A. Fyles

The relationship between elevated interstitial fluid pressure and blood flow in tumors : A bioengineering analysis

PURPOSEnTo examine the hypothesis that elevated interstitial fluid pressure (IFP) is a cause of reduced blood flow in tumors.nnnMATERIALS AND METHODSnA physiologic model of tumor blood flow was developed based on a semipermeable, compliant capillary in the center of a spherical tumor. The model incorporates the interaction between the tumor vasculature and the interstitium, as mediated by IFP. It also incorporates the dynamic behavior of the capillary wall in response to changes in transmural pressure, and the effect of viscosity on blood flow.nnnRESULTSnThe model predicted elevated tumor IFP in the range of 0 to 56 mmHg. The capillary diameter in the setting of elevated IFP was greatest at the arterial end, and constricted to between 3.2 and 4.4 microm at the venous end. This corresponded to a 2.4- to 3.5-fold reduction in diameter along the length of the capillary. The IFP exceeded the intravascular pressure distally in the capillary, but vascular collapse did not occur. Capillary diameter constriction resulted in a 2.3- to 9.1-fold steady-state reduction in tumor blood flow relative to a state of near-zero IFP.nnnCONCLUSIONnThe results suggest that steady-state vascular constriction occurs in the setting of elevated IFP, and leads to reduced tumor blood flow. This may in turn contribute to the development of hypoxia, which is an important cause of radiation treatment failure in many tumors.

Expression of hypoxia-inducible factor-1α in cervical carcinomas: correlation with tumor oxygenation ☆

PURPOSEnTo investigate the relations between hypoxia-inducible factor-1 (HIF-1), tumor oxygenation, and clinical correlates in patients with locally advanced carcinoma of the uterine cervix.nnnMETHODS AND MATERIALSnBiopsies from 42 patients with invasive cervical carcinoma and previous polarographic O2 measurements were assessed for the expression of HIF-1alpha using digitized microscopic imaging and analysis.nnnRESULTSnThe HIF-1alpha expression levels ranged from <0.1% to 10.7% of the total tumor area; the positive staining was localized exclusively to the nuclei. Three distinct arrangement patterns of HIF-1alpha-positive cells in relation to blood vessels were identified using spatial image mapping: (1) most HIF-1alpha-positive cells were located within the typical oxygen diffusion distance in tissue (< or =150 microm to the nearest blood vessel); (2) most HIF-1alpha-positive cells were located in the vicinity (< or =60 microm) of the blood vessels; and (3) no apparent spatial relationship was found between HIF-1alpha-positive cells and blood vessels. A statistically significant association was found between HIF-1alpha expression and tumor oxygenation (Spearman correlation coefficient = 0.4, p <0.01), as determined with the Eppendorf pO2 histograph. No correlation was found between the level of HIF-1alpha expression and patient outcome, using disease-free survival as the end point.nnnCONCLUSIONnOur results suggest that HIF-1alpha expression may represent a useful biologic marker for hypoxia in uterine cervical cancer.

Interstitial fluid pressure in cervical carcinoma

Interstitial fluid pressure (IFP) is elevated in many animal and human tumors. The authors assessed tumor IFP and its relation to tumor oxygenation in a prospective clinical study of patients with cervical carcinoma.

Proliferation measurements with flow cytometry Tpot in cancer of the uterine cervix: Correlation between two laboratories and preliminary clinical results

PURPOSEnTo assess the prognostic value of the pretreatment potential doubling time (Tpot) in carcinoma of the uterine cervix, relative to other established clinical factors.nnnMETHODS AND MATERIALSnFifty-two patients with cervical cancer were studied prospectively from March 1991 to October 1993. Pretreatment evaluation included examination under anesthesia and tumor biopsy 6 h following the intravenous administration of bromodeoxyuridine (200 mg). Tpot was determined by deriving the labeling index (LI) and S-phase synthesis time (Ts) using flow cytometry. Six patients were not evaluable and excluded. The remaining 46 patients (average age 55 years) were treated uniformly with radical radiation therapy. There were 39 squamous carcinomas and 7 adenocarcinomas. Fédération Internationale de Gynécologie et dObstétrique (FIGO) stages were: Ib and IIa, 12 patients; IIb, 18 patients; III and IV, 16 patients. The median external beam dose was 50 Gy (range, 45-52.8 Gy) delivered in 25 fractions. The median intracavitary dose was 40 Gy (range. 25.5-40 Gy) delivered with a single line source to a point 2 cm lateral of the midline, with a mean dose rate of 0.71 Gy/h. The median overall treatment time was 45 days (range, 34-73 days). As of July 31, 1994, 12 patients had died of disease, and the average follow-up for alive patients was 1.4 years (range, 0.5-3.3 years).nnnRESULTSnThere were 27 tumors with diploid deoxyribonucleic acid (DNA) content and 19 tumors were aneuploid. The median and mean Tpot for the 46 patients were 5.5 and 6.6 days, respectively [range, 2.0-25.6 days; coefficient of variation (CV), 74%]. For 25 patients where Tpot measurements were performed at two separate laboratories, there was a fair correlation (r = 0.74), but systematic differences were detected suggesting that the lack of agreement was not simply due to intratumoral variation. To date, 30 patients remained disease free, while 8 patients had pelvic failure and 9 patients developed distant metastases as the first failure site (1 patient developed both at the same time). In univariate analysis, the only significant prognostic factor for disease-free survival was tumor size (p = 0.004). A short Tpot (or high LI) and long overall treatment time (OTT) were weakly associated with poorer disease-free survival, although not statistically significant (1/Tpot, p = 0.14; LI, p = 0.23; OTT, p = 0.04). Age, FIGO stage, hemoglobin level, S-phase fraction, DNA ploidy, and Ts were not associated with disease-free survival. Multiple regression analysis was not performed because of the relatively small number of patients and short follow-up.nnnCONCLUSIONSnTpot values determined with current techniques by different laboratories cannot be used interchangeably for the purpose of therapy decisions. Vigorous quality assurance and standardization of the laboratory procedures and analysis methods are important to reduce interlaboratory variation. In this uniformly treated group of patients with cancer of the uterine cervix, traditional clinical prognostic factors remain the most important. Preliminary data suggest that the flow cytometry-determined Tpot and labeling index predict for disease-free survival, although a larger number of patients with longer follow-up is required to assess the true prognostic significance of these assays and to determine if their effect is independent of other clinical factors.

Effect of distributional heterogeneity on the analysis of tumor hypoxia based on carbonic anhydrase IX.

Immunohistochemistry (IHC) is used extensively to assess markers for prognosis and sensitivity to novel anticancer agents, as well as in the routine clinical assessment of cancers. Yet, although it is well known that tumors are highly heterogeneous, the resulting sampling error in the measurement of histological markers is often ignored, particularly in basic scientific studies. In this paper, we tested the hypothesis that the optimization of tissue sampling to compensate for heterogeneity improves the correlation between histological measurements of the intrinsic hypoxia marker carbonic anhydrase IX (CAIX) and global tumor oxygenation status. The study was based on a group of 24 patients with invasive cervical carcinoma from whom multiple biopsies were obtained at the time of direct pO2 assessment within the tumor, done as part of a research study. Measurements were made by image analysis of multiple deep sections cut through these biopsies, labeled for CAIX using both immunofluorescence and immunohistochemical techniques, and included tissue microarray (TMA) simulations. Variance and correlation analysis showed that the size of the tissue sample (biopsy or TMA core) was the major factor affecting accuracy of measurement in the sample. Sampling of multiple biopsies/cores also improved the global tumor assessment, provided that these were sufficiently separated in space. Optimization of sampling resulted in an improved correlation of CAIX staining with tumor pO2 measurements, consistent with the hypothesis. However, CAIX was inferior to pO2 measurements as a tool for patient stratification. Improved analytical methods to account for intratumoral heterogeneity are needed to provide reliable measurements of molecular markers.

Tumour proliferation and apoptosis in human uterine cervix carcinoma II: correlations with clinical outcome

PURPOSEnThe prognostic value of tumour proliferation and apoptosis measurements were studied prospectively in patients with carcinoma of the uterine cervix, relative to other established clinical factors.nnnMATERIALS AND METHODSnThe labelling index (LI) for bromodeoxyuridine was determined by flow cytometry (fc) and also by immunohistochemistry. Apoptosis was assessed histologically using morphological criteria. Patients were treated with radical radiation therapy (RT).nnnRESULTSnThe median/mean LI-fc were 6.7%/7.9% (range 1.52-3.9%). The median/mean apoptosis index (AI) were 1.0%/1.6% (range 0-6.8%). To date, 27 patients have died of disease, and the median follow-up for alive patients is 3.2 years (range 0.4-6.0 years). Among 64 patients who completely responded to treatment, 25 patients have relapsed (six pelvic, 17 distant and two pelvic and distant). In univariate analysis, the most significant factors for disease-free survival (DFS) were large tumour size (P=0.0001), low haemoglobin (P=0.01 ), LI-fc (DFS 67% for LI < 7%, 33% for LI > or = 7%, P=0.03), and T(pot) (DFS 66% for T(pot) > 5 days, 35% for T(pot) < or = 5 days, P=0.04) Stage, overall treatment time (OTT), S-phase fraction, ploidy, T(s), LI by histology, mitotic index, and AI were not significant. Multivariate analysis (Coxs model) showed that the only significant prognostic factors for DFS were tumour size and OTT. However, for small tumours (diameter < 6 cm), either a high LI-fc ( > or = 7%) or a high AI ( > 1%) was associated with poorer DFS, whereas patients with larger tumours (diameter > or = 6 cm) fared poorly regardless of LI-fc and AI.nnnCONCLUSIONSnTumour size was the most important prognostic factor in cervix carcinoma. Although none of the biologic parameters have independent prognostic significance when the effect of initial tumour size was taken into account, our data suggests that LI and AI may be useful in discriminating outcome for patients with smaller tumours when managed by radical RT. These findings support the hypothesis that rapidly proliferating tumours are less likely to be controlled with a conventional course of RT.

Role of mitomycin C in the development of late bowel toxicity foloowing chemoradiation for locally advanced carcinoma of the cervix

PURPOSEnTo determine if the inclusion of mitomycin C (MMC) in chemoradiation protocols for locally advanced cervical cancer (LACC) significantly enhances the development of serious (Grade 3) late bowel toxicity (SLBT).nnnMETHODS AND MATERIALSnThe incidence of SLBT in 154 patients with LACC entered in six consecutive chemoradiotherapy protocols between February 1982 and June 1987 was determined. Fifty-four patients who were treated with MMC, 5-fluorouracil (5-FU), and radiation were compared to 100 patients who received similar treatment without MMC. Univariate and multivariate analyses assessed the effect of the following parameters on the development of SLBT: (a) external beam dose, (b) rectal and rectosigmoid dose, (c) paraaortic radiation, (d) intracavitary dose and dose rate, (e) volume of tissue irradiated to a total dose of 60 Gy, (f) International Federation of Gynecology and Obstetrics stage, (g) age, (h) number of courses of 5-FU, (i) previous abdominopelvic surgery, (j) split versus continuous radiation, and (k) administration of MMC.nnnRESULTSnThe overall incidence of SLBT was 15.6%: 14 of 54 (26%) versus 10 of 100 (10%) for patients who did or did not receive MMC, respectively (p = 0.009). Multivariate analysis revealed the administration of MMC as the only factor predictive for the development of SLBT (p = 0.012, odds ratio = 3.15; 95% confidence interval 1.3-7.7). A significant reduction in SLBT was observed with the elimination of MMC from the chemoradiation protocols despite dose escalation of both radiation and 5-FU. No increase in overall survival was observed in patients receiving MMC, 5-FU, and radiation compared with 5-FU and radiation alone.nnnCONCLUSIONnThe inclusion of MMC in these chemoradiation protocols for LACC is associated with significant enhancement in serious late bowel toxicity.

Interrelationship of proliferation and hypoxia in carcinoma of the cervix.

PURPOSEnIn human cervix cancer treated with radiotherapy, we have previously shown from separate groups of patients that tumor hypoxia and proliferation rate as measured by bromodeoxyuridine (BrdU) labeling index (LI) are important determinants of clinical outcome. We now examine the relationship of these two pre-treatment predictive assays in 43 patients studied prospectively from 1994-98 where both tests were performed for each patient.nnnMATERIAL AND METHODSnNewly diagnosed patients with carcinoma of the cervix were examined under anesthesia for staging purposes. Patients were given BrdU (200 mg) by intravenous route prior to the procedure. Tumor oxygenation was measured with the Eppendorf pO2 histograph. Biopsy of tumor was then performed and the BrdU LI was obtained by flow cytometry. The degree of tumor hypoxia for each tumor was expressed as median pO2 values, and as the percentage of pO2 readings <5 mm Hg (HP5).nnnRESULTSnThe median age was 53 years (range 23-79 years). There were 32 squamous, and 11 non-squamous carcinomas. FIGO stages were: IB and IIA, 8; IIB, 17; IIIB, 18; with a median tumor size of 6 cm (range 2-10 cm). The patients received uniform treatment with radical radiation therapy. There were 22 diploid and 21 aneuploid tumors. The median LI, pO2, and HP5 were 8.0%, 5.4 mm Hg, and 46.8%, respectively. Tests for linear associations showed no significant correlation between median pO2 vs. LI (r = 0.078, p = 0.62), and HP5 vs. LI (r = -0.14, p = 0.38).nnnCONCLUSIONSnThe clinical outcome in this group of patients is immature, but these results suggest that tumor hypoxia and proliferation measurements are independent and potentially complementary predictive assays in cervix carcinoma. Further investigations are required to examine the distribution of proliferating tumor cells and its relationship with hypoxic tumor cells in tissue sections with the use of immunohistological techniques and image analysis systems.

Tumor proliferation and apoptosis in human uterine cervix carcinoma I: correlations between variables

PURPOSEnParameters for tumor proliferation and apoptosis were studied prospectively in 84 previously untreated patients with a diagnosis of carcinoma of the uterine cervix.nnnMATERIALS AND METHODSnTumor proliferation was assessed by in vivo labeling with bromodeoxyuridine (BrdU), followed by a biopsy of the tumor 4-10 h thereafter during an examination under anesthesia. The potential doubling time (Tpot) was obtained by deriving the BrdU labeling index (LI) and S-phase duration (Ts) using flow cytometry. The LI for BrdU and its staining pattern were also determined immunohistochemically. Apoptosis was assessed histologically using morphological criteria.nnnRESULTSnSeven patients were excluded and the FIGO stages of the remaining 77 patients were as follows: IB and IIA, 20 patients; IIB, 29 patients; IIIB and IV, 28 patients. The median tumor diameter was 6 cm. There were 61 squamous cell, 11 adeno- and five adenosquamous carcinomas. Of the 63 patients in whom the tumor grade could be determined, 37 were well or moderately well differentiated and the remaining 26 were poorly differentiated. The median mitotic index (MI) was 0.7%. There were 43 diploid and 34 aneuploid tumors. Median values for Ts and S-phase fraction (SPF) were 9.9 h and 16%, respectively. The median BrdU LI by flow cytometry (LI-fc) was 6.7%. There was a significant correlation between LI-fc and LI by histology, although values for the latter (median 11.1%) were consistently higher than those determined by flow cytometry by a factor of 1.5. The median Tpot value was 5.0 days. The median apoptotic index (AI) was 1.0% and AI correlated positively with LI-fc. Median values for LI-fc increased with increasing tumor size and were 5.1%, 6.4%, 7.5% and 11.0% for tumors measuring < or = 4 cm, 4-6 cm, 6-8 cm and > 8 cm, respectively. The remaining proliferation parameters, however, showed no correlation with tumor size, stage, grade or histologic type.nnnCONCLUSIONSnIn carcinomas of the cervix, tumor proliferation is positively associated with apoptosis and tumor size. These findings suggest that parameters for tumor proliferation and apoptosis are associated with tumor progression and may thus be predictive of clinical outcome.

Circulating Human Papillomavirus DNA as a Biomarker of Response in Patients With Locally Advanced Cervical Cancer Treated With Definitive Chemoradiation

PurposeTo determine whether plasma human papillomavirus (HPV) DNA predates clinical recurrence and compare its accuracy with 3-month fluorodeoxyglucose positron emission tomography (FDG-PET) in locally advanced cervical cancer.MethodsThis prospective multicenter study accrued 23 women with stage IB to IVA cervical cancer planned for definitive chemoradiation therapy (CRT). Plasma HPV DNA was measured serially by digital polymerase chain reaction, and FDG-PET was performed at 3 months post-CRT.ResultsOf the 19 women with HPV+ cervical cancer included in this analysis, 32% were stage IB, 58% IIB, and 10% IIIB/IVA. Median follow-up was 24 months (range, 18 to 30 months). All patients had detectable plasma HPV DNA before treatment. Six patients had detectable plasma HPV DNA at the end of CRT, and three of them developed metastases at 3 months. Of the 13 patients with undetectable plasma HPV DNA at end of CRT, to date, only one has developed recurrence. Six of those 13 patients had a positive 3-month FDG-PET w...