Vital Tumor Tissue Research Articles

Discriminating Vital Tumor from Necrotic Tissue in Human Glioblastoma Tissue Samples by Raman Spectroscopy

Vital and necrotic glioblastoma tissues were studied by Raman microspectroscopy to identify possibilities for the development of an in vivo Raman method for real-time intraoperative brain biopsy guidance. The histologic malignancy grade of gliomas depends on the presence of parameters such as endothelial proliferation and necrosis, which are often not evenly distributed within the tumor. Because tissue samples obtained by stereotactic surgery are relatively small, sampling errors may easily occur by missing these crucial features. Although necrosis is important for grading, specimens containing only necrosis are diagnostically useless. Raman microspectroscopic mapping experiments were performed on unfixed cryosections of glioblastoma, obtained from 20 patients. After spectral acquisition, a clustering analysis was performed, resulting in groups of similar spectra. Each cluster was assigned a color, and pseudo-color Raman maps of the tissue sections were constructed. After the Raman experiments, the tissue sections were stained for histopathologic analysis, enabling identification of the histologic origin of the Raman spectra and assignment of the Raman spectral clusters to either vital or necrotic tissue. A classification model for discrimination between vital and necrotic tumor tissue based on linear discriminant analysis was developed. The classification model was evaluated using independent Raman data obtained from nine other tissue sections and yielded 100% accuracy. Information about the biochemical differences between necrosis and vital tumor was obtained by the analysis of difference spectra. Necrotic tissue was found to consistently contain higher levels of cholesterol (-esters). This in vitro result indicates that Raman spectra contain the information to distinguish vital glioblastoma from necrosis and makes Raman spectroscopy a powerful candidate for guidance of stereotactic brain biopsy.

Sequential MRI and MR spectroscopy in embolized meningiomas: correlation with surgical and histopathological findings.

The preoperative embolization of meningiomas is commonly used to facilitate surgery. The purpose of this study was to evaluate the morphological and metabolic changes in embolized meningiomas and to correlate the results with surgical and histopathological findings. In a prospective study, 36 patients with intracranial meningiomas were included. The extent of devascularization was assessed by angiography and MR volumetry. MRI and MR spectroscopy (MRS) were performed before and sequentially after embolization. At surgery, blood loss was measured and intraoperative duplex-mode ultrasound was applied to identify avascular tumor portions. Histopathological specimens were evaluated for the histological subtype, localization and extent of necrotic tumor portions. Postembolization MRI revealed a variable pattern of secondary revascularization and devascularization with an early onset following embolization. In all patients, peripheral secondary enhancement was present which histopathologically represented a thin layer of vital tumor tissue. MRS revealed lactate in devascularized areas immediately after embolization. Lipids were not observed before the 3rd day after embolization and were always associated with avascular and soft tissue at the time of surgery. Embolized meningiomas feature a variable dynamic with the potential for revascularization and secondary devascularization. Lipid signals indicate avascular and soft tissue at surgery. In case of delayed surgery, MRI and MRS should be performed in order to exclude revascularization and to establish the fatty degeneration of the meningioma.

Vascular endothelial growth factor expression, angiogenesis, and necrosis in renal cell carcinomas.

Rapidly growing tumors often develop necrosis. In the present study the expression of vascular endothelial growth factor (VEGF) was investigated and compared to microvessel density and necrosis of renal cell carcinomas. In the tumor-host interface the microvessel density was significantly increased compared to central tumor areas. Tumor necrosis was associated with a decrease of microvessel density and an increase of the VEGF protein expression within the perinecrotic rim. VEGF protein was focally upregulated in vital tumor tissue. An increase of the apoptotic rate of endothelia and vital tumor tissue in tumors with necrosis could not be detected. VEGF(121,165) mRNA was decreased in proliferatively active carcinomas compared to less proliferative tumors. Multicellular renal cell cancer spheroids as a model of chronic hypoxia developed central apoptosis but no necrosis. VEGF was upregulated in the spheroid. Tumor microvessels expressed matrix metalloproteinase -2 and -9 and an incomplete pericyte covering in comparison to tumor-free tissue indicating immature active angiogenesis. We conclude that highly proliferative renal cell carcinomas outgrow their vascular supply and develop chronic hypoxia inducing a decrease of proliferation and an increase of VEGF expression. However, chronic hypoxia does not cause significant necrosis or apoptosis. Tumor necrosis is more likely induced by acute hypoxia due to immature microvessels. Furthermore, VEGF expression associated with concomitant tumor necrosis may help identify renal cell carcinomas susceptible to antiangiogenic therapy.

Grading of Tumor Regression in Non-small Cell Lung Cancer: Morphology and Prognosis

Different types of multimodality therapy, including chemoradiotherapy and surgery, increasingly are being used for the treatment of patients with locally advanced non-small cell lung cancer (NSCLC; stages IIIA and IIIB). In this context, the applicability of a morphologic regression grading and its prognostic value were investigated. In a multicenter phase II trial, 54 patients with locally advanced NSCLC received neoadjuvant bimodality treatment (ie, two cycles of ifosfamide, carboplatin, and etoposide, followed by twice-daily radiation up to 45 Gy with simultaneous administration of carboplatin and vindesine). Forty patients underwent resections. Using the corresponding resection specimens of the primary and regional lymph nodes, the following regression grading was established: grade I, no regression or only spontaneous tumor regression; grade II, morphologic evidence of therapy-induced tumor regression with at least 10% (grade IIa) or < 10% (grade IIb) vital tumor tissue; and grade III, complete tumor regression with no evidence of vital tumor tissue. Regression grading then was correlated with the survival time. Three tumors were classified as regression grade I, 10 were classified as regression grade IIa, 20 were classified as regression grade IIb, and 7 were classified as regression grade III. Patients with tumors of regression grades IIb or III showed significantly longer survival times than those with tumors of regression grades I or IIa (median survival time, 36 vs 14 months, respectively; 3-year survival rate, 52% vs 9%, respectively; p = 0.02). These survival times were also compared for patients who had undergone complete resection (median survival time, not reached vs 23 months, respectively; 3-year survival rate, 56% vs 11%, respectively; p = 0.03). The presurgical clinical response after patients had received neoadjuvant multimodality therapy had no predictive value in assessing the extent of therapy-induced tumor regression in the resection specimen. After neoadjuvant therapy of patients with NSCLC, the proposed tumor regression grading was of predictive value for long-term survival. Beyond the achievement of complete tumor resection (R0), a therapy-induced tumor regression of < 10% of vital tumor tissue is pivotal for superior long-term outcomes.

Therapy monitoring with 2-(18F)-FDG positron emission tomography after neoadjuvant radiation treatment of mouth carcinoma

Combined protocols of radiation therapy and surgical resection, as applied in advanced oral cancer, rely on objective and early assessment of treatment response to radiation therapy. Non-responders require immediate radical salvage surgery even in spite of substantial operative risks, while complete or subtotal response may give reasons for continuing the conservative approach. Therefore, we investigated radiation response by FDG-PET for early monitoring of oral cancer. In 30 patients with advanced stages of oral cancer (Table 1), FDG-PET (Siemens, ECAT EXACT 922) was performed within 4 weeks after completion of preoperative radiation therapy (36 Gy). SUV of tumor regions were compared to the histologic degree of tumor regression in complete resection specimens. Statistic evaluation included correlation analysis of SUV vs tumor regression and ROC analysis for SUV cut-off values. While low FDG accumulation was found in tumors with histological complete remission (2.3 +/- 0.4) as well as in cases of residual tumor (3.4 +/- 1.8), high FDG uptake was a rather specific indicator of vital tumor tissue (Figure 2). Significant correlation (p = 0.045) between postradiotherapeutic FDG-uptake and histological tumor regression was recognized. A SUV > 2.75 as a clinically practicable threshold value for the identification of residual vital tumor resulted in a specificity of 88%, sensitivity of 68%, a positive predictive value of 94% and a negative predictive value of 50% (Figure 3). Based on our actual follow-up data we could not confirm a significant correlation between postradiotherapeutic SUV and patients' survival. Within a standardized protocol, FDG-PET recognize treatment response to radiation therapy in oral squamous cell carcinoma with a reasonable specificity and thus provides a basis for further therapeutic decisions. An increased SUV (> 2.75) may be the rational to justify an aggressive surgical approach even when patients face substantial surgical or anesthesiological risk. However, the posttherapeutic pattern of glucose uptake varies with the applied treatment modalities and has to be explored for the protocol applied.

Apoptotic cell death induced by local brain hyperthermia in a rat glioma model.

Hyperthermia has been shown to inhibit glioma growth both in vitro and in vivo, and has been reported to induce apoptosis of a variety of cells. We investigated the role of apoptosis in tumor cell death following hyperthermia in a rat glioma model representing human glioblastoma. Apoptotic cell death was evaluated by terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) and hematoxylin and eosin (H & E) staining. We also examined c-Jun expression immunohistochemically. Apoptotic cell death in rat brain tumors that grew after implantation of C6 glioma cells showed regional differences. In all rats, apoptotic cells, characterized by extreme chromatin condensation and fragmented nuclei with apoptotic bodies in H & E-stained sections, were observed in the gliomas' necrotic cores. TUNEL-positive cells were observed in the border zones between necrotic and vital tumor cells. Before hyperthermia, TUNEL-positive cells were sporadically distributed in the vital tumor tissue. After hyperthermia, the number of TUNEL-positive cells in the peripheral region of the tumor mass increased significantly, reached a peak after 6 h and returned to the basal level within 24 h (P < 0.01). C-Jun protein immunoreactivity was not observed in the cells at the tumor periphery. These data indicate that significantly apoptotic cell death unrelated to c-Jun expression occurs after hyperthermia, and that this form of cell death may be the mechanism of tumor regression following hyperthermia treatment of intracranial gliomas.

Regression grading of neoadjuvant non-small-cell lung carcinoma treatment

In the scope of a multi-center-study 35 resection specimens from patients with locally advanced non-small cell lung cancer after neoadjuvant chemotherapy and radiation were processed histologically and graded according to the following regression grading system: grade I: no or only slight, in general spontaneous tumor regression, grade IIa: incomplete tumor regression with more than 10% and grade IIb less than 10% vital tumor tissue as well as grade III: complete tumor regression. In 15 patients with grade II a to III tumor regression roughly concentric foci of various size with a sequence of central tumor necrosis, narrow foam cell rim, vascular granulation tissue and peripheral scar formation were demonstrated as characteristic feature of response to neoadjuvant therapy. In patients with grade IIb to III tumor regression ("responders") median survival time of 27.9 months was significantly longer than in patients with grade I to II a tumor regression ("non-responders") with a median survival time of 12.7 months.

Detection of vital germ cell tumor cells in short-term cell cultures of primary tumors and of retroperitoneal metastasis--clinical implications.

By establishing short-term cell cultures derived from retroperitoneal metastasis after neoadjuvant chemotherapy, our aim was to improve the diagnosis and prognosis in patients with advanced testicular germ cell tumors. The histological evaluation of surgically removed metastatic tissue by retroperitoneal lymphadenectomy (RLA) is extremely complicated after previous chemotherapy, but knowledge of persistence of vital tumor cells in residual lesions is of great prognostic value and therapeutic consequence in patients with testicular germ cell tumors. We therefore investigated whether vital tumor tissue could be detected in short-term cell cultures derived from such metastatic lesions by measuring the concentration of the tumor markers beta human chorionic gonadotropin (beta HCG) and alpha-1 fetoprotein (AFP) in cell culture supernatants. We initially demonstrated the specificity of the determination in cell cultures of human transitional-cell carcinoma cell lines, human foreskin fibroblasts and normal testicular tissue. In a group of 20 patients with untreated primary testicular germ cell tumors, detection of beta HCG and AFP was increased about threefold in cell culture supernatants in comparison to the serum concentration. Finally, we prepared primary cell cultures from surgically removed retroperitoneal metastasis of 12 patients with testicular germ cell tumors after chemotherapy. The serum concentrations of beta HCG and AFP of all patients were at normal values when RLA was performed. However, pathologically increased concentrations of beta HCG (3/3) and AFP (2/3) in cell culture supernatants were found in 3 of 12 cell cultures. Interestingly, these three patients with a pathological increase in beta HCG and AFP as determined in the supernatant of the short-term cell cultures had tumor progression within a mean follow-up of 3 +/- 1 months (P < 0.01), whereas 9 of 12 patients who had no pathological increase in beta HCG and AFP as determined in the supernatant of the short-term cell culture were in complete remission (CR) after a mean follow-up of 40 +/- 11.6 months.

Kleinzelliges Bronchialkarzinom nach Chemotherapie Morphologische Befunde

Resection specimens of 14 patients with small cell bronchial carcinoma after neoadjuvant chemotherapy were processed histologically and graded according to a three-step regression grading system: grade I, no or only slight tumor regression; grade II, incomplete tumor regression; grade III, complete tumor regression without vital tumor tissue. In five patients with either no vital tumor tissue or only small tumor remnants in the resection samples, a typical sequence of central fresh tumor necrosis, foam cell rim, vascular granulation tissue and peripheral scar formation was seen. This morphological finding may be interpreted as a characteristic, but unspecific parameter of good response to preoperative chemotherapy. The presence of vital tumor rims surrounding the capillary bed with intermingled necrotic foci, however, argues in favor of spontaneous tumor regression, which is commonly observed in small cell lung cancer.

Morphometric studies on treated and untreated heterotransplanted malignant tumours.

Short-term therapy effects on heterotransplanted tumours are important to know in order to study the action of anticancer treatment. They are, however, difficult to assess due to interference from such factors as edema, vascular swelling and necrosis. Non-invasive methods such as NMR can be of some use, but the applicability is limited since background noise from the host animal organism may confound measurements. We studied a series of Cisplatine-treated bladder carcinomas heterotransplanted to nude mice and their untreated controls in order to illustrate short-term chemotherapy effect. We applied simple unbiased morphometric methods on formalin-fixed and paraffin-embedded material in order to obtain an unbiased estimate of the volume of the whole tumour and the volume of vital tumour tissue on day 1 and day 3 after treatment, at which time no gross changes in tumour size were evident. Furthermore, we estimated the number of tumour cells in the tumours. The estimate was based on measurement of the mean nuclear volume and the nuclear volume fraction in combination with the measured volume of vital tissue in the tumour. The pronounced therapy effects seen in this experimental system may be of potential interest in monitoring effects at the clinic.

Critical checking of the radiological diagnosis of "complete remission" and "partial remission" following induction chemotherapy of small-cell lung cancer in the light of postoperative histological examination.

A multimodal therapy concept for small-cell lung cancer, which for patients with established pretherapeutic homolateral lymph-node metastases (N2) prescribes induction chemotherapy with subsequent resection as well as supplemental chemo- and radiotherapy, provided the opportunity to evaluate histologically the radiological diagnoses "complete remission" and "partial remission" using resection specimens. In 17 patients a 75% to 100% reduction in tumor size was achieved according to radiological diagnosis. Predictions of "no evidence of disease" or "evidence of disease" were only correct in ten cases. In the remaining seven cases, histology showed the radiological findings to be incorrect. This gives a 77% sensitivity for radiological diagnosis with no specificity. Moreover, differentiation between therapy effect on the primary tumor and on the N2 metastases gives similar results: sensitivity 64% and 67% respectively, specificity 33% and 25% respectively. It is concluded that, particularly after the tumor responds well to therapy, radiological techniques are unsuitable for establishing a diagnosis of "no evidence of disease" or "evidence of disease" in small-cell lung cancer. This is because on the one hand the radiological methods available do not permit clear differentiation between vital tumor tissue and necrosis or fibrosis, while on the other hand groups of vital tumor cells beyond the resolution power of X-ray technology will escape detection.

Combined radiotherapy and surgery in the treatment of neck node metastases from squamous cell carcinoma of the head and neck.

A prospectively recorded series of 107 patients with clinical neck node metastases from head and neck squamous cell carcinomas, treated in 1983-1988, and with initial local control, is evaluated. Eighty-eight patients received preoperative, and were operated 4-6 weeks after radiotherapy, and 19 received postoperative radiotherapy. Forty-four of the neck specimens in the preoperatively treated patients showed vital tumor tissue, 7 with positive and 37 with negative resection margins. Nine of the latter 37 patients died due to regional recurrence. Twenty-three of the preoperatively treated patients had no palpable residual tumor following radiotherapy, but histological examination showed vital tumor tissue in five, of whom two had N1 neck disease. The overall regional failure rate was 19%. Eleven patients (10%) died from local recurrence and 11 from distant metastases. Forty-one patients (38%) are alive without evidence of disease and three (3%) alive with disease (mean observation time 30 months). Combined treatment is recommended for all cases of neck node metastases.

In vivo NMR T2 relaxation of experimental brain tumors in the cat: A multiparameter tissue characterization

Experimental gliomas (F98) were inoculated in cat brain for the systematic study of their in vivo T 2 relaxation time behavior. With a CPMG multi-echo imaging sequence, a train of 16 echoes was evaluated to obtain the transverse relaxation time and the magnetization M(0) at time t = 0. The magnetization decay curves were analyzed for biexponentiality. All tissues showed monoexponential T 2, only that of the ventricular fluid and part of the vital tumor tissue were biexponential. Based on these NMR relaxation parameters the tissues were characterized, their correct assignment being assured by comparison with histological slices. T 2 of normal grey and white matter was 74 ± 6 and 72 ± 6 msec, respectively. These two tissue types were distinguished through M(0) which for white matter was only 0.88 of the intensity of grey matter in full agreement with water content, determined from tissue specimens. At the time of maximal tumor growth and edema spread a tissue differentiation was possible in NMR relaxation parameter images. Separation of the three tissue groups of normal tissue, tumor and edema was based on T 2 with T 2 (normal) < T 2 (tumor) < T 2 (edema). Using M(0) as a second parameter the differentiation was supported, in particular between white matter and tumor or edema. Animals were studied at 1–4 wk after tumor implantation to study tumor development. The magnetization M(0) of both tumor and peritumoral edema went through a maximum between the second and third week of tumor growth. T 2 of edema was maximal at the same time with 133 ± 4 msec, while the relaxation time of tumor continued to increase during the whole growth period, reaching values of 114 ± 12 msec at the fourth week. Thus, a complete characterization of pathological tissues with NMR relaxometry must include a detailed study of the developmental changes of these tissues to assure correct experimental conditions for the goal of optimal contrast between normal and pathological regions in the NMR images.

Pulmonary metastases: Pathological anatomy

In most cases, the histopathological examination of pulmonary metastases will lead to vital clues indicating the primary tumor even after years of disease-free intervals. When evaluating 344 metastases of 100 patients, correlation to the known primary tumor was possible in 98% of the cases. Important additional information may be gained by regression grading of metastases, for example after chemotherapy. In 28 metastases of 4 patients suffering from primary tumors of the testicle, no vital tumor tissue could be demonstrated. The age peak of patients with surgically removed pulmonary metastases was between 40 and 50 years, varying widely, however, depending on the primary lesion. The size of the resected metastases was between 1 and 100 mm, with a peak of 6-10 mm. The examination of early metastases enhanced our knowledge concerning the different phases of metastatic spread such as embolization, implantation, metastatic growth with neoangiogenesis, spontaneous or therapeutically induced tumor regression and local reactions of the pulmonary tissue. The characterization of tumor tissue using, among others, immunohistochemical techniques will lead to clinical and therapeutic consequences.

Two-exponential analysis of spin-spin proton relaxation times in MR imaging using surface coils

Proton relaxation time measurements were performed on a standard whole body MR imager operating at 1.5 T using a conventional surface coil of the manufacturer. A combined CP CPMG multiecho, multislice sequence was used for the T 1 and T 2 relaxation time measurements. Two repetition times of 2000 ms (30 echoes) and 600 ms (2 echoes) with 180°-pulse intervals of 2τ = 22 ms were interleaved in this sequence. A two-exponential T 2 analysis of each pixel of the spin-echo images was computed in a case of an acustic neurinoma. The two-exponential images show a “short” component ( T 2s) due to white and gray matter and a “long” component ( T 2s) due to the cerebrospinal fluid. In the fatty tissue two components with T 2s = 35 ± 3 ms and T 2L = 164 ± 7 ms were measured. Comparing with Gd-DTPA imaging the relaxation time images show a clear differentiation of vital tumor tissue and cerebrospinal fluid.

Results of adjuvant surgery in patients with stage III and IV nonseminomatous testicular tumors after cisplatin‐vinblastine‐bleomycin chemotherapy

From January 1978 to April 1983, 53 patients were treated with cisplatin-vinblastine-bleomycin chemotherapy because of advanced nonseminomatous testicular tumor (NSTT). After the chemotherapy, the serum tumor markers were back to normal in 41 patients, of whom 35 were eligible for surgical removal of the residual tumor. In four patients, vital tumor tissue was found in the residual tumor. Salvage chemotherapy resulted in complete remission. Residual mature teratoma was encountered after the chemotherapy in 15 of the 25 patients with a teratomatous component and in one of the ten patients without a teratomatous component in the primary tumor. On completion of the study, 38 of the 53 patients (72%) are still alive, with a median follow-up of 65 months. Subdivided by tumor volume, survival is found to amount to 92% for small-volume disease, 67% for large-volume disease, and 64% for very-large-volume disease. Six patients (11%) developed a recurrence in the course of the follow-up. Exploratory laparotomy after remission induction chemotherapy is necessary in all patients with a teratomatous component in the primary testicular tumor who have become tumor marker negative, irrespective of the roentgenographic findings of the retroperitoneum. Patients without a teratomatous component in the primary tumor should have exploratory laparotomy only in case of roentgenographic evidence of retroperitoneal residual tumor. A thoracotomy is needed only in the presence of roentgenographic evidence of pulmonary residual lesions.

Binding of five monoclonal anti-CEA antibodies with different epitope specificities to various carcinoma tissues.

Five monoclonal antibodies that recognize different epitopes on carcinoembryonic antigen (CEA) were reacted with tissue sections of various carcinoma specimens. The vital, non-necrotic tissues of those carcinomas of the oesophagus, pancreas, colon and rectum, medullary thyroid, ovary and cervix in which CEA related epitopes were detectable bound all five antibodies to a similar degree. In 3/5 lung carcinomas, 2/10 mammary carcinomas and 1/5 gastric carcinomas, a significantly different binding of the monoclonal antibodies by vital tumour tissue was present as determined by three independent investigators. In necrotic tissue areas, heterogeneity of antibody binding was more common. In a previous investigation, it was shown that normal granulocytes and liver tissue differentially bind the monoclonal anti-CEA antibodies, indicating the presence of crossreacting antigens. The equivalent binding of the five monoclonal anti-CEA antibodies to most of the carcinomas tested suggests that this binding is due to the presence of complete CEA molecules and not only of cross-reacting antigens.