Native fluorescence spectroscopic evaluation of chemotherapeutic effects on malignant cells using nonnegative matrix factorization analysis.

The native fluorescence spectra of retinoic acid (RA)-treated and untreated human breast cancerous cells excited with the selective wavelengths of 300 nm and 340 nm were measured and analyzed using a blind source separation method namely Nonnegative Matrix Factorization (NMF). The results show that the fluorophores of human malignant breast cells change their compositions when they are treated with RA. The reduced contribution from tryptophan, NADH and flavin to the fluorescence of the treated breast cancerous cells was observed in comparison with that of the untreated cells. The results indicate that the decrease of adenosine triphosphate (ATP) in the RA-treated cells. The possible clinical applications of this native fluorescence study are discussed.

Subsurface tumor progression investigated by noninvasive optical second harmonic tomography.

Nonlinear optical imaging with femtosecond (10(-15)-second) laser technology was used to evaluate the subsurface tumor progression in control, dysplasia, and cancerous 7, 12-dimethylbenz[a]anthracene-treated hamster cheek pouch mucosa tissues. Two-dimensional images of hamster cheek pouch mucosa tissues were obtained by scanning the second harmonic signal at various sagittal and axial positions. The spatial mapping of the second harmonic signals showed depth differentiation between normal, dysplasia, and a more advanced cancerous state. This nonlinear optical method offers a noninvasive in situ imaging tool to the medical community.

In vivo native cellular fluorescence and histological characteristics of head and neck cancer.

Native cellular fluorescence (NCF) represents the innate capacity of tissues to absorb and emit light of a specified wavelength. The ability to define the relationship of in vivo NCF with biological characteristics of neoplastic disease may allow for an improved understanding of the clinical course of disease. Head and neck cancers from 35 patients were evaluated in vivo for NCF characteristics using a xenon lamp-based spectrometer coupled to a handheld fiberoptic probe. Spectral assessment was limited to lambda 450-nm emission characteristics, in which tissues were excited at various wavelengths, ranging from lambda 290 nm to lambda 415 nm, and the intensity of lambda 450 nm emission was recorded. Each cancer was subsequently biopsied and assessed for histological differentiation by a pathologist who was blinded to NCF analysis. Considerable variation in spectral characteristics between head and neck cancers was identified, which was determined, in part, by NCF characteristics of the normal mucosa from the same patient. Poorly differentiated tumors were more likely than well- or moderately differentiated tumors to have lower excitation maxima (P < 0.05 by ANOVA). Most significantly, the tumor differentiation status, as well as the probability of demonstrating recurrent disease, could also be related to the NCF characteristics of the patient's normal mucosa from the same site within the upper aerodigestive tract. NCF analysis may represent an effective tool to identify biological characteristics of head and neck tumors in vivo without the need for invasive biopsies. Results suggest the need to explore the determinants of NCF characteristics expressed by clinically normal mucosa.

Native fluorescence spectroscopy of normal and malignant epithelial cells.

Native fluorescence spectroscopy of normal human oral and malignant epithelial cells was studied under uv excitation. Differences were observed in the excitation spectra between normal and malignant epithelial cells for 340 nm emission. The observed differences may be utilized for both discrimination and changes associated with the amino acid residues in the cellular proteins.

Native cellular fluorescence and its application to cancer prevention.

Native cellular fluorescence (NCF) represents the innate capacity of tissues to absorb and emit light of specified wavelengths. Recent advances in optical engineering and computer technology have provided the, opportunity to measure NCF characteristics of various tissues in vivo. This report will briefly review the current status of NCF analysis of various neoplastic tissues. The status of investigations involving the upper aerodigestive tract will be discussed. Though initial results demonstrate that neoplastic tissues can be discriminated from normal mucosa by NCF analysis, the biologic basis of this difference remains uncertain. This report will also emphasize that the ability to screen for cancer in aerodigestive mucosa may be enhanced through the assessment of multiple emission and excitation wavelengths. The true nature of the cellular fluorophores responsible for these mucosal spectral characteristics should be more fully defined in coming years.

Development of infants and toddlers with clefts from birth to three years of age.

OBJECTIVE: The purpose of this study was to use caregiver report measures to describe the developmental status of infants and toddlers with clefts. METHOD: Developmental assessment data were obtained on 186 infants and toddlers with cleft lip (n = 48), cleft palate (n = 46), and cleft lip/palate (n = 92) at one of the following age categories: 5 months (n = 47), 13 months (n = 46), 25 months (n = 47), and 36 months (n = 46). Developmental assessment measures used were the Kent Infant Developmental Scale and the Minnesota Child Development Inventory, both caregiver reports. Data were analyzed in separate 2-between ANOVAs (age x cleft type) for each developmental domain according to developmental assessment measure. Further, results were examined relative to the normative sample. RESULTS: The ANOVA results indicated that at 5 months, lower motor and self-help developmental quotients (DQs) were evident compared to the 13-month-old level. When compared to the normative sample, the 5-month-old infants exhibited 'at-risk/delayed' development on the motor, self-help, and cognitive domains, and as reflected on their full-scale scores, depending on the cleft type. Infants at 13 and 25 months were within normal limits in all developmental domains, with the exception of the 13-month-old infants with cleft palate, who demonstrate 'at-risk' development in the motor domain. At 36 months of age, all toddlers demonstrated significantly lower developmental performance in the fine motor, gross motor, and expressive language domain compared to the 25-month-old toddlers. Toddlers with cleft palate exhibit 'at-risk/delayed' development in the expressive language domain at 36 months. CONCLUSION: Data are discussed relative to the events surrounding team management of clefts, including surgery, middle-ear problems, and feeding difficulty.

Innate cellular fluorescence reflects alterations in cellular proliferation.

BACKGROUND AND OBJECTIVE: The objective of this study was to examine the question of whether unique spectral patterns were associated with cell proliferation and could be identified by comparing the fluorescence pattern of slow to rapid growing cells. STUDY DESIGN/MATERIALS AND METHODS: Three in vitro model systems, (A431 cells inhibited by EGF, serum-starved 3T3 fibroblasts, and normal oral epithelial cells exposed to TGF beta), were analyzed using fluorescence spectroscopy. Growth status was monitored by cell number, 3H-thymidine incorporation, and flow cytometry. RESULTS: The excitation spectra (lambda ex 240-430 nm, lambda em 450 nm) effectively distinguished slow and rapid growing cells in all three systems. Statistical analysis of the ratios of the main broad peak (320-350 nm) to a point on the down-slope of the curve at 370 nm was statistically significant. Ratios in the emission scan (lambda ex 340 nm, lambda em 360-660 nm) could separate slow and rapid growing A431 and oral epithelial cells (P = 0.0001 and P = 0.023, respectively), but not slow and fast growing 3T3 cells (P = 0.56). CONCLUSION: Innate cellular fluorescence has the potential to discriminate proliferating and nonproliferating cell populations.

Native cellular fluorescence identifies terminal squamous differentiation of normal oral epithelial cells in culture: a potential chemoprevention biomarker.

Native cellular fluorescence (NCF) is being investigated as an intermediate endpoint biomarker for chemoprevention. Oral epithelial cells were cultured under three conditions to identify a spectral pattern for epithelial differentiation: cells maintained in serum-free keratinocyte growth medium were the least differentiated (KGM cells); cells switched to DMEM/F12 plus 10% FCS were intermediate in differentiation (DMEM/F12/FCS cells); DMEM/F12/FCS cells switched to serum-free DMEM/F12 plus 0.8 M NaCl to induce cornified envelopes were the most differentiated (DMEM/F12/NaCl cells). The differentiation status was characterized using immunohistochemistry and electron microscopy. NCF analysis was able to distinguish terminally differentiated epithelial cells (DMEM/F12/NaCl) from those less differentiated cells (KGM, DMEM/F12/FCS) in several emission (lambda ex 340 nm, lambda em 360-660 nm; lambda ex 365 nm, lambda em 400-700 nm; lambda ex 420 nm, lambda em 440-800 nm) and excitation scans (lambda ex 200-360 nm; lambda em 380 nm, lambda ex 240-430 nm; lambda em 450 nm, lambda ex 250-460 nm, lambda em 480 nm; lambda ex 270-500 nm, lambda em 520 nm). The ability to discriminate terminal differentiation in this in vitro model supports the concept of using NCF as an intermediate biomarker to monitor in vivo mucosal differentiation.

Native cellular fluorescence of neoplastic upper aerodigestive mucosa.

OBJECTIVE: To identify alterations in the biochemical composition and histoarchitectural structure of the lining epithelium that signal malignant transformation in carcinogen-exposed upper aerodigestive mucosa by quantitation of native cellular fluorescent properties. DESIGN: Case series. SETTING: Head and Neck Service, Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY. PATIENTS AND METHODS: Thirty-one patients with previously untreated mucosal neoplasias of the oral cavity and pharynx were studied by means of a hand-held fiberoptic probe attached to a xenon lamp-based fluorescent spectrometer. Fluorescence analyses of the lesions and contralateral normal sites were performed in each patient on the basis of specific emission and excitation wavelengths. Differences between normal tissue and neoplastic mucosa were tested for statistical significance by paired t test and Hotelling's T2 test. RESULTS: The ratios of fluorescence intensities of neoplastic mucosa and contralateral normal sites were significantly different in three of the four fluorescence scans (excitation of 300 nm and emission of 320 to 580 nm; excitation of 340 nm and emission of 360 to 660 nm; and excitation of 200 to 360 nm and emission of 380 nm) when analyzed individually (P < .05). The ratios in the scans with excitation of 240 to 430 nm and emission of 450 nm were not significantly different. All four scans, when analyzed together, demonstrated significant differences between normal and neoplastic tissues (P < .01). CONCLUSIONS: Neoplastic mucosa of the upper aerodigestive tract within an individual patient will express native cellular fluorescent properties in vivo that differ from those of normal upper aerodigestive epithelia. This may represent a noninvasive screening method for head and neck squamous cell cancers.

Native cellular fluorescence can identify changes in epithelial thickness in-vivo in the upper aerodigestive tract.

BACKGROUND: Change in epithelial thickness is part of the neoplastic transformation process of the upper aerodigestive tract. The quantitation of native cellular fluorescence (NCF) may represent a noninvasive means of distinguishing such a change. PATIENTS AND METHODS: Nineteen patients with squamous neoplasms and 12 surgical specimens from cancer patients were analyzed for NCF using a hand-held fiber optic probe attached to a fluorescent spectrometer. Tumors and normal sites were analyzed for fluorescence, and tissue samples were obtained. Ratios of intensities of various emitted wavelengths were computed to quantitate and compare various spectral patterns. These ratios were then correlated with mucosal thickness. RESULTS: The 330 nm peak in the excitation scan (lambda Ex 200 to 360 nm, lambda Em 380 nm) was lost in the tumors compared with the normal sites. The 390 nm peak in the emission scan (lambda Ex 340 nm, lambda Em 360 to 660 nm) was also lost. The 290 nm/330 nm ratio in the in-vivo excitation scan (lambda Ex 200 to 360 nm, lambda Em 380 nm) correlated with changes in epithelial thickness. The 390/450 ratio in the emission scan (lambda Ex 340 nm, lambda Em 360 to 660 nm) correlated negatively with the mean epithelial thickness. CONCLUSIONS: Native cellular fluorescence analysis can identify changes in neoplastic tissues, including changes in epithelial thickness.

Detecting retinoic acid-induced biochemical alterations in squamous cell carcinoma using intrinsic fluorescence spectroscopy.

Intrinsic fluorescence spectroscopy offers a new method for diagnosing head and neck cancers. By establishing a unique spectral fingerprint for benign tissue, one can readily identify subtle changes in tissue based on altered spectral patterns. The authors applied this technology to a multicellular tumor spheroid (MTS) model and obtained baseline spectral data. A cohort of MTS was treated with the chemopreventive agent retinoic acid (RA) to determine its effect on tumor cells. Excitation and emission spectroscopy were performed on the samples. Spectroscopic scans demonstrated consistently that RA-treated MTS exhibit a decrease in the peak associated with reduced nicotinamide-adenine dinucleotide (NADH) and an increase in the peaks associated with flavins, tryptophan, and cytokeratins when compared to controls. These findings are suggestive of alterations in cellular electron transport, an increase in proteins incorporating tryptophan, and a decrease in adenosine triphosphate (ATP) in the RA-treated cells. A discussion of the potential clinical applications of intrinsic fluorescence spectroscopy is included.

Clinical correlates of circulating immune complexes and antibody reactivity in squamous cell carcinoma of the head and neck. The Department of Veterans Affairs Laryngeal Cancer Study Group.

PURPOSE: To evaluate the correlation between the presence and titer of host-derived antibody reactivity, circulating immune complexes, and clinical course and prognosis in patients with squamous cell carcinoma of the head and neck (SCCHN). MATERIALS AND METHODS: Serum samples, obtained from untreated patients with squamous cell carcinoma of the larynx entered onto a multiinstitutional trial, were evaluated for the presence of elevated circulating immune complexes (221 patients) and host-derived antibody directed against two SCCHN cell lines (107 patients). RESULTS: Patients had significantly elevated levels of circulating immune complexes as measured by C1q binding compared with normal controls. Patients with higher levels of circulating immune complexes were less likely to respond to chemotherapy. No correlations were noted between immune complex levels and stage of disease, nodal status, site of disease, recurrence, or survival. Evaluation of native antibody titers for their relationship to clinical correlates showed no statistically significant associations. In sera subjected to immune complex dissociation, patients with moderately or poorly differentiated tumors had significantly higher antibody titers when compared with patients with well-differentiated tumors. Because marked variation in the increase of antibody titers following immune complex dissociation was noted, the ratio of immune complex-dissociated to native antibody titer was examined. Patients with a high ratio had a lower proportion of complete and partial responses to chemotherapy. CONCLUSION: Our results support the conclusion that the formation of tumor-associated immune complexes in patients with SCCHN is associated with a decreased response to chemotherapy.

Serum and acute phase protein modulation of the effector phase of lymphokine-activated killer cells.

An understanding of the role that immunomodulatory factors play in the effector phase of lymphokine-activated killer (LAK) activity is essential for the development of biologic response modifiers for use in the treatment of advanced carcinoma. Fifteen head and neck cancer patients were studied. Single-donor killer cells activated by recombinant interleukin-2 (10 U/mL) and induced in either a complete medium or complete medium plus a 10% autologous serum solution were used. Effector phase solutions of 25% autologous serum were used in chromium 51 release assays to determine sera immunomodulation of LAK cell cytotoxicity. Both K562 and squamous carcinoma (MDA686-Ln) tumor cell lines were tested. Significant effector phase inhibition (EPI) of cytotoxicity occurred in 40% of studied patients. Seventy percent of patients with stage III or IV or recurrent disease exhibited EPI, whereas only 20% of patients with stage I or II disease and 30% of controls did so. EPI of cancer patient serum correlated directly with alpha 1-antitrypsin, alpha 1-acid glycoprotein, and C-reactive protein (CRP) levels (MDA686-Ln targets) (r = 0.6, 0.7, and 0.6, respectively) (P < .02). Neither EPI against K562 targets nor EPI in control patients correlated with acute phase protein levels. These findings suggest that advances in in vivo immunomodulatory therapy will be dependent upon further elucidation of serologic inhibition of the effector phase of the LAK cell phenomenon. The relationship between LAK cell recognition and EPI requires further investigation.

Serologic determinants of survival in patients with head and neck cancer: validating a clinical prediction model.

Quantitative measurements of serum C1q-binding macromolecules (C1qBM) and immunoglobulin A (IgA) were done on 162 patients using previously described methodology. The measurements were compared to a previously described head and neck cancer population. Using the Cox Proportional Hazards model, the prognostic implications regarding high C1qBM and subsequent death with disease (P = .02), and regional recurrence (P = .0094) were validated, but not our previous IgA-related prognostic implications. When both study populations were combined, C1qBM was predictive of survival in those patients treated with induction chemotherapy (P = .0001). C1qBM was not a significant predictor of survival in patients treated with surgery plus postoperative radiation therapy in either this second "test" population or in the original "training" population. The findings demonstrate the confounding influence of treatment modalities and the importance of model validation.

Detection of regulatory factors of lymphokine-activated killer cell activity in head and neck cancer patients treated with interleukin-2 and interferon alpha.

Interleukin-2 (IL-2) and interferon-alpha (INF-alpha) are biologic modifiers that have met with limited clinical success in the treatment of human malignancies. We conducted a phase 2 trial of IL-2-IFN-alpha in patients with advanced or unresectable squamous cell carcinoma of the upper aerodigestive tract. Two patients were analyzed sequentially for serum induction phase-blocking factors of lymphokine-activated killer (LAK) cell activity in their therapy. Serum also modulated LAK activity independent of autologous or allogeneic effector cells. Significantly inhibitory serum samples were stable in multiple freezings and thawings. Heat-treating the inhibitory serum, at 56 degrees C for 30 minutes, only partially removed the serum inhibitory capacity. Sequential analysis of p55 and p75, subunits of IL-2 receptors, showed that absence of effector cell lytic activity was associated with markedly decreased fluorescence of the IL-2Rp75 subunit only. No significant alteration of the IL-2Rp55 subunit occurred with therapy. These studies support the theory that lymphocyte and multiple serum factors, developing during IL-2-IFN-alpha therapy, regulate the induction of in vitro LAK activity. Further understanding of these factors may lead to improvements in biologic modifier therapy.

Acute-phase proteins in patients with head and neck cancer treated with interleukin 2/interferon alfa.

Circulating acute-phase proteins may mediate adverse reactions in patients receiving biologic response modifiers, including inhibition of immune responsiveness and clinical toxic effects. Nine patients with unresectable head and neck squamous cell carcinoma were prospectively examined for levels of acute-phase proteins during interleukin 2/interferon alfa immunotherapy and for clinical toxic effects. Simultaneous determination of the in vitro immunomodulatory capacity of autologous serum on the induction of lymphokine-activated killer cells was assessed in 4-hour chromium release assays. Of the seven acute-phase proteins analyzed, haptoglobin and C-reactive protein levels were elevated before therapy was started. Toxic events leading to cessation of interleukin 2/interferon alfa therapy had a high correlation with elevated C-reactive protein and lowered C3 component of complement levels. No relationship was noted between serum levels of acute-phase proteins and induction inhibition of lymphokine-activated killer cell cytotoxicity. The role of C-reactive protein and complement degradation products in mediating interleukin 2/interferon alfa toxicity requires further investigation.

Immunomodulation of the induction phase of lymphokine-activated killer activity by acute phase proteins.

Effective treatment of head and neck cancer with biologic response modifiers may be benefitted by an understanding of in vivo factors capable of modulating the lymphokine-activated killer (LAK) cell phenomenon. Eighteen patients with squamous cell carcinoma of the head and neck were studied. Killer cells from each patient, activated by recombinant interleukin-2 (10 U/ml), were induced in either complete medium alone or complete medium plus 10% autologous serum solution and analyzed. Cytotoxicity against both K562 and squamous cell carcinoma (MDA686-Ln) cell lines was determined by use of standard chromium-release assays. The immunomodulatory capacity of serum was correlated with levels of various acute phase proteins. Autologous serum significantly inhibited the induction phase of the LAK phenomenon in 61% of patients and stimulated it in 22%. No patients with early stage I or II disease had significant inhibition of induction. No direct correlation between inhibition and serum acute phase protein levels were seen. An inverse relationship was seen between the C3 component of complement and induction inhibition (r = -0.6). These findings suggest that advances of in vivo immunomodulatory therapy will require elucidation of mechanisms of serologic inhibition of the induction phase of the LAK phenomenon. Such studies may lead to serologic modification to enhance treatment efficacy of biologic response modifiers.

Circulating C1q-binding macromolecules and their relationship to radiographic characteristics of laryngeal cancer.

Circulating macromolecules capable of binding the first component of complement (C1qBM) may represent subcellular components of tissue/tumor debris generated from rapidly proliferating invasive disease. Thirty-eight patients were randomly selected from 74 untreated patients with laryngeal cancer on the basis of disease stage and C1qBM levels. C1qBM levels were correlated with computed tomographic evidence of tumor necrosis and/or thyroid cartilage destruction. Results show that patients with stage III/IV disease with tissue necrosis and/or cartilage invasion had demonstrably higher C1qBM levels than did individuals with similarly staged disease with no evidence of these radiographically defined characteristics (120 +/- 81 micrograms/mL vs 18 +/- 15 micrograms/mL); the strongest association was reflected by the area of necrosis within regional lymph metastases. Elevated C1qBM level in patients with stage III/IV laryngeal cancer thus reflects highly aggressive disease, which is less responsive to therapeutic intervention.

Serologic determinants of survival in patients with squamous cell carcinoma of the head and neck.

Specific circulating serum proteins may reflect unique properties governing the growth and progression of head and neck cancers. One hundred three previously untreated patients with squamous cell carcinoma of the head and neck were prospectively evaluated for serum IgA, IgG, and IgM and C1q-binding macromolecules. Immunoglobulins were assessed by the immunoturbidimetric technique. C1q-binding macromolecules (C1qBM) were measured utilizing the iodine-125 assay of Zubler et al (J Immunol 1976; 116: 232-5). Neither the level of serum immunoglobulins nor C1qBM values were correlated with the primary site, AJC (American Joint Committee on Cancer) stage of disease, or size of primary lesion. Likewise, comparison of serum IgA with C1qBM values demonstrated that these laboratory parameters were independent variables (r = 0.15 by Pearson linear regression). Univariate statistical analysis, utilizing the Cox proportional hazard model, showed serum IgA and C1qBM values to each contribute significantly to the ability to predict survival in patients with advanced squamous cell carcinoma of the head and neck (p = 0.01 and 0.003, respectively). Furthermore, multivariate analysis reveals that both C1qBM and serum IgA levels contribute significantly to the hazards model beyond staging in predicting survival (p less than 0.001). Predictive results were most apparent in patients with stage IV disease and related to the probability of both regional and distant metastatic recurrences. Conversely, serologic analysis provided no information in patients who were staged early. These results support pretreatment multiparametric serologic analysis of patients with squamous cell carcinoma of the head and neck.

Significance of C1q-binding macromolecules within the head and neck cancer patient.

Elevated levels of macromolecules, within the peripheral blood of head and neck cancer patients, capable of binding the first component of complement (C1qBM) in vitro have prognostic implication. Namely, elevated levels of C1qBM have been associated with nonresponse to induction chemotherapy. In this investigation, a series of in vitro studies regarding the biological properties of C1qBM were combined with a longitudinal analysis of 112 previously untreated head and neck cancer patients. Our purpose was to shed light on the biological significance of this circulating macromolecule, a substance composed, in part, of IgG and IgM. A potential confounding influence of C1qBM with induction chemotherapy, which could contribute to observed prognostic findings, was negated by two in vitro observations: the macromolecule failed both to bind the chemotherapeutic agents cisplatin, bleomycin, and 5-fluorouracil and to impede the cytotoxic effect of these same drugs on a cultured human head and neck cancer cell line. The clinical relevance of C1qBM was reinforced by the observation that elevated levels predicted a high probability of death with disease (P = 0.005 by Cox's proportional hazards model). The prognostic implication was independent of the use of induction chemotherapy, i.e., patients with high C1qBM levels treated with multimodality therapy not composed of anticancer drugs did equally poorly. Thus, the prognostic significance of C1qBM in patients undergoing induction chemotherapy appears independent of drug effect and appears reflective of tumors that are more rapidly progressive and potentially less responsive to therapeutic intervention, including combinations of surgery, radiation, and/or chemotherapy.