciplinary approach, advocated by many authors, although promising, has failed to demonstrate increased survival rates. Limitations of this review include the lack of a clear definition of NE-PCa and consequently, the lack of strong evidence provided by a large series with long-term follow-up. CONCLUSIONS: Supported from this extensive review, we propose it is worthwhile to investigate a new multimodal therapeutic approach to address advanced NE-PCa starting from a debulking (with radical intent) of the disease plus epigenetic therapy with stem cell differentiation stage factors (SCDSFs). In addition immunotherapy can be used to treat the cancer presenting phenotype in association with chemomodulation plus ablative therapies, in case of advanced or recurrent diseases. SCDSFs may be utilized to regulate cancer stem cells and possible new phenotypes could also be associated with ablative therapies. Hormonal deprivation, radiotherapy, chemotherapy, ex vivo vaccines and targeted therapies could also be used and reserved in case of failure.

Neuroendocrine tumors of the lung: hystological classification, diagnosis, traditional and new therapeutic approaches.

Lung neuroendocrine tumors are neoplasms originating from bronchopulmonary neuroendocrine cells, usually Kulchitsky cells, loaded with argentaffin granules. They account for 20-25% of all primitive lung tumors, the most common being the small-cell undifferentiated carcinoma. They include different tumors, from tumors of low-grade malignancy, especially the typical carcinoids, with high survival rates after surgical therapy, to the high-grade malignancy tumors, especially small-cell undifferentiated carcinomas. The latter have very few indications for surgical treatment with a low survival rate, even after multimodal therapy. The aim of this review is to describe the present knowledge and discuss possible new developments in the management of pulmonary neuroendocrine tumors. The authors examine and discuss in particular the role that surgical techniques should have in the treatment of small-cell lung cancer in opposition to a nihilism position that has limited therapies to non-surgical approaches. The critical review of this attitude opens the door to a more aggressive approach. In the meantime the review shows that it might be possible to include the new minimally invasive percutaneous ablative techniques as cryosurgery, thermotherapy and irreversible electroporation within a modern and flexible framework. The authors also present the hypothesis that cancer stem cells (CSC) are at the basis of recurrences of small-cell lung cancer (SCLC) and therefore that the issue is of difficult solution with the conventional oncologic approach considering the chemo-resistance of CSC to drugs. For these reasons an epigenetic therapy based on differentiation factors is proposed alongside the usual surgical and chemo-radiation protocols.

Cryoablation induces necrosis and apoptosis in lung adenocarcinoma in mice.

This study evaluated cryoablation on subcutaneously transplanted tumors of lung adenocarcinoma LA795 in T739 mice in vivo, in an effort to assess the feasibility of cryoablation in treatment of NSCLC. Subcutaneously transplanted lung adenocarcinoma LA795 was implanted into T739 mice yielding tumors of approximately 2.5 cm in diameter. Following cryoablation, the various modes of cell death were studied: necrosis in the central frozen zone by light microscopy and apoptosis in periphery of the frozen zone by in situ end labeling (TUNEL). Bc1-2 and bax expression were detected by immunohistochemical SABC procedures, and the cleavage and activation of Caspase 3 and PARP in peripheral zone by Western blot. We find that in central cryoablated zone, necrosis was the dominant mode of cell death occurring at three hours and four days post-thaw. The first three-hour necrosis peak involved approximately 47% of the tumor while the four-day peak increased in volume to 68% of the tumor. In peripheral cryoablation zone, definite cell apoptosis could be observed by morphological examination under light microscope and TUNEL staining, peaking at 8-16 h after cryoablation. Immunohistochemical results yielded little change in bcl-2 protein expression before and after cryoablation. However, bax protein expression was up-regulated significantly after cryoablation. In addition, cleavage and activation of Caspase-3 and PARP occurred in the peripheral freeze zone after the treatment. It indicated that Cryoablation efficiently induces cell death both by necrosis and apoptosis. Cryoablation appears to induce apoptosis in the peripheral freeze zone through the intrinsic mitochondrial caspase pathway based on bax upregulation. This observation allows us to suggest that cryoablation may be combined with chemotherapy to increase cancer destruction.

Laparoscopic cryomyolysis: an alternative to myomectomy in women with symptomatic fibroids.

BACKGROUND: The purpose of this study was to determine whether cryomyolysis may present an alternative valid surgical procedure to hysterectomy or myomectomy for selected women with symptomatic fibroids who wish to preserve their uterus but do not desire future pregnancies. METHODS: Sixty-three women with symptomatic fibroids who refused either myomectomy or hysterectomy, requesting a conservative surgery for myomata, underwent laparoscopic cryomyolysis using a 3- to 5-mm or 8-mm cryoprobe (CRYOcare system). RESULTS: Our study showed a mean (+/-standard deviation) decrease of myoma volume of 60.3% (+/-20.7) and complete symptom relief in 83.6% (p < 0.001) of patients after a 12-month follow-up from cryomyolysis. No significant intra- or postoperative complications were noted. CONCLUSION: Cryomyolysis is an effective laparoscopic procedure for obtaining myoma shrinkage and symptom relief in women with symptomatic fibroids who desire to preserve their uterus.

[Ultrasound-guided cryosurgery of the prostate: short- and long-term experience]. / Criochirurgia ecoguidata della prostata: esperienza a breve e medio termine.

We have assessed 24 patients consecutively treated with cryosurgery and chosen according to the guidelines of the European Study Group of Urologic Cryosurgeons. Of the 24 patients (average age about 70, range 61-79), all were not considered candidates for radical prostatectomy, 9 (37%) were clinical stage cT2 N0M0, 15 (63%) cT3 N0M0 who had not received any prior treatment, except 1 patient (61 years old) who was treated with TCT and successive recurrence of the disease (cT2). Of the 24 chosen patients, 13 (55%) were over the age of 71, 11 (45%) had important factors of co-morbidity and an elevated risk of surgery (ASA 3). The average PSA was of 19.3 ng/ml (range 2.2-61). Gleason score was 2-5 in 9 cases, 6-7 in 14 and 8-10 in 1 case. In the follow-up, we evaluated serum PSA every 3 months and transrectal ultrasound and the echoguided prostatic biopsies at 6, 12 and 24 months. Post-operative complications included: ecchymosis and edema of external genitals (16/24), fever > 38 degrees C (1/24), sloughing syndrome (6/24), urinary tract infections (10/24) acute urine retention (1/24). In 2 cases, 6 months after treatment, a transrectal resection was carried out. After a follow-up at 6 months, the PSA was 0.4 ng/ml (range 0.1-0.9), in 1 case. In positive core biopsy out of 6 showed neoplastic cells with fibrous tissue; the patient had a PSA of 0.58 ng/ml. At 12 months there were 11 assessable patients. The average PSA was 0.3 ng/ml (range 0.1-0.9). At 24 months there were 4 assessable patients, 1 of 4 showed serum PSA level of 4 ng/ml and cancer in apical biopsy. Erectile dysfunction was assessed on 8 patients affective before surgery: 1 referred to sufficient erections at penetration (1/8, 12.5%). After removal of the catheter, 4 of the 20 patients suffered stress and urge incontinence with the use of 1 pad a day. In 1 case, 18 months from surgery, slight stress incontinence was found (1 pad/day). Cryoablation is an efficient method and is given to slight post-operative morbidity and no intra-operative mortality, also in patients with high risk for open surgery. Indications may be found in patients with the following conditions: older than 72 years, severe co-morbidity and high risk for surgery, neoplasia at high risk of progression, and disease recurrence after radiotherapy. Our case history is at the moment encouraging and a larger number of cases as well as longer follow-up are needed.

[Role of transrectal ultrasonography in the follow-up of patients treated with prostatic cryosurgery]. / Ruolo dell'ecografia transrettale nel follow-up dei pazienti sottoposti a criochirurgia prostatica.

Cryosurgery of prostate is a minimally invasive treatment for localized cancer. Imaging techniques (transrectal ultrasound (TRUS) or magnetic resonance) have been proposed to evaluate tumor persistence/recurrence after cryosurgical treatment other than serum PSA and prostate biopsies. Actually, criteria to identify cancer after cryosurgical ablation are not well assessed. Aim of this study is to evaluate the clinical significance and role of TRUS in detecting tumor within the former prostate gland after cryoablation. We evaluated ultrasound (US) features and imaging, serum PSA and biopsies in 20 patients treated by cryosurgery at our Hospital with a mean follow-up of 18 months. Twenty patients (mean age 70 years, PSA 25.9 ng/ml, clinical stage: 10 T2 N0M0 and 10 T3 N0M0) were followed up for a mean of 16 months. Ultrasound findings (gland volume, capsule, hypoechoic area, post-voided urine volume, seminal vesicles) were correlated to PSA levels (every 3 months) and prostate biopsies (6, 12 and 24 months). All cases were evaluated by the same ultrasound scanner (Eidos, Hitachi-5-6.5 MHz) and by two operators. Prostate capsule was interrupted by hypo-hyperechoic areas in all cases. Transition zone was no more recognizable. Ultrasound findings showed in all cases hypoechoic areas, but US did not identified tumor recurrence in 2 patients. During follow-up, PSA below 0.5 ng/ml was recorded in 75% of cases. We detected tumor in 2 cases, respectively 12 and 18 months after cryosurgery: in the first case few residual cancer cells within fibrous tissue were found in 1 out of 6 biopsies at 6 months (PSA 0.58 ng/ml), in the second one, tumor with viable normal prostatic glands was found in one biopsy of the apex at 18 months (PSA 4.0 ng/ml). TRUS showed several anaechoic foci with necrotic tissue and coalescence of liquid areas in 2 patients (one developed acute urinary retention). Actually, serum PSA is the best marker in order to detect clinically significant tumor after cryosurgery. Hypoechoic areas and capsule interruptions observed by ultrasound imaging of prostate gland after cryosurgery are not correlated with tumor recurrence or tumor persistence. TRUS is only indicated for ultrasound-guided biopsies during follow-up and to confirm urologic complications.

[Echography in minimally invasive surgery]. / L'ecografia in chirurgia mininvasiva.

Thanks to the great development of laparoscopic surgery and his continuous technical evolution, echography plays more and more important role in the pre- and intraoperative diagnosis. In the hepato-pancreatic pathology the use of the laparoscopic echography reduces clearly the role of laparotomic exploration. In laparoscopic surgery of rectal-colon, the echography is more specific and sensitive in compared with pre-operative MR and CT to individualize liver metastasis, to locate them. This allows the treatment of such lesions through the cryosurgery. In pancreatic lesions such method plays a non releasable role in tumors staging, giving essential elements to the surgeon to operate and for a better definition of operative strategy. Also in the adrenal masses surgery, laparoscopic echography reveals very useful especially in the anatomical structures identification (i.e. renal vein and entrance of the left adrenal vein) in the patients already operated or obese. In the preoperative study of rectal tumors the use of rotating and transrectal probes allows to define the degree of infiltration of the lesion and to perform a mini-invasive treatment through endoscopic transanal microsurgery with the radiochemotherapy. Thus echography in mini-invasive surgery has an unreplaceable role both in diagnosis as in evaluation of the parameter of therapeutical approach.

Ultrasound-guided laparoscopic cryoablation of hepatic tumors: preliminary report.

The purpose of this ongoing prospective study is to evaluate the feasibility, safety, and efficacy of a total laparoscopic approach for ultrasound-guided cryoablation of primary and secondary hepatic tumors. Of 56 patients who underwent ultrasound-guided cryoablation, a total laparoscopic approach was employed in 18 (5 men, 13 women; mean age 48.6 years, range 35-77 years). Fifteen patients were included for secondary hepatic tumors and three for primary hepatic tumors. Selection criteria were the presence of three or fewer nodules, less than 40% liver volume replaced by tumor, and absence of extrahepatic disease. Altogether 28 lesions were confirmed by intraoperative laparoscopic ultrasonography and were treated; 25 by ultrasound-guided laparoscopic cryoablation and 3 by laparoscopic wedge resection. After cryoablation, surface parenchymal splits with bleeding from the frozen tissue were observed in six patients and required conversion to open surgery in two patients whose lesions were located in segment 8. No major complication and no mortality were observed. One or more minor complications occurred in nine patients; they included pleural effusion (n = 8, 44.4%), subdiaphragmatic fluid collection ( n = 3, 16.6%), worsening hepatic insufficiency in a cirrhotic patient (n = 1, 5.5%), and wound infection in a patient converted to open surgery (n = 1, 5.5%). The mean hospital stay was 6.4 days (range 3-14 days). At a mean follow-up of 10.8 months (range 5-16 months) all patients are alive and 14 are disease-free, as demonstrated by normalization of tumor markers and negative magnetic resonance imaging. In carefully selected patients total laparoscopic ultrasound-guided cryoablation is feasible and safe. A longer period of follow-up is required to evaluate the efficacy of the procedure and its impact on survival.

Histopathology of the frozen prostate. The microscopic bases of prostatic carcinoma cryoablation.

In the last few years percutaneous cryoablation surgery of the prostate has been re-introduced as an alternative means to treat prostatic carcinoma. Advantages of the technique include local effectiveness in eradicating tumors, minimal morbidity rate and lower costs when compared to radical surgery. We report a study documenting the histopathological changes seen in 317 biopsy specimens obtained from 30 patients (age range 59-83 years, median 73 years) treated with cryosurgical ablation for prostate cancer. Pre- and postoperatory assessment was inclusive of plain clinical, laboratory and instrumental data (digital rectal examination, transrectal ultrasound scan, serum prostatic specific antigen concentration) and systematic biopsies obtained from conventional and modified prostate sextants. Fifteen patients had tumors extending through the prostate capsule (pT3 and pT4). Six patients had stage PT1 tumors and 9 had stage pT2. Tissues were sampled at 3, 6 and between 12-18 months postoperatively. The histologic findings, in decreasing order of frequency, were: full core fibrosis, necrosis, granulation tissue, basal cell hyperplasia, cell swelling, hemosiderin deposits, chronic inflammation, thick nerves and prostatic hyperplasia. Necrosis was of the coagulative type, sometimes associated with nuclear debris, and seen at relatively short interval from cryotherapy. Fibrosis with hyaline qualities was seen especially at 12-18 month interval. The presence of necrosis, as well as granulation tissue, hemosiderin deposits and cell swelling, strongly correlate to intervals from cryosurgical ablation. Residual tumor tissue was focal (0.5-1 mm) and recognizable in 9 cores from 4 patients (13.3%) sampled especially from the prostatic apex. Incipient tumor necrosis was seen in 11 cores, without particular distribution. These findings indicate that cryosurgery results in distinctive changes in both tumoral and non-tumoral prostate tissue. Knowledge of the histopathologic patterns is important since it provides the clinicians with information on treatment efficacy or failure, and could assist in the selection of larger groups of patients eligible to cryosurgical ablation.

Iontophoresis of drugs in the bladder wall: equipment and preliminary studies.

Iontophoresis is the active transport of ions into tissues by means of an electric current: Ji = -D(i)delta Ci/delta chi + DizeECi/kT. Where Ji is the total ionic flux, D(i) the diffusion coefficient, Ci the concentration, z the valency, and E the electric field. The first expression on the right side of the equation is Fick's law of diffusion and approaches zero for bladder mucosa, which leads to uncertain results following intravesical administration of various therapeutic agents. The application of an electric field will potentially accelerate drug administration into the bladder wall in a controllable manner. To evaluate this concept, an appropriate source of electric current and electrodes was fabricated; then, studies were conducted in human cadaveric bladders and clinical trials in human subjects. Ionized dyes were applied in duplicate to 10 fresh cadaveric bladders. Electric currents (3.5-5.0 mA) were applied for 20 min to 10 solutions, and no current was used in 10 controls. Twenty-eight patients had 100 ml solutions of 1% mepivacaine or lidocaine with epinephrine infused into their bladders prior to endoscopic resections. Twenty-two patients received currents of 10-20 mA for 10-20 min, and 6 controls had either no drugs or a current of reverse polarity applied. Visually and on microscopy, the 10 control cadaveric bladder surfaces demonstrated only faint staining of the surface mucosa whereas the experimental surfaces showed full-thickness staining of the mucosa extending into the muscularis. The 6 control patients required supplemental anesthesia or abandonment of the operative procedure. Of the 22 experimental subjects, 16 tolerated procedures with up to 25 g of tissue removed by diathermic resection.

Intra-renal reflux.

A new model for the study of intra-renal reflux (IRR) is proposed. The renal pelvis of human kidneys, either obtained from cadavers or removed surgically, was injected, at increasing pressures, with dye solutions to investigate intra-renal reflux. To reproduce physiological conditions as closely as possible, arterial perfusion was performed, either continuously or by means of a peristaltic pump, so that a predetermined pressure in the vascular system could be obtained. Comparison was made between results obtained by this technique and the results reported in the literature and previously recorded by the present authors without any perfusion of the vascular system. Our results show significant diffusion of the vascular system. Our results show significant differences in the threshold of IRR compared to data from experiments in which vascular perfusion was not simultaneously performed. It can be concluded that, under physiological conditions, intrarenal flux occurs at pressures of 40 cm H2O, and pyelovenous reflux at pressures of 60-70 cm H2O.