Citoinclusão de capa leucocitária e medula óssea de cães: padronização da técnica / Cell block of canine buffy coat and bone marrow in paraffin blocks: technique standardization

A técnica de citoinclusão é amplamente utilizada e reconhecida por profissionais da área da saúde, em particular médicos patologistas, porém as informações sobre a aplicação desse método em medicina veterinária ainda são raras. Análises citológicas de medula óssea e da capa leucocitária (porção do sangue formada por concentrados de leucócitos) são amplamente utilizadas no diagnóstico de patologias de origem neoplásica e não neoplásica e de agentes infecciosos. Embora a importância do uso da técnica seja amplamente reconhecida, não há registro da utilização de amostras citológicas de medula óssea e capa leucocitária na confecção de citoinclusões em blocos de parafina, como meio de diagnóstico no segmento médico veterinário. Assim, este trabalho tem como objetivo elaborar um protocolo de citoinclusão em parafina para amostras citológicas de medula óssea e capa leucocitária de cães. Foram submetidas à técnica de citoinclusão 110 amostras de capa leucocitária e 44 de medula óssea de cães portadores ou não de enfermidade sistêmica, sendo que em 68% destas, tanto para a capa leucocitária quanto para a medula óssea, as amostras se mostraram viáveis. A utilização do álcool 95% como fixador e de etapas no processamento histológico de 20 minutos (álcool absoluto, xilol e parafina, três banhos cada) foi crucial para a qualidade dos cortes histológicos e para a análise microscópica dos espécimes corados pela hematoxilina-eosina. A separação mecânica da capa leucocitária e a centrifugação do aspirado de medula óssea foram eficientes e de baixo custo no preparo das citoinclusões. Ressalta-se a importância na padronização da técnica de citoinclusão, em particular para amostras de capa leucocitária e medula óssea, visando à obtenção de espécimes de qualidade independentemente das limitações de equipamentos...

The cell block technique is widely used and recognized by health professionals, but lacks in information regarding the specific contribution of this method to veterinary medicine. The cytology of bone marrow and buffy coat (cellular portion of the hole blood) are widely used in the diagnosis of neoplastic and non-neoplastic diseases, and also for the detection of infectious agents. Although the importance of these samples is widely recognized, there is no information about the use of buffy coat and bone marrow samples in the cell block procedure among the research material used for this paper. This work aims for the cell block standardization for canine buffy coat and bone marrow samples. We collected 110 buffy coat samples and 44 bone marrow samples for the cell block preparation, and 68.2% of buffy coat and bone marrow proved to be viable at the end of the procedure. The 95% ethanol fixatives along with the 20 minute processing steps (absolute ethanol, xilol and paraffin, 3 of each) were crucial for the quality of the material both in microtomy and optical microscopy. Mechanical separation of the buffy coat proved to be easy and cheap and was used to compose the cell block technique. In this research we emphasized the importance of cell block standardization in order to develop and easy, inexpensive and reproducible method, regardless of any of the professionals' limitations...

Combined cytoreductive laser therapy and immunotherapy for palliation of metastatic renal cell carcinoma to the head and neck.

Interleukin-2 (IL-2) remains the mainstay of treatment for metastatic renal cell carcinoma (RCC), but minimally invasive surgical techniques have provided new options for the combined treatment of RCC. Two patients with metastatic RCC to the head and neck treated by combined laser-induced thermal therapy and IL-2 were described in this case report. Both patients had an extended survival compared to the historical survival of 10 months for metastatic RCC but eventually succumbed to progressive disease. The authors' initial experience with metastatic RCC suggests that laser thermoablation and immunotherapy in selected patients with metastatic RCC is warranted as a palliative treatment, but a larger study with long-term follow-up is necessary to determine the effectiveness of this approach.

Intratumoral hypericin and KTP laser therapy for transplanted squamous cell carcinoma.

OBJECTIVES/HYPOTHESIS: To test intratumoral photodynamic therapy (IPDT) as a new treatment for squamous cell carcinoma in a preclinical tumor model. STUDY DESIGN AND METHODS: Human P3 squamous carcinoma cells were transplanted subcutaneously in athymic nude mice and allowed to grow into 300- to 500-mm3 tumors. Hypericin dye at 1 microg/gm of body weight was injected intratumorally (IT) or intravenously (IV). After 4 hours hypericin biodistribution was assessed in ethanol extracts from tissues by fluorescence spectroscopy. IPDT also was tested by KTP laser fiberoptic insertion in tumors 4 hours after IT dye injection compared to KTP532 laser therapy alone (532 nm, 1W, 40-60 J, 0.6-mm fiber). RESULTS: Hypericin concentration in tissues was as follows: (IT vs. IV) for tumors (3660 vs. 135 ng dye/gm tissue), lung (760 vs. 6345), liver (75 vs. 935), blood (65 vs. 480) compared to skin (465 vs. 110) or muscle (335 vs. 80) adjacent to the squamous cell tumors. Four hours after dye injection, the tumor exhibited bright orange fluorescence when excited by KTP 532-nm green laser light. The IPDT-treated tumors had a 3.32+/-0.32-mm radius of cell destruction when H&E-stained sections were examined compared with 2.5+/-0.38 mm for the laser only control group (n = 10, P = .003). CONCLUSIONS: This pilot study indicates laser IPDT with hypericin induces a significant increase in tumor necrosis compared with laser alone and may be useful as a less invasive adjuvant treatment for recurrent or inoperable human squamous cell cancers of the head and neck.

Combined cisplatinum and laser thermal therapy for palliation of recurrent head and neck tumors.

In recent years endoscopically controlled laser-induced thermal therapy (LITT) has been increasingly accepted as a minimally invasive method for palliation of advanced or recurrent head and neck or gastrointestinal cancer. Previous studies have shown that adjuvant chemotherapy can potentiate endoscopic laser thermal ablation of obstructing tumors leading to improved palliation in advanced cancer patients. Eight patients with recurrent head and neck tumors volunteered to enroll as part of an ongoing phase II LITT clinical trial, and also elected to be treated with systemic chemotherapy (cisplatin, 80 mg/m(2)) followed 24 h later by palliative laser thermal ablation. Laser treatments were repeated in patients with residual disease or recurrence for a total of 27 LITT sessions. Four of the 8 patients treated with laser thermal chemotherapy remained alive after a median follow-up of 12 months. Of the 12 tumor sites treated, complete responses were located in the oral cavity (3), oropharynx (1), hypopharynx (1), maxillary sinus (1), and median survival for these patients was 9.5 months. This initial experience with cisplatinum-based laser chemotherapy indicates both safety and therapeutic potential for palliation of advanced head and neck cancer but this must be confirmed by longer follow-up in a larger cohort of patients.

Combined intratumor cisplatinum injection and Nd:YAG laser therapy.

OBJECTIVES/HYPOTHESIS: Interstitial laser therapy (ILT) has become useful for tumor palliation in patients with advanced head and neck cancer. Cisplatinum chemotherapy also is a frequent adjuvant treatment for recurrent tumors, but systemic toxicity limits application. Intratumor cisplatinum injection combined with ILT may improve therapy of these recurrent tumors with reduced toxicity. STUDY DESIGN: Prospective. Tumor transplants were injected with cisplatinum in a gel implant before ILT to evaluate treatment response and toxicity in a preclinical study. METHODS: UCLA-P3 human squamous cell carcinoma tumors were grown as subcutaneous transplants in nude mice and treated by intratumor injection of 2 mg/mL cisplatinum in a slow-release, collagen-based gel carrier 4 hours before interstitial implantation of Nd:YAG laser fiberoptics to induce local tumor hyperthermia. Treatment efficacy and toxicity were followed for 12 weeks after combined drug and laser therapy compared with ILT alone. RESULTS: Combined cisplatinum gel and ILT was a significant improvement (P < .01 by chi-square test) and induced 57% complete responses without regrowth in 21 transplanted tumors compared with only 24% in 21 tumors after ILT alone during 12-week follow-up. Recurrences in both cases appeared to result from nonuniform laser energy delivery within tumors via the implanted fiberoptic tip. CONCLUSIONS: The results of this experimental combined cisplatinum and ILT study suggest it may be possible to improve treatment of advanced head and neck cancer by intratumor injection of gel implants containing the drug followed by interstitial Nd:YAG laser hyperthermia.

Laser and cisplatinum for treatment of human squamous cell carcinoma.

OBJECTIVE: Interstitial laser therapy (ILT) with the neodymium:yttrium-aluminum-garnet (Nd:YAG) (1064 nm) laser via fiberoptics is becoming a more precise, minimally invasive alternative for thermoablation of unresectable or recurrent head and neck neoplasms, but recurrence is often seen at the margin. Combining intratumor chemotherapy with interstitial laser should be most effective using drugs activated by thermal energy. The objective of the current study was to test intratumor cisplatinum (cis-diaminedichloroplatinum [CDDP]) injections given in conjunction with laser therapy as an experimental approach for improved treatment of squamous cell carcinoma (SCC). METHODS: Human SCC tumors were grown as subcutaneous transplants in nude mice and injected with CDDP (0.4 to 1.2 mg/g) in water or in collagen-based gel carrier with epinephrine (epi-gel) followed by ILT via 0.6-mm fiberoptics coupled to an Nd:YAG laser (1064 nm/180 J). RESULTS: Tumors injected with CDDP epi-gel exhibited a partial response with two- to fourfold tumor delay compared with aqueous drug or untreated SCC transplants during 10 weeks' follow-up. Combined drug and laser therapy significantly (P < .01) decreased tumor volume, with recurrence in only 25% of animals tested compared with 78% tumor regrowth after ILT alone. CONCLUSION: These initial results suggest that laser chemotherapy may become an effective treatment for advanced head and neck cancer.

Palliative laser therapy for recurrent head and neck cancer: a Phase II clinical study.

OBJECTIVES: Laser therapy is becoming a more precise, minimally invasive alternative for tumor ablation. Recent reports confirm successful palliation of pain and functional disabilities in patients with advanced deep carcinoma of the head and neck using interstitial laser phototherapy (ILT). STUDY DESIGN, PATIENTS, AND METHODS: The current study describes an ongoing Phase II trial of neodymium/yttrium-aluminum-garnet (Nd:YAG) laser therapy for palliation of advanced head and neck cancer. A total of 40 advanced cancer patients have been entered into this protocol (25 men and 15 women). RESULTS: Nineteen of these patients had no evidence of recurrence after ILT with an average follow-up of 11 months (range, 2 to 24 mo). Currently, 19 of these patients are alive, 14 with tumor remission and six with persistent disease. A total of 79 tumor sites received ILT with 43 (54.5%) completely ablated. Stratified by tumor site, ILT led to a complete response in 21 of 24 in the oral cavity, eight of 28 neck tumors, four of 10 in skin, and 10 of 17 in other sites. The procedure was well tolerated in most cases and was repeated at intervals in patients with residual disease or recurrences for a total of 118 laser treatments (average, 2.95 treatments per patient). CONCLUSIONS: The results suggest that ILT can be performed safely and repeated as needed, and may be less costly than conventional surgery for head and neck cancer. However, additional follow-up is needed to obtain convincing evidence of long-term therapeutic benefits.

Improved photochemotherapy of malignant cells with daunomycin and the KTP laser.

Laser photochemotherapy of malignancies may become an effective palliative treatment for advanced had and neck cancer using light-sensitive, chemotherapeutic drugs activated in tumors via interstitial laser fiberoptics. Previously, it was reported that cultured human P3 squamous cells incubated 2 hours with daunomycin (Dn) exhibited tenfold enhanced cytotoxicity after exposure to argon laser light at 514 nm. This short-term uptake leads to drug localization in cytoplasmic and membrane sites prior to nuclear accumulation and daunomycin topoisomerase inhibition. In the current study phototoxicity of Dn-sensitized human cancer cells was tested using broad-spectrum white light compared to monochromatic green-wavelength light. Drug uptake and laser energy levels were optimized for maximum synergy. To test light-enhanced chemotherapy in vitro, the kinetics of cell uptake and toxicity of daunomycin was measured at 1, 2, and 5 microg/ml in three human tumor cell lines: P3 squamous-cell carcinoma, M26 melanoma, and TE671 fibrosarcoma. After 2 hr Dn uptake, all cell lines were tested for phototherapy response by exposure to 300- to 900-nm visible light from a xenon lamp or monochromatic 532-nm green light from a KTP laser. When the KTP laser output was varied from 0 to 120 Joules in Dn-sensitized tumor cells, a linear phototherapy response was seen with energy as low as 12 J inducing drug phototoxicity. These results provide evidence that daunomycin cytotoxicity is enhanced when exposed to 532-nm laser illumination in the three tumor types tested and confirm that the response is related to both energy level and drug dose.

Anthrapyrazoles and interstitial laser phototherapy for experimental treatment of squamous cell carcinoma.

Interstitial laser therapy (ILT) is an effective palliative treatment for advanced head and neck cancer, but recurrence often is seen at the margin. The objective of the current study was to test combined drug and laser therapy as an experimental approach for improved treatment of human squamous cell carcinoma (SCCA). Human SCCA tumor transplants were grown in nude mice and injected with the photosensitive anthrapyrazole CI-941 before ILT. Intralesional drug injections alone at levels ranging from 60 to 1200 microg/gm of tumor induced a growth delay at the higher doses, but recurrence was seen in all 35 tumors tested. SCCA tumor transplants injected with 240 microg/gm CI-941 followed after 4 hours by ILT with the KTP532 laser led to a complete response rate of 72% (21/29) compared with 45% (13/29) for ILT alone. Laser chemotherapy was a significant improvement compared with ILT when partial and complete responses were combined (P < 0.03). The results provide preclinical evidence that laser chemotherapy may become a useful minimally invasive treatment for advanced squamous cell carcinoma of the head and neck.

KTP laser and neutral red phototherapy of human squamous cell carcinoma.

Neutral red (NR) is a cationic, nontoxic vital dye employed as a histologic stain for proliferating cells; it has been used clinically for photodynamic treatment of herpes simplex virus lesions. NR is selectively taken up and concentrated by mitotic cells, an important characteristic for more effective antineoplastic agents. In the present study, UCLA-SO-P3 human squamous carcinoma cells displayed minimal toxicity when incubated with up to 50 microg/ml NR in the absence of light. However, cells incubated with greater than 0.5 microg/ml NR followed by exposure to KTP laser light at 532 nm exhibited nearly 100% tumor cell death. The degree of cell toxicity was proportional to NR dose and laser light fluence. This study demonstrates that NR is an excellent cancer cell photosensitizer in vitro, and, after adding additional in vivo preclinical testing, may prove to be a useful agent in photodynamic destruction of head and neck tumors.

Laser chemotherapy of human carcinoma cells with three new anticancer drugs.

A new experimental therapy for squamous carcinoma was tested by sensitizing human tumor cells with light-sensitive anticancer drugs followed by laser illumination at visible or infrared wavelengths. The anthrapyrazole DUP-941 and the isoquinoline derivative DUP-840 were compared with the dianthraquinone hypericin. P3 human squamous carcinoma cells were incubated for 2 h with the drugs at escalating doses ranging from 5 to 100 micrograms/ml, then exposed to visible green 532-nm or infrared 1064-nm light at 300 J output from a KTP/Nd:YAG laser. Tumor cell toxicity measured by in vitro MTT viability assays was minimal after DUP-840 uptake but was slightly enhanced by infrared laser emissions. By contrast, the strong tumoricidal effects seen after DUP-941 uptake were amplified over 10-fold by 532-nm light and up to 2-fold by 1064-nm light. Hypericin-sensitized tumor cells were killed after 532 nm irradiation even at the lowest drug dose but were not affected by 1064-nm illumination. The results suggest that laser chemotherapy with drugs sensitive to photothermal energy could become a useful new treatment modality for cancer.

Cisplatinum and interstitial laser therapy for advanced head and neck cancer: a preclinical study.

BACKGROUND AND OBJECTIVE: Direct intratumor injection of cisplatinum (CDDP) and laser therapy were tested for improved treatment of squamous cell carcinoma (SCCA). STUDY DESIGN/MATERIALS AND METHODS: Human SCCA tumors were grown as s.c. transplants in nude mice and injected with CDDP (0.4-1.2 mg/gm) in water or in collagen-based gel carrier with epinephrine (epi-gel), followed by interstitial laser therapy (ILT) via 0.6 mm fiberoptics (532 nm/300 J). RESULTS: Tumors injected with CDDP epi-gel exhibited a partial response with 2-4-fold tumor growth delay, compared to aqueous drug or untreated SCCA transplants during 10-week follow-up. Combined drug and laser therapy significantly decreased tumor volume with recurrence in only 25% (2/8) of animals tested, compared to 66% tumor regrowth (10/15) after ILT alone. CONCLUSION: These initial results suggest laser chemotherapy may become an effective treatment for advanced head and neck cancer.

Hypericin: a new laser phototargeting agent for human cancer cells.

Laser activation of anthracycline-related drugs combines chemotherapy with photoablation for improved treatment. Hypericin, a structurally related anthraquinone, was tested for laser activation and cytotoxicity in human cancer cells. Viability of P3 squamous cell carcinoma cells incubated with 1 to 20 microgram/mL hypericin was reduced by more than 95% after 1 minute exposure at 4 degrees C to an argon laser (514 nm, 5 W), a KTP-532 laser (532 nm, 5 W), or a 20-A xenon lamp. Viability was reduced over 90% in six human carcinoma, sarcoma, and melanoma cell lines by this combined treatment, but only trace toxicity was seen after separate exposure to hypericin or light alone. These results show that hypericin is a sensitive agent for phototherapy of human cancer cells in vitro and indicate that this drug may be useful for tumor targeting via minimally invasive imaging-guided laser fiber optics.

Laser photochemotherapy with anthracyclines on cultured human squamous carcinoma cells.

A new treatment for cancer has been tested in vitro using light-sensitive anthracyclines followed by laser photoactivation, as described by several investigators. We previously reported 10-fold enhanced laser killing after 2 hours of incubation with daunomycin by cultured human carcinoma cells. This short-term uptake leads to drug localization in cytoplasmic and membrane sites prior to nuclear accumulation and topoisomerase inhibition. In the present study, daunomycin was incubated for 2 or 24 hours with P3 squamous carcinoma cells to directly compare cytoplasmic vs. nuclear drug targeting before and after KTP-532 laser activation. Monolayer cultures of the P3 cells sensitized with daunomycin for 2 hours, then chilled (4 degree C), and exposed to the KTP laser (532 nm, 94.2 J/cm2) had a 2- to 10-fold increased therapeutic response compared with drug or laser alone when measured by MTT tetrazolium assays. After 24 hours of incubation with daunomycin, the chemotherapeutic response of P3 tumor cells was amplified 2-fold by laser exposure. The results suggest that daunomycin and laser treatment can be combined for improved therapy of human cancer.

Interstitial laser photochemotherapy with new anthrapyrazole drugs for the treatment of xenograft tumors.

Photodynamic therapy (PDT) with lasers and new dyes has gained popularity in recent years as a minimally invasive technique with high tumoricidal effects in vitro and in some cancer patients. However, because new laser dyes are not FDA approved at present, the clinical evaluation of PDT may be years away. During the past 6 years we have used laser alone for photothermal ablation in both preclinical studies and in a large number of patients with an observed 60% tumor response rate. The 40% treatment failure led us to explore the possibility of combined therapy with lasers and standard chemotherapeutic drugs. We have recently tested a promising preclinical alternative using implantation of a bare 600-microns KTP 532 laser fiberoptic in multiple tumor sites 30 min after intratumor injection of the anthrapyrazole DUP-941. As a control, this drug was injected in 3 sites of P3 human squamous cell tumor transplants in nude mice, which led to tumor stasis without regression. Similar 400-600 mm3 tumors exposed to laser illumination alone (0.8 W for 5 sec) at multiple sites resulted in tumor regrowth after 10 weeks in 80% of the animals. However, combining interstitial laser illumination with intratumor DUP-941 injections led to complete tumor regression in 85% of the mice. We propose that intratumor drug injection followed by interstitial laser fiberoptic treatment represents a potentially useful new method for tumor ablation in advanced cancer patients.

Laser photochemotherapy: a less invasive approach for treatment of cancer.

The effectiveness of combining surgery with chemo- and radiation therapy in treatment of human cancer provides a useful model for further development of new multimodality approaches including laser photochemotherapy. Laser endoscopy often is a useful treatment for obstructive tumors in airways, but interstitial laser fiberoptics is becoming a more precise, minimally invasive alternative for ablation of unresectable or recurrent neoplasms. Combining intratumor chemotherapy with laser energy delivery via interstitial fiberoptics should be most effective using drugs activated by photothermal energy. A number of investigators have shown that anthracyclines and cis-platinum are likely candidates for light or heat activation in cancer cells. An advantage of anthracyclines is their dual role as antitumor drugs and as photosensitizers. Because they are effective chemotherapy agents without photoactivation, two approaches are possible to increase tumor responses. Maximum tolerated dose followed by photoillumination via laser fiberoptics can be used to obtain better tumor palliation. Improved treatment response to lower intratumor drug levels after laser activation also should reduce systemic toxicity. Preclinical studies and recent case reports from several groups suggest photochemotherapy with currently approved drugs and lasers may soon become an attractive alternative for treatment of recurrent tumors in cancer patients.

Hypericin uptake in rabbits and nude mice transplanted with human squamous cell carcinomas: study of a new sensitizer for laser phototherapy.

Tissue uptake and biodistribution of hypericin was measured in rabbits and in nu/nu mice xenografted with P3 human squamous cell carcinoma to assess the value of this dye as an in vivo sensitizer for laser photoinactivation of solid tumors. Hypericin has absorption maxima at 545 and 590 nm with a fluorescence emission peak at 640 nm in ethanol. Dye uptake after intravenous injection was tested at 4 and 24 hours in rabbit tissues by ethanol extraction and quantitative fluorescence spectrophotometry. Maximum dye levels were seen at 4 hours in most vascular organs with lung having fivefold higher uptake than spleen followed by liver, blood, and kidney. Mice were examined after 2, 4, 6, 8, and 24 hours and after 3 and 7 days for dye uptake. The peak concentration of hypericin in murine organs was reached at 4 hours with uptake per gram of tissue as follows: lung > spleen > liver > blood > kidney > heart > gut > tumor > stomach > skin > muscle > brain. Elimination of hypericin was rapid in most murine organs with residual dye under 10% of maximum by 7 days compared to 25% to 30% retention for the squamous cell tumors and several normal tissues. These results suggest that hypericin may be a useful photosensitizer for KTP/532 laser interstitial therapy of human cancer.